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  1. Supplementary Sidebars Supplementary Sidebar 1.1 Supplementary Sidebar 1.2 Supplementary Sidebar 1.3 Supplementary Sidebar 1.4 Supplementary Sidebar 1.5 Supplementary Sidebar 2.1 Supplementary Sidebar 2.2 Supplementary Sidebar 3.1 Supplementary Sidebar 3.2 Supplementary Sidebar 3.3 Supplementary Sidebar 3.4 Supplementary Sidebar 3.5 Supplementary Sidebar 4.1 Supplementary Sidebar 4.2 Supplementary Sidebar 4.3 Supplementary Sidebar 4.4 Supplementary Sidebar 5.1 Supplementary Sidebar 5.2 Supplementary Sidebar 5.3 Supplementary Sidebar 6.1 Supplementary Sidebar 6.2 Supplementary Sidebar 6.3 Supplementary Sidebar 6.4 Supplementary Sidebar 6.5 Supplementary Sidebar 7.1 Supplementary Sidebar 7.2 Supplementary Sidebar 7.3 Supplementary Sidebar 7.4 Supplementary Sidebar 7.5 Supplementary Sidebar 7.6 Supplementary Sidebar 7.7 Supplementary Sidebar 8.1 Supplementary Sidebar 8.2 Supplementary Sidebar 8.3 Supplementary Sidebar 8.4 Supplementary Sidebar 8.5 Supplementary Sidebar 8.6 Supplementary Sidebar 9.1 Supplementary Sidebar 9.2 Supplementary Sidebar 9.3 Supplementary Sidebar 9.4 Supplementary Sidebar 9.5 Supplementary Sidebar 10.1 Supplementary Sidebar 11.1 Supplementary Sidebar 11.2 Supplementary Sidebar 11.3 Supplementary Sidebar 11.4 Supplementary Sidebar 11.5 Supplementary Sidebar 11.6 Supplementary Sidebar 11.7 Supplementary Sidebar 12.1 Supplementary Sidebar 12.2 Supplementary Sidebar 12.3 Supplementary Sidebar 12.4 Supplementary Sidebar 12.5 Supplementary Sidebar 12.6 Each female cell can access information only from a single X chromosome Reproductive cloning demonstrates the extraordinary efficiency of the DNA repair apparatus The network of miRNA-controlled genes Knocking down gene expression with shRNAs and siRNAs Gene cloning strategies Commonly used histopathological techniques The complicated conventions for classifying and naming tumors Is SV40 responsible for the mesothelioma plague? Maintenance of KSHV and HPV genomes in episomal and chromosomal form Re-engineering the retrovirus genome for gene therapy Classic Kaposi’s sarcoma appears to be a familial disease Viruses like RSV have very short lives Endogenous viruses can explain tumor development in the absence of infectious viral spread Boveri and Hansemann independently hypothesized genetic abnormality as the cause of cancer cells’ malignant behavior Southern and Northern blotting Genes undergo amplification for a variety of reasons Making anti-Src antibodies presented a major challenge The protozoan roots of metazoan signaling Lateral interactions of cell surface receptors Systematic surveys of phosphotyrosine and SH2 interactions The complexities of understanding RTK signaling The rationale for multi-kinase signaling cascades Non-canonical Wnt signaling The Hippo pathway and control of stem cell proliferation Heterozygosity in the human gene pool Which is more likely—LOH or secondary, independent mutations? The polymerase chain reaction makes it possible to genetically map tumor suppressor genes rapidly The MSP reaction makes it possible to gauge methylation status of promoters Ubiquitylation tags cellular proteins for destruction in proteasomes Krebs cycle enzymes and cancer development Homologous recombination allows restructuring of the mouse germ line The origins of embryonic stem cells Plasticity of the cell cycle clock Chromatin immunoprecipitation (ChIP) Some tumors increase Id concentrations by de-ubiquitylating them Specific targeting of cell cycle regulators by E3 ubiquitin ligases The major puzzle surrounding the RB gene: retinoblastomas UV-B radiation, HPV, and cutaneous squamous cell carcinomas The TUNEL assay Dominant-negative functions of mutant p53 alleles: functional interactions between p53 and its p63 and p73 cousins Some mutant p53 alleles cause highly specific tumors Autophagy is critical to post-fertilization development The use of the TRAP assay and adaptations thereof permits the rapid and quantitative assessment of the levels of the catalytic activity of telomerase enzyme in eukaryotic cells Monoclonal antibodies and fluorescence-activated cell sorting (FACS) How does multi-step tumor progression actually take place? Symbiosis between distinct subpopulations within a tumor Comparative genomic hybridization Are rodent carcinogen tests reliable indicators of danger to humans? Does saccharin cause cancer? How does diet affect colon cancer incidence? Hematopoiesis as a model for the organization of many kinds of tissues Stem cell pools may explain the protective effects of pregnancy The conserved-strand mechanism and protection of the stem cell genome Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular genome How does red meat cause colon cancer? A convergence of bacterial, yeast, and human genetics led to the discovery of hereditary non-polyposis colon cancer genes 1 2 3 4 6 7 9 11 12 14 16 17 18 20 21 22 23 24 25 26 28 29 30 31 32 33 34 35 36 38 41 43 44 45 46 47 48 50 52 53 54 55 56 57 59 61 62 63 64 65 67 69 72 75 76 77
  2. Supplementary Sidebar 12.7 Supplementary Sidebar 13.1 Supplementary Sidebar 13.2 Supplementary Sidebar 13.3 Supplementary Sidebar 13.4 Supplementary Sidebar 13.5 Supplementary Sidebar 13.6 Supplementary Sidebar 13.7 Supplementary Sidebar 13.8 Supplementary Sidebar 13.9 Supplementary Sidebar 13.10 Supplementary Sidebar 14.1 Supplementary Sidebar 14.2 Supplementary Sidebar 14.3 Supplementary Sidebar 14.4 Supplementary Sidebar 14.5 Supplementary Sidebar 14.6 Supplementary Sidebar 14.7 Supplementary Sidebar 14.8 Supplementary Sidebar 14.9 Supplementary Sidebar 14.10 Supplementary Sidebar 14.11 Supplementary Sidebar 14.12 Supplementary Sidebar 14.13 Supplementary Sidebar 14.14 Supplementary Sidebar 15.1 Supplementary Sidebar 15.2 Supplementary Sidebar 15.3 Supplementary Sidebar 15.4 Supplementary Sidebar 15.5 Supplementary Sidebar 15.6 Supplementary Sidebar 15.7 Supplementary Sidebar 15.8 Supplementary Sidebar 15.9 Supplementary Sidebar 15.10 Supplementary Sidebar 15.11 Supplementary Sidebar 15.12 Supplementary Sidebar 15.13 Supplementary Sidebar 15.14 Supplementary Sidebar 15.15 Supplementary Sidebar 15.16 Supplementary Sidebar 16.1 Supplementary Sidebar 16.2 Supplementary Sidebar 16.3 Supplementary Sidebar 16.4 Supplementary Sidebar 16.5 Supplementary Sidebar 16.6 Supplementary Sidebar 16.7 Supplementary Sidebar 16.8 Supplementary Sidebar 16.9 Supplementary Sidebar 16.10 Supplementary Sidebar 16.11 Homology-directed repair Localization of growth factors is important for proper heterotypic interactions Ongoing heterotypic signaling in carcinomas Certain highly advanced tumors provide exceptions to the generally observed dependence of carcinoma cells on stroma Myofibroblasts predict clinical progression of cancer A technique for separating stromal from epithelial cells Microvessel leakiness dooms many forms of anti-cancer therapy: optimizing anti-angiogenic treatments The temporary nature of vessel regression created by anti-angiogenesis therapy Kaposi’s sarcoma cells hold the record for the number of documented heterotypic signals they receive Effects of an anti-VEGF-R monoclonal antibody on the growth of a human tumor xenograft Fibroblasts are heterogeneous and can change dynamically in response to signals Visualization of the dynamics of pathfinding fibroblasts followed by squamous cell carcinoma cells Metastasizing cancer cells often take on hitchhikers while traveling through the blood Instruments for detecting circulating tumor cells Hidden micrometastases are revealed through organ transplantation Wolves in sheep’s clothing: when carcinoma cells invade the stroma TGF-β works in conflicting ways during tumor progression Dynamics of EMT induction: the EMT may be controlled in some cancer cells exclusively by their own genomes An example of an EMT relatively late in embryonic development Relatively rapid metastatic dissemination of advanced primary tumor cells Our cells devote an enormous number of genes to regulating protein degradation Peritoneovenous shunts provide dramatic support for the seed and soil hypothesis Tooth extractions may occasionally become exceedingly painful Tumor stem cells further complicate our understanding of the metastatic process Does Darwinian evolution accommodate metastasis-specific alleles? Rearrangements of chromosomal DNA segments generate a vast array of antigen-binding domains in antibodies and T-cell receptors Virus-infected cells may not always be recognized by the immune system Bizarre tumors reveal how cancer cells can become infectious agents Mice have proven to be far more useful for tumor biologists than chickens An HPV vaccine protects against many cervical carcinomas An unexpected type of anti-p53 reactivity is often found in cancer patients Immune recognition of tumors may be delayed until relatively late in tumor progression Some paraneoplastic syndromes reveal defective tolerance and overly successful immune responses to tumors TSTAs can arise as by-products of chemical and physical carcinogenesis Are melanomas more antigenic than other tumors? Strategies for cloning genes encoding melanoma TATAs Anti-CD47 therapies hold promise in treating lymphomas and other hematopoietic malignancies Cancer cells may thwart extravasation by circulating T cells Herceptin can be modified to potentiate cancer cell killing Bone marrow transplantation and the treatment of hematopoietic malignancies Whole genome sequencing allows a new attack on tumor cells Modern cancer therapies have had only a minor effect on the overall death rate from the disease Prostate cancers usually do not require aggressive intervention—a tale of two countries Clinical practice and our understanding of disease pathogenesis have often been poorly aligned, leading to sub-optimal, often tragic outcomes The ability to assign tumors to specific disease subtypes is critical to the success of drug development p53 germ-line polymorphisms and somatic mutations can complicate the induction of apoptosis by drugs Ras function can be inhibited by interfering with the enzymes responsible for the maturation of the Ras protein Chemical synthesis, compound libraries, and high-throughput screening Evolution can generate huge collections of structurally similar proteins Large-scale screen of the inhibitory effects of a drug on various kinases Epidermal growth factor receptor expression levels predict little about a tumor’s susceptibility to receptor antagonists Akt/PKB function is controlled by multiple upstream signals 78 79 80 81 82 83 84 86 88 89 90 92 93 96 98 99 100 101 102 103 104 105 106 107 108 109 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 131 132 134 135 138 140 142 143
  3. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.1 Each female cell can access information only from a single X chromosome In the simplest depiction of sex chromosome behavior, each male cell relies on the information carried by its single X chromosome, while each female cell is able to consult both of its X chromosomes. Given the large number of genes present on the X chromosome, this disparity would create substantial physiologic differences between male and female cells, since female cells would, in general, express twice as much of each of the products specified by the genes on their X chromosomes. This problem is solved by © 2014 Garland Science the mechanism of X-inactivation. Early in embryogenesis, one of the two X chromosomes is randomly inactivated in each of the cells of a female embryo. This inactivation silences almost all of the genes on this chromosome and causes this chromosome to shrink into a small particle termed the Barr body (Figure S1.1). Subsequently, all descendants of that cell will inherit this pattern of chromosomal inactivation and will therefore continue to carry the same inactivated X chromosome. Accordingly, the female advantage of carrying redundant copies of X chromosome–associated genes is only a partial one. Figure S1.1 X chromosomes as Barr bodies One of the two X chromosomes in each cell of an early female embryo is randomly inactivated and remains inactivated in all lineal descendant cells. It is visible in the interphase nuclei of these cells, where it remains condensed and associated with the nuclear membrane (white arrow). (From B.P. Chadwick et al., Semin. Cell Dev. Biol. 14:359–367, 2003.) TBoC2 b1.10/s1.01 1
  4. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.2 Reproductive cloning demonstrates the extraordinary efficiency of the DNA repair apparatus The efficiency of the complex apparatus that repairs DNA (see Chapter 12), the resulting suppression of somatic mutations, and the consequent integrity of the genomes of somatic cells have all been illustrated dramatically by the successes of animal cloning in recent years. In the much-celebrated case of the sheep Dolly, the nuclei from cells taken from the breast tissue of her “mother” were removed and implanted in egg cells (oocytes) whose own nuclei had previously been eliminated. The resulting cells, containing donor nuclei and recipient egg cytoplasm, were then induced to proliferate and to form embryos (Figure S1.2). The fact that an essentially normal sheep (Dolly) was born many months later, having developed from one of these embryos, demonstrated that the genome in one of © 2014 Garland Science her mother’s mammary gland cells carried no mutations that compromised normal embryologic development. The implied intactness of the genome in the breast cells of Dolly’s mother indicates a high degree of integrity in DNA replication. During embryonic development, the genome of the fertilized egg that was destined to develop into Dolly’s mother was copied and recopied dozens of times during the cycles of cell growth and division that intervened between the initial formation of the fertilized egg and the formation, many months later, of the mother’s breast tissue. This recopying process, involving DNA replication and a repair apparatus that operated to weed out mutant DNA sequences, was highly effective in preserving an intact somatic cell genome that was capable of programming normal organismic development. Dolly’s “mother” cells prepared from mammary gland single cell prepared cells fuse by electric shock unfertilized sheep egg haploid nucleus removed egg induced to divide early embryo embryo transferred to womb of second sheep Dolly is born enucleated egg Figure S1.2 Reproductive cloning and genomic integrity In the initially reported reproductive cloning procedure, the nucleus of an unfertilized egg was removed and the nucleus from a somatic cell—a mammary gland cell from Dolly’s “mother”—was introduced into the enucleated egg. The resulting cell was diploid, having received its entire genetic complement from the mammary gland cell originating with Dolly’s “mother.” Being diploid, it was genetically equivalent to a fertilized egg. This reconstituted egg cell was induced to divide by chemical means and proceeded to generate an embryo that could then be implanted in the womb of a foster “pseudo-pregnant” ewe—a ewe whose physiologic state was comparable to that of a naturally pregnant ewe but had been induced in this case by hormonal treatment. This implanted embryo could then develop into a newborn, albeit at low efficiency. Successful generation of a healthy newborn revealed that the donor somatic cell from Dolly’s “mother’s” mammary gland carried the genome of the species in an essentially intact, functional form. This donor mammary gland cell initially arose after many successive cell divisions during the development of Dolly’s “mother” from a natural fertilized egg. The ability of this cell’s genome to generate Dolly indicated that these many successive cell divisions did not affect its integrity. TBoC2 b1.13/s1.02 2
  5. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.3 The network of miRNA-controlled genes Research into the functions of microRNAs makes it increasingly apparent that the molecules are key components of myriad regulatory circuits within cells. Indeed, it is plausible that almost all regulatory circuits rely on one if not several miRNAs in order to modulate or fine tune the signals that they are © 2014 Garland Science processing. The image presented here (Figure S1.3) presents the state-of-the-art in miRNA research in 2008 that focused on miRNAs that affect signaling pathways relevant to cancer pathogenesis. Subsequently reported research has generated a far more elaborate network of interactions. been demonstrated by 2008 to play a role in modulating various Figure S1.3 OncomiRs and cancer pathogenesis cancer-related cell-biological phenotypes; this class of microRNAs Characterizations of microRNAs have revealed that they play have been dubbed oncomiRs. The complexity of this microRNAimportant roles in modulating the expression of proteins in a TBoC2 n1.108/s1.03 regulated circuitry is growing progressively. (From Melanoma wide variety of subcellular regulatory circuits, including those that Molecular Map Project; see http://www.mmmp.org/MMMP/public/ are deregulated during the course of cancer development. This biomap/viewBioMapImage.mmmp) map illustrates the involvement of a series of microRNAs that had 3
  6. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.4 Knocking down gene expression with shRNAs and siRNAs The discovery of microRNAs (miRNAs), as described in Section 1.10, led to an understanding of the complex molecular machinery that allows their processing from primary transcripts in the nucleus and their dispatch into the cytoplasm. There they inhibit the functioning of thousands of cellular mRNA species. At the same time, these discoveries enabled the development of novel experimental strategies in which exogenous miRNAs could be introduced into cells through the use of viral vectors or transfection, whereupon these introduced RNAs exploit the cellular miRNA-processing machinery to produce mimics of endogenously synthesized miRNAs; the mimics then proceed to shut down the operations of targeted mRNA species, partly by blocking translation and partly by driving the degradation of these mRNA molecules (Figure S1.4). This ability to shut down the expression of proteins of expressed genes within cells has become an invaluable tool for cancer researchers who wish to demonstrate the essential role that one or another protein plays in a cell-biological process under study. Extending the earlier nomenclature that described the inactivation of genes in the mouse germline—termed knockout (see Supplementary Sidebar 7.7)—the new procedure has come to be called “knockdown.” For example, knockdown of the expression of a certain © 2014 Garland Science pro-apoptotic protein may protect a cell from apoptosis, demonstrating the essential role that the protein plays in orchestrating apoptosis in such a cell. Similarly, knockdown of the expression of a growth factor receptor may render a cell unresponsive to that factor, demonstrating the receptor’s key role in conferring such responsiveness. While highly useful in principle, in practice the application of this technique has proven challenging in the case of certain genes being targeted for knockdown. Thus, a targeted mRNA may not respond significantly for reasons that are unclear, necessitating the screening of multiple vectors, each targeting a distinct nucleotide segment in the 3ʹ untranslated region of an mRNA under study; in some cases, four, five, or even six alternative siRNAs or shRNAs must be tested before an experimenter discovers one that can reduce protein expression by 90% or more. Independent of these difficulties in achieving significant knockdown is the opposite problem: some siRNAs and shRNA have been found to affect far more mRNAs than their intended target—the phenomenon of “off-target effects.” Off-target effects often make it impossible to draw rigorous conclusions from the results of these experiments because of the confounding effects on other mRNAs and proteins operating in unrelated signaling pathways. 4
  7. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg lentiviral or retroviral vector CYTOPLASM NUCLEUS polIII integrated provirus Drosha pre-shRNA © 2014 Garland Science Figure S1.4 Suppression of RNA function by RNA interference The process of RNA interference (RNAi) operates normally in cells to produce microRNAs (miRNAs) that suppress mRNA function, as described in Section 1.10 and Figure 1.20. This process can be exploited experimentally to suppress targeted mRNAs. Two major strategies can be used. In the first, a retrovirus or lentivirus vector (see Supplementary Sidebar 3.3) is constructed that uses an RNA polymerase III promoter to drive expression of a transcript that serves as precursor of an shRNA (small hairpin RNA). The resulting preshRNA is processed by the Dicer enzyme into an siRNA (small interfering RNA) of 19–21 nucleotides, which is reduced to a single-stranded RNA (ssRNA). The ssRNA associates with the Argonaut protein (Ago2) and other proteins to form a RISC (RNA-induced silencing complex), which anneals to partially homologous target sequences that are usually located in the 3ʹ untranslated region (3’UTR) of certain mRNAs. This results either in the cleavage of the mRNA or inhibition of its translation, blocking in both cases expression of the mRNA and thus the gene encoding this mRNA. Because the retroviral or lentiviral provirus is integrated into the cell chromosome and is transmitted heritably to the progeny of an initially infected cell, the expression of a targeted mRNA can be suppressed stably over many successive cell generations. As an alternative strategy, an siRNA of a particular configuration can be chemically synthesized and transfected into a cell. While this is easier than the procedure described above, the targeted mRNA will be suppressed only transiently, since there is no mechanism to ensure the continued synthesis of the siRNA in the descendants of the initially transfected cell. Dicer RISC inhibition of translation mRNA cleavage s1.04 5
  8. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 1.5 Gene cloning strategies Gene cloning strategies first became practical in the late 1970s, and since that time a diverse array of strategies has been developed. © 2014 Garland Science Figure S1.5 illustrates the logic of gene cloning that is common to many of these strategies. fragment genome introduce fragments separately into vector arms of vector amplify each fragment separately in a bacterial colony identify and amplify inserted fragment of interest genome segment (carrying gene of interest) has been cloned Figure S1.5 Molecular cloning of genes Many versions of the gene cloning procedure have been developed since this technology was first invented in the 1970s. Pictured is an outline of one version. The genome from an organism (e.g., humans) is fragmented into relatively small segments, often several tens of kilobases long (top); each DNA segment is linked to the DNA of a vector (gray arms), yielding a recombinant vector. Each recombinant vector is then inserted independently into a bacterium, which is expanded into a colony containing many thousands of bacteria. The bacterial colony bearing the vector with the gene of interest is identified and the vector is retrieved, yielding millions of copies of this genomic fragment (bottom right). TBoC2 b1.21/s1.05 6
  9. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 2.1 Commonly used histopathological techniques Several techniques are used frequently to mark structures in the fields viewed through a light microscope in order to resolve tissue architecture, the subcellular structures Figure S2.1 Some frequently used techniques for staining histopathological sections While certain modern microscopic techniques allow visualization of tissues at various depths of focus (sometimes termed the “Z axis”), the great majority are limited to viewing at a single depth, either in cell monolayers or in tissue sections. The latter are prepared by sectioning tissues that have been stabilized by certain fixatives, such as formaldehyde, glutaraldehyde, or ethanol, which permeabilize cells and cross-link molecules in the biological sample prior to its being embedded in paraffin wax and sectioned; this embedding increases the mechanical strength of the sample, thereby facilitating sectioning. Alternatively, a tissue sample may be frozen and sectioned. In both cases, the resulting tissue sections are then deposited on glass slides. The magnification of the images (e.g., 20×) is then represented as the size of an object in an image of a microscopic field divided by its actual size; this factor may then be further increased or decreased as the initially viewed image is printed or presented in digital form. (A) Hematoxylin and eosin (H&E) staining reveals complex tissue architecture in this section of a human basal-like carcinoma of the breast (40×). Hematoxylin stains nuclear structures, notably the histones in the chromatin, blue; in contrast, eosin stains cytoplasm, connective tissue, and extracellular structures, such as extracellular matrix, pink. Note the two major types of tissue organization here: the carcinoma cells have large dark purple nuclei and are assembled in continous aggregates while the nonneoplastic cells forming the loose, fibrous stroma have small very dark nuclei. (B) Use of Masson’s trichrome stain allows a variety of structures to be visualized with a single staining procedure. In this micrograph (20×) of experimentally transformed human mammary epithelial cells growing as a tumor xenograft in a mouse, erythrocytes (red blood cells) appear bright red, collagen appears blue, cytoplasms appear light pink, and cell nuclei appear dark pink (C) Immunohistochemistry (IHC) depends on the ability to conjugate (link) an antigen-specific antibody with an enzyme, usually horseradish peroxidase; this enzyme uses added hydrogen peroxide (H2O2) to oxidize an added chemical substrate, usually generating a dark brown or black product. In this image (40×), the section of a human basal-like breast tumor has been immunostained using an antibody that binds the p53 tumor suppressor protein, which, in mutant form, accumulates in high concentrations in cell nuclei, revealing them here as dark ovals. The section was then stained with hematoxylin which was used here as a counterstain to reveal remaining structures in the tumor, seen here as light blue. Nuclei stained with hematoxylin but not with the anti-p53 peroxidase-conjugated antibody are mostly normal, non-neoplastic cells of the tumor-associated stroma. (D) Normal human mammary gland tissue has been © 2014 Garland Science within individual cells, proteins that form these various structures, and even specific genes borne by chromosomal DNA (Figure S2.1). stained here using the technique of immunofluorescence (IF), in which antigen-specific antibodies have been conjugated to fluorescent dyes (sometimes termed fluorophores) that emit light at various wavelengths after being excited with light of different, specific wavelengths. This allows the staining with multiple distinct antigen-specific antibodies (in this case three) that enables the concomitant visualization of multiple distinct antigens and thus the cells that express them. In this micrograph (63×), cells expressing the CD44 antigen have been stained with a conjugated antibody that generates a pink/purple signal, cells expressing the CD24 antigen have been stained with an antibody that generates a blue signal, while cells expressing α-smooth muscle actin have been stained with an antibody that generates a green signal. The blue mammary epithelial cells are arrayed as the lining of ducts that have a dark lumen (cavity) in their center. (E) An alternative combination of antibodies allows this visualization of human basal-like breast cancer at 40× magnification. In this case, the anti-p53 antibody has been conjugated to a fluorescent dye emitting a blue signal, an antibody recognizing the BRCA1 protein has been conjugated with a dye emitting a red signal (yielding small red dots in cell nuclei), while an antibody recognizing the PTEN tumor suppressor protein has been stained with an antibody that emits green light. This image indicates that the neoplastic cells with pinkish blue nuclei generally have lower levels of the BRCA1 protein than do the non-neoplastic cells of the tumor stroma, which lack mutant p53 protein and thus the pinkish blue nuclei. (F) In this micrograph (63×), immunofluorescence staining has been combined with fluorescence in situ hybridization (FISH), which allows the detection of DNA sequences in the section by annealing (hybridizing) sequence-specific DNA probes that have been coupled with specific fluorescent dyes; the combined staining is sometimes termed immunoFISH. Here, an anti-p53 antibody stains carcinoma cell nuclei dark blue, while specific DNA probes have detected DNA sequences associated with the centromeres of Chromosome 10 (CEP10, red) and Chromosome 17 (CEP17, green), as well as the genes encoding the PTEN (yellow) and BRCA1 (light blue) proteins. (G) Monkey kidney cells growing in monolayer culture were forced to express a protein that caused them to undergo cell–cell fusion, generating a multinucleated cell, sometimes termed a polykaryon or syncytium (center). The nuclei were stained with DAPI (4’,6’-diamidino-2phenylindole), a dye that emits blue fluorescence when it binds to dsDNA (double-stranded DNA), while the actin cytoskeleton was stained with phalloidin, which binds actin filaments and in this case has been linked to the green fluorescent dye FITC (fluorescein isothiocyanate). (A,C–F, courtesy of F. Martins and K. Polyak. B, courtesy of S. McAllister. G, courtesy of S. Rozenblatt.) 7
  10. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) (B) (C) (D) (F) © 2014 Garland Science (E) (G) 8
  11. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 2.2 The complicated conventions for classifying and naming tumors There are several hundred distinct cell types forming the specialized tissues in the human body. Human tumors that arise in the cells of these various tissues exhibit a wide spectrum of phenotypes that defy categorization by any single classification scheme that utilizes either purely morphological or purely molecular criteria. Consequently, human tumors are currently classified by a combination of their histological, clinical, and molecular features. A purely molecular taxonomy may well emerge in the future, but for the moment we are forced to deal with the complex classification schemes that have developed over the past century. The rationale behind the currently used approaches is outlined below. The great majority of human tumors arise from epithelial tissues. While the term cancer is generally used to refer to all malignant human tumors, the term carcinoma specifically refers to human tumors that arise from epithelia. The term adenocarcinoma is a further subdivision of epithelial cancers that arise from single-cell-layer or two-cell-layer epithelia that share key features in their form and function. For example, these epithelia form glands in which the epithelial cells exhibit an apical– basal polarity, with the apical end oriented toward the lumen (the central space enclosed by the gland) and the basal end oriented away from the lumen. In general, these cells secrete substances into the lumen and some may also absorb substances from it. This explains why the prefix adeno- (which is derived from the Greek word for gland) is used to distinguish the above tumors from other carcinomas that arise in epithelia—such as those of the skin and bladder—that act as a protective barrier rather than facilitator of molecular transport. Benign epithelial tumors are in general referred to as adenomas. It is notable that the majority of human cancers are adenocarcinomas arising from lung, breast, prostate, pancreas, liver, colon, uterus, ovary, and so forth. Precisely why adenocarcinomas account for such a disproportionate share of human tumors is not easily explained by invoking either the proportion of the human body that these epithelia represent or the proliferation rate of their constituent cells. Nonepithelial tumors are dramatically different from epithelial tumors in several important aspects. Thus, when referring to © 2014 Garland Science these other tumors, a uniform terminology, comparable to the adenoma and adenocarcinoma terminology used for epithelial tumors, is often replaced by a differentiation state-based system, that is, one that is based on the degree of differentiation and tumor progression. For example, benign nonepithelial tumors that arise from the mesenchymal cells of the stroma are designated by adding the suffix -oma to the name of the normal differentiated tissue that they resemble; hence, lipoma is the term for fat-cell tumors. The suffix -sarcoma is used for the corresponding malignant tumors—liposarcoma, in this example— that appear to arise in the same tissue. The hematopoietic neoplasms are classified based upon their physical appearance, specifically, lymphomas (solid masses) or leukemias (individual cells suspended in the blood), regardless of their benign or malignant behavior. Much of this terminology developed independently for each tissue type and reflects the history of pioneering research for each neoplasm. By now, the terms in common use are so embedded in clinical oncology that they are essentially impossible to change. Regardless of these quirks, these broad categories can teach important lessons about the nature of tumors that arise in different tissue types. For example, there is a great difference between benign epithelial and stromal tumors, both of which may be called “-omas.” Epithelial adenomas are often intermediaries in a multi-step progression that begins with fully normal cells and ends with malignant adenocarcinomas, as is discussed in Chapter 11. In contrast, certain benign stromal “-omas” may persist stably in a benign state without further progression and may thus behave very differently from malignant sarcomas arising in the same tissues. In addition, there is a great difference in the types of genetic lesions that drive malignancy in these two broad classes. Most nonepithelial tumors are typically associated with a single, dominant genetic event, such as a specific gene translocation. Often, these translocations are so uniform (“recurrent”) among different patients and so distinctive that their presence is determined in order to help make a definitive diagnosis of the specific type of tumor being examined. Examples of these tumors are listed in the accompanying Table S2.1 below. In contrast, it is generally very difficult to find any single mutation or translocation that is uniformly present in most cases of a given epithelial cancer type, with the K-ras mutations Table S2.1 Examples of malignant nonepithelial tumors and their characteristic genetic lesions Tumor Translocation Involved genes CML t(9;22)(q34;q11) Bcr-Abl Ewing’s sarcoma t(11;22)(q24;q12) EWS-FLI1 Clear-cell sarcoma t(11;22)(p13;q12) EWS-WT1 Myxoid chondrosarcoma t(9;22)(q22;q11-12) EWS-ATF1 Alveolar rhabdomyosarcoma t(2;13)(q35;q14) t(1;13)(q35;q14) PAX3-FKHR PAX7-FKHR Synovial sarcoma t(X;18)(p11.2;q11.2) SYT-SSX1; SSX2 Myxoid liposarcoma t(12;16)(q13;p11) CHOP-TLS(FUS) Congenital fibrosarcoma t(12;15)(q13;q25) ETV6-NTRK3 Text and table courtesy of Tan A. Ince. 9
  12. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg in pancreatic carcinomas and HER2/neu amplifications in a subset of breast cancers being rare exceptions. Epithelial malignancies are typically driven by multiple, accumulated genetic events that are present in different combinations in different tumors and follow the well known “multi-hit” model of stepwise progression to cancer (see Chapter 11). Tumors that arise in the nervous system represent yet another special category. The cells of the brain and peripheral nerves are derived from the specialized neural ectoderm of the early embryo (see Figure 2.5) and thus differ from non-neural epithelia and mesodermal derivatives (that is, stromal and hematopoietic cells). Due to their distinct embryologic origin, the neuroectodermal tumors that arise in the central and © 2014 Garland Science peripheral nervous system are considered to be a fourth category of tumors. Nature knows no fixed rules, however, and there are some tumors that are difficult to classify from this simple perspective. For example, choroid plexus cells that line the ventricles of the brain are also derived from the neural ectoderm, but they give rise to tumors with an epithelial phenotype. Finally, some tumors can develop into subpopulations of neoplastic cells exhibiting distinct cell phenotypes, yielding admixtures of epithelial and even stromal cell types. The prototypical example of this class of tumors is provided by teratomas, which are composed of tissues from multiple germ cell layers and hence are suspected to arise from germ or stem cells. 10
  13. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.1 Is SV40 responsible for the mesothelioma plague? The mesothelium is the membranous outer lining of many internal organs and derives directly from the embryonic mesoderm. Mesotheliomas—tumors of various mesothelial surfaces, notably the pleural membranes of the chest—were virtually unknown in the United States in the first half of the twentieth century. Beginning in about 1960, however, their incidence began to climb steeply. By the end of the century, the annual incidence in the United States approached 2500 and was attributed mostly to asbestos exposure, specifically a subtype termed crocidolite. The disease is seen frequently among those who have worked with asbestos, which was used as a heatresistant insulating material until being banned in the 1980s. However, 20% of mesothelioma patients have no documented exposure to asbestos, a fact that has provoked a search for other etiologic (causative) agents. SV40 is a plausible etiologic agent of mesothelioma. By 2003, forty-one laboratories across the world had reported SV40 DNA, RNA, or protein in mesothelioma cells. Traces of the virus are otherwise rarely found in human tumors, with the exception of certain types of brain tumors. Cultured human mesothelial cells are readily infected by SV40, and this infection leads rapidly to their immortalization, that is, to the ability of these cells, which usually have limited proliferative potential in culture, to multiply indefinitely. © 2014 Garland Science The presence of SV40 contamination in poliovirus vaccine stocks has raised concerns that mesothelioma may have been induced in many individuals as an unintended side effect of poliovirus vaccination. However, there are many individuals with mesotheliomas who are highly unlikely to have been exposed to these vaccines. Moreover, the viral T-antigen protein, whose presence is invariably observed in SV40-transformed cells (see Section 3.6), is rarely detected in mesothelioma tumors. Also, attempts at demonstrating viral DNA have usually yielded DNA segments that are indicative of laboratory artifacts, for example, contamination of mesothelioma tumor samples by laboratory stocks of SV40 or by recombinant DNA plasmids engineered to include segments of the viral genome. The discoveries of such contaminations provide increasing ammunition for those who are skeptical of SV40’s role in mesothelioma pathogenesis. As many as 85% of humans are known to be infected with two viruses that are closely related to SV40—JC and BK—and antisera that recognize the capsids of these viruses cross-react with the capsid of SV40, explaining many of the claims that SV40 is often present in human tissues. In immunosuppressed individuals, notably AIDS patients, JC virus can cause a fatal brain degeneration—progressive multifocal leukoencephalopathy (PML). And in patients who are immunosuppressed because they are organ transplant recipients, BK virus has been implicated in the causation of certain carcinomas (see, for example, Figure 3.14B). 11
  14. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.2 Maintenance of KSHV and HPV genomes in episomal and chromosomal form Both herpesvirus and papillomavirus genomes are able to persist for years in a virus-infected tissue in an episomal form, that is, as molecules that are not integrated into the host-cell chromosomal DNA. In the case of Kaposi’s sarcoma herpesvirus (KSHV), this is achieved through the actions of its LANA protein (Figure S3.1A), which binds to terminally repeated sequences in the viral genome as well as to the histone molecules forming the core nucleosome (see Figure 1.19). This enables maintenance of the viral genome even though direct covalent linkage between viral and host DNAs is absent. A similar dynamic operates in human papillomavirus (HPV)infected tissues. Following initial infection, HPV-infected cells and viral genomes in general are cleared from the infected tissue within a period of weeks or months by the immune system. However, in a minority of infected individuals, the HPVs persist, establishing chronic infections in epithelial tissues and maintaining their genomes, once again, in an episomal state, apparently for years if not decades. These viral infections sometimes trigger low- and then high-grade squamous intraepithelial lesions (LSILs, HSILs) of the cervix; the number of episomal HPV genomes per cell generally increases during this progression and, depending on the subtype of HPV virus, may reach more than several hundred copies per infected cell. On rare occasion, these chronic infections may trigger the onset of frank malignancies—cervical carcinomas; squamous cell carcinomas triggered by HPV can also arise in the oropharynx. In the genomes of the cervical carcinoma cells, oncogenic fragments of the HPV genomes are invariably found in an integrated state, while the episomal HPV genomes are generally no © 2014 Garland Science longer detectable. This integration is achieved by mechanisms similar to those used by SV40 to establish its genome in cells that it has transformed (see Figure 3.16/n3.103) and involves nonhomologous recombination between viral genomes and hostcell chromosomal sequences. This chromosomal integration, as was the case with SV40, ensures the perpetuation of the HPV genome fragments in descendant cells. The HPV genomes seem to be integrated preferentially in chromosomal “common fragile sites”—chromosomal regions (>100) scattered throughout normal genomes and exhibiting relatively high genetic instability, including susceptibility to dsDNA breakage. This integration has a second functional consequence: the molecular steps that lead to integration usually result in the deletion or inactivation of certain viral genes that are needed for viral replication of episomal viral DNA, and of other genes whose products operate to repress expression of the virus-encoded oncoproteins (Chapters 7 and 8). Without this repression, the viral oncoproteins are expressed by the integrated viral genomes at far higher levels, thus driving the progression of pre-neoplastic virus-infected cells into carcinoma cells. The repression of HPV oncoprotein expression is the task of the viral E2 protein, which has a second, ostensibly related function: it acts to link the unintegrated viral genome to a cellular protein that is bound to chromatin during mitosis (Figure S3.1B). This ensures the tethering of the viral genome to chromosomes during mitosis (Figure S3.1C). When this tethering is blocked experimentally, the viral genomes are unable to “hitchhike” with the chromsomes during mitosis and consequently are largely left in the cytoplasmic portion of the mitotic cells, which leads to their rapid loss following multiple successive cellular growthand-division cycles. 12
  15. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) © 2014 Garland Science (B) DNA N-terminal domain of E2 C-terminal domain of E2 H3 Brd4 chromatin protein H2A C N hinge H4 LANA N α1 α2 α3 binding to viral DNA H2B (C) E2 functional ~175 on chromosomes E2 blocked Figure S3.1 Tethering of episomal viral genomes to host-cell chromatin Certain DNA viruses maintain chronic infections through their ability to maintain their genomes stably but in an unintegrated (episomal) configuration over many successive cell generations. They do so by synthesizing viral proteins that are able to physically link the viral nucleoprotein to the chromatin of the infected host cell when that cell passes through mitosis, ensuring transmission of the viral episomal genomes to both daughter cells. (A) In the case of KSHV/ HHV-8, the agent of Kaposi sarcoma, the virus makes a protein termed LANA (latency-associated nuclear antigen; blue), that is able, on the one hand, to associate with a nucleosome at the interface between its H2A and H2B histones (see Figure 1.19) and, on the other, to bind terminal repeat sequences in the viral DNA genome. (B) Human papilloma virus type 16 (HPV-16) uses the N-terminal domain of its E2 protein (light green) to tether the episomal viral DNA genome to the Brd4 chromatin-remodeling protein (blue), which is bound, in turn, to cellular chromatin during mitosis, enabling the viral genome (tethered to the C-terminal domain of E2) to “hitchhike” with the cellular chromosomes during mitosis. (C) The association of HPV-16 genomes with metaphase chromosomes can be gauged using HPV-16-specific DNA probes in FISH (fluorescence in situ hybridization) analyses. The chromosomes are stained here in red, while the viral genomes are detected as yellow dots if they are located above the metaphase chromosomes and as green dots if they are not located above the chromosomes. In the upper panel, the E2 protein is functioning normally in chromosomal tethering, whereas in the lower panel, its ability to bind to the Brd4 chromatin-associated protein has been blocked. As indicated, enumeration of the viral genomes above chromosomes reveals a drastic decrease in the metaphase chromosome-associated viral genomes when E2 tethering function is blocked. (A, from A.J. Barbera et al. Science 311:856–861, 2006. B and C, upper, from E.A. Abbate, C. Voitenleitner, and M.R. Botchan. Mol. Cell 24:877–889, 2006. C, lower, courtesy of C. Voitenleitner and M.R. Botchan.) ~5 on chromosomes TBoC2 s3.01 13
  16. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.3 Re-engineering the retrovirus genome for gene therapy The technology of gene therapy has depended on the ability to insert genes into cells or tissues that lack functional versions of those genes, with the intent of reversing the genetic and thus phenotypic defects. The technique of transfection, in which naked DNA is introduced into cells via a variety of techniques (for example, see Figure 4.1) is relatively inefficient and the introduced DNA is integrated into the chromosomal DNA of the recipient cell in a haphazard fashion, resulting in multiple copies of introduced DNA, some of which Figure S3.2 Construction of a retroviral vector While a variety of retroviral-like genomes have been used to construct viral vectors, the basic schemes used in all of these constructions are quite similar. (A) The dsDNA provirus of a retrovirus—the product of reverse transcription—is introduced into a cloning plasmid and modified subsequently by the standard techniques of recombinant DNA. Note that the provirus contains two identical copies of a segment (red) termed the LTR (long terminal repeat) at its ends— products of the reverse transcription process. The presence of these LTRs is critical for the transcription of the provirus by the cellular RNA polymerase II. Restriction enzymes are then used to cleave out viral genes (dashed lines) and to replace them with a gene-of-interest (orange) that will be transduced by the future retroviral vector. In addition, a gene encoding a selectable marker (purple) is introduced. Once expressed in a vector-infected cell, this marker gene can be used to ensure that this cell will be protected from the toxic effects of an applied drug (for example, neomycin), while uninfected cells lacking this marker gene (and thus the remainder of the vector’s genome) will be killed off. This strategy can guarantee that the only surviving cells in a large population of cells that have been exposed to this re-engineered virus will be those cells that have been successfully infected by the viral vector. (B) The introduction of two reading frames into this vector—that encoding the geneof-interest—creates a problem, since in general, the translation of a mammalian mRNA can only be initiated at one site near its 5ʹ end. Hence, when this bicistronic provirus (carrying two genes) is transcribed into an mRNA, ribosomes may only be able to translate the 5ʹ-proximal reading frame of this mRNA. This problem can be addressed in at least two ways. Thus, the selectable marker can be introduced in place of the viral env gene; following transcription of a normal retroviral provirus, a certain proportion of the primary pre-mRNA transcripts are routinely processed by splicing in a fashion that causes the viral gag and pol genes to be excised, thereby juxtaposing the env gene (or a gene introduced into its place) with the 5ʹ end of the resulting mRNA. By replacing the viral env gene with an introduced gene-ofinterest, spliced mRNA can then serve as a template for translation by ribosomes, since this gene has now been juxtaposed with the 5ʹ end of the mRNA. A more frequently used strategy, shown here, is to introduce an IRES (internal ribosome entry site; blue) between the two introduced genes just upstream of the (in this case) selectable marker. This IRES segment overrides the normal mechanisms preventing ribosomes from initiating translation at more than one site of an mRNA template; instead, in the presence of an IRES segment, a ribosome can now initiate translation in the middle of an mRNA in addition to its normal 5ʹ-proximal site of initiation. In principle, if this provirus is introduced into a cell (for example, via gene transfer/transfection, see Figure 4.2), it can be transcribed by the cellular RNA polymerase II and thus create a polyadenylated RNA molecule that is exported into the cytoplasm. However, this © 2014 Garland Science are fragmented at random sites during the integration process. Many of these problems are addressed by the use of retrovirus genomes as vectors to transduce (carry) cloned DNA sequences into recipient cells (Figure S3.2). These vectors take advantage of the high efficiency of integration of retroviral DNA and the resulting stable establishment in the recipient cell of integrated proviruses that carry intact copies of the introduced DNA. This technology is limited, however, by the carrying capacity of a retroviral vector, which is only able to accommodate four to five kilobases in introduced sequences in its genome. RNA cannot leave the cell because it cannot be packaged by viral proteins, since some and perhaps all of the viral protein-encoding genes have been deleted during the construction of this viral vector. (The strategy of leaving these genes present and intact in the re-engineered viral genome is not viable, since retroviral virions can only package an RNA molecule of ~10 kb in length. Hence, viral genes must be excised to make room for inserted genes-ofinterest and selectable marker genes.) This problem necessitates co-transfecting the re-engineered vector DNA together with wild-type proviral DNA, which expresses the requisite virion proteins. Such co-transfection can ensure that many of the cells in a population exposed to these two DNAs will take up and express both DNAs. In such co-transfected cells, the viral proteins expressed by the wild-type viral genome can now package the RNA transcribed from the re-engineered vector DNA, resulting in infectious virions that can then be used to infect a variety of target cells, thereby transducing (introducing) the gene-of-interest into these cells. (C) The above strategy is effective in transducing genes-of-interest into cells that previously did not express them. However, it has a major drawback: these initially infected cells may become co-infected with wild-type viral genomes, and the resulting co-infected cells may then release progeny particles that can spread further in a population or in the body of an infected animal during a second infectious cycle. This creates a major problem if the transduced gene-of-interest happens to be a gene that may be toxic or oncogenic, since subsequently occurring, endless rounds of infection might, in principle, spread this gene in an uncontrollable fashion, thereby creating a biohazard. Alternatively, the infectious spread of the wild-type virus may also create a hazard, since nontransforming retroviruses may be tumorigenic if they create large numbers of infected cells and thereby inadvertently activate by insertional mutagenesis infected proto-oncogenes (see Section 3.11). In order to prevent these possibilities, a strategy has been devised that allows the viral vector to be introduced into an initially infected cell but precludes further infectious spread from that cell to yet other cells in second and subsequent rounds of infection. This strategy depends on the creation of “packaging cells,” which express the three viral mRNAs (encoding the viral gag, pol, and env proteins) from three unlinked viral genes, none of which is part of a provirus. Such cells can package the re-engineered viral vector RNA, because they synthesize all the proteins required for assembly of an intact virion and thus for virion infectivity. However, when another cell is subsequently exposed to the infectious virions leaving this cell, this cell will acquire the re-engineered vector genome and will express its genes, but this cell will be unable to generate a new generation of infectious virions, since no intact viral genomes were ever co-introduced into such cells. Therefore this infected cell, although genetically altered, will not become the source of infectious virus particles that can launch subsequent rounds of infection. 14
  17. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) gag MLV RNA 5′ pol MLV RNA (B) env AAAAAA...3′ dsDNA LTR reverse transcription LTR introduce IRES segment dsDNAs LTR © 2014 Garland Science LTR gene of interest excise viral genes selectable marker LTR LTR LTR IRES LTR introduce gene of interest + viral vector with introduced sequences gene of interest LTR wild-type proviral DNA co-transfect engineered vector with wild-type proviral DNA LTR transcription of transfected proviral DNA introduce selectable marker gene selectable marker gene LTR packaging of both RNAs into infectious virions LTR (C) engineered viral vector DNA mRNAs expressed by packaging cells 5′ transfect vector DNA into packaging cells that express wild-type viral mRNAs from three unlinked genes 5′ env gag 5′ AAAAAA...3′ pol AAAAAA...3′ AAAAAA...3′ infectious virions that can transduce vector into cells secondary spread from initially infected cells cannot occur because these virions lack the gag, pol and env genes TBoC2 s3.02 15
  18. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.4 Classic Kaposi’s sarcoma appears to be a familial disease Some virus-induced malignancies are limited largely to small subpopulations and thus resemble familial cancer syndromes. We will encounter many of these syndromes in Chapters 7, 8, 9, and 12. There, we will learn that inheritance of mutant alleles of tumor suppressor genes or DNA repair genes can create a strong, inborn cancer predisposition. Prior to the onset of the AIDS epidemic, the disease of Kaposi’s sarcoma (KS)—a malignancy of cells related to those forming the endothelial lining of lymph ducts—was confined largely to small subpopulations, notably men of Mediterranean and Jewish descent. This resembled a familial cancer, in which cancer-predisposing alleles were present only in the gene pools of certain ethnic subpopulations. After the onset of the AIDS epidemic, however, KS became 1000-fold more common, and at least this form of KS could be associated with an infectious agent—human herpesvirus-8 (HHV-8), also known as KSHV. This virus, along with a number of other infectious agents, is an opportunistic pathogen that thrives in the bodies of those lacking a functional immune system. Because of the AIDS epidemic in Africa, KS has now become the fourth most common infection-induced cancer worldwide. The virology of HHV-8 failed to explain how the “classic,” pre-AIDS KS is transmitted in immunocompetent populations even though these tumors could also be associated with HHV-8 infections. Moreover, it was unclear why this disease should be confined to small populations rather than spreading, like other viral infections, through large populations. Recent research has revealed that different substrains of HHV-8 (as defined by sequence polymorphisms in viral DNAs) are present in different subpopulations of Jews; one substrain predominates among Ashkenazic Jews (of recent European descent), while a second is common among Sephardic Jews (of North African and Middle Eastern descent). Both populations have infection rates that are © 2014 Garland Science as much as 10 to 20 times higher than in non-AIDS Western populations. Provocatively, within these subgroups, the transmission of specific HHV-8 substrains is correlated with inheritance of certain mitochondrial DNA polymorphisms far more strongly than with inheritance of Y-chromosome polymorphic markers. Mitochondrial DNA is transmitted maternally, indicating that maternal transmission of virus (occurring possibly via saliva) has played a major role in creating pockets of disease in family lineages that are likely to extend back to founder populations that existed more than 2000 years ago. [Another maternal transmission route may explain the high incidence of adult T-cell leukemia (caused by the HTLV-I retrovirus) in southern Japan (see Section 3.12).] Unexplained by these findings is the fact that KS is confined in the non-AIDS populations to males— a mystery that remains unresolved. Hence, certain geographically and ethnically localized malignancies, such as classic KS and adult T-cell leukemia, are actually due to viruses that spread poorly “horizontally” (that is, from one adult to another) but can be transmitted “vertically” (between parent and offspring) through long-term, intimate contact. This echoes the behavior of certain strains of mice that have high rates of breast cancer. As first shown in Bar Harbor, Maine, in 1933, transmission of disease from parent to offspring occurred when females of high-incidence strains were mated to males of low-incidence strains, but not following the reverse matings. Also, when female pups of high-incidence strains were transferred to low-incidence foster nursing mothers within 24 hours of birth, only 8% eventually developed breast cancer, compared with a 92% incidence exhibited by mice that had been nursed by mothers from a high-incidence strain. This led to the conclusion that this breast cancer susceptibility was transmitted from one generation to the next by a milk-borne infectious agent, which was later identified as mouse mammary tumor virus (MMTV). 16
  19. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 3.5 Viruses like RSV have very short lives Circumstantial evidence indicates that infection of a chicken cell by a virus like avian leukosis virus (ALV) resulted in the introduction of a copy of the chicken c-src gene into the ALV genome, creating the hybrid virus that we term RSV. This recombination event seems to have occurred only once in the twentieth century; that is, all src-containing chicken retroviruses appear to be the descendants of the RSV that arose on one occasion in a chicken coop in about 1909. The discovery of RSV depended on a fortuitous circumstance: Peyton Rous was able to study the virus only because a Long Island, New York, chicken farmer brought him a prized, tumor-bearing hen with the hope that Rous would be able to cure the bird of its cancer (see Figure 3.1B). © 2014 Garland Science The presence of the acquired src gene in the viral genome confers no obvious growth advantage on this virus, and RSV is not naturally spread from animal to animal. Moreover, similarly configured retroviruses, which also carry both viral and cellular sequences in their genomes, spread poorly from one host animal to another, often because they have deleted essential viral replication genes from their genomes in order to accommodate acquired oncogenes of cellular origin. For these reasons, it seems clear that hybrid retroviruses such as RSV arise through rare genetic recombination events within infected animals, create tumors in these animals, and usually disappear when these animals die, unless an alert chicken farmer or slaughterer happens to discover the tumors and pass them on to an interested virologist. 17
  20. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.1 Endogenous viruses can explain tumor development in the absence of infectious viral spread The exposure of a mouse or chicken to a retrovirus often results in the infection of a wide variety of cell types in the body including, on occasion, the cells in the gonads—ovaries or testes. Infections of cells in these organs can result in the integration of a retroviral provirus (see Section 3.7) into the chromosomes of cells that serve as precursors to either sperm or egg. Such proviruses become established in a genetic configuration that m.m.dom. m.m.mol. transmission of infectious virus particles is equivalent to that of the cellular genes carried by the sperm or egg. Therefore, when these gametes participate in fertilization, the provirus can be transmitted to a fertilized egg and thus to all of the cells of a resulting embryo and adult (Figure S4.1). This provirus might now become ensconced in the genome of all animals that descend from the initially infected animal. Such a provirus would be termed an endogenous virus to distinguish it from viruses that are transmissible from one individual to the next via infection. Mus musculus outbred subspecies Mus musculus laboratory strains (B) m.m.cas. m.m.mus. (A) a b c d e f g h i j k kb 9.4 infection of germ cells, e.g., in testes 6.6 4.4 production of sperm carrying a provirus production of a zygote carrying a provirus 2.3 2.0 development of animal carrying a provirus in all of its cells (C) 5′ LTR 3′ LTR K10 probe transmission via germ line to descendants 1 2 3 4 5 6 7 8 9 10 12q14 transcriptional activation of an endogenous virus in one cell 11q22 3q24 spread of virus via infection of cells throughout the body 108b 109 115 © 2014 Garland Science Figure S4.1 Origin of endogenous retroviruses (A) These viral genomes arise when retroviruses (red dots) succeed in establishing a systemic infection in an organism (for example, a mouse) and infect, among other cells, a precursor cell of gametes—sperm or eggs. Once a resulting provirus (green rectangle) becomes integrated in the genome of a gamete (in this case sperm), it may be introduced into the genome of a fertilized egg and thereafter be distributed to all cells (green dots) of the organism arising from this zygote. This organism can in turn transmit the provirus to its descendants via the normal route of sexual reproduction. Activation of expression of the endogenous provirus in an animal (red dot) can lead to infectious spread throughout the body, viremia, and eventually leukemia (bottom). (B) The presence of endogenous retrovirus (ERV) genomes can be detected by probing the genomic DNA of a species using the DNA of an infectious retrovirus as the probe. Shown is a Southern blot (see Figure S4.3) of the ERV genomes present in the DNAs of a variety of mouse strains and subspecies. In this case, only the subclass of ERV genomes related to xenotropic murine retroviruses is being probed. Each band in a gel channel represents a restriction fragment in a cell genome that carries part or all of an ERV genome. The variability of ERV integration sites from one lab strain to another indicates that numerous novel ERVs have been integrated into the mouse germ line since the speciation of Mus musculus, from which all these strains derive. (C) In contrast to the mouse ERV genomes, those detected (using as a probe a fragment of the clone of a human ERV, above) in a collection of human DNAs show rather similar integration sites across the species, indicating their germ-line integration before the emergence of the human species; the polymorphic differences (black arrows) are largely the results of recombination between the pair of long terminal repeat (LTR) sequences at the ends of individual ERV proviruses and resulting deletion of the intervening stretches of proviral DNA. (B, from K. Tomonaga and J.M. Coffin, J. Virol. 73:4327–4340, 1999. C, from J.F. Hughes and J.M. Coffin, Proc. Natl. Acad. Sci. USA 101:1668–1672, 2004.) 18
  21. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Endogenous proviruses that are actively transcribed in an animal’s tissues may create a viremia and, following millions of replication cycles, may induce cancer in that animal. Such disease-inducing endogenous proviruses are therefore disadvantageous and, during the course of evolution, will be eliminated from a species’ gene pool. This explains why the endogenous proviruses found in the germ lines of most species are, with rare exception, either transcriptionally silent or incapable of encoding infectious virus particles. Careful examinations of the genomes of a variety of mammalian and avian species demonstrate the presence of numerous endogenous retroviral genomes, most of which are clearly relics of germ-line infections that occurred in the distant evolutionary past. Having resided for millions of years in a species’ germ line, most have suffered so many mutations that they are no longer able to specify infectious virus particles. However, a small subset of endogenous viral genomes, notably those that have been recently inserted into a species’ germ line, may remain genetically intact. Given the proper stimulus, these previously latent proviruses may suddenly be transcribed in one or another cell, generating virus particles that spread via infection from this cell throughout the body and eventually trigger some type of malignancy, usually of hematopoietic cells (see Section © 2014 Garland Science 3.11). For example, the high rate of leukemia in mice of the AKR strain is attributable to the frequent spontaneous activation of an endogenous murine leukemia provirus, the subsequent infectious spread of virus throughout the mouse, viremia, and, finally, via insertional mutagenesis, the activation of a protooncogene and the eruption of a leukemia. Such germ-line transmission of viral genomes was thought to be nothing more than a laboratory curiosity until reports, beginning in 2006, revealed that certain people carry loads of human herpesvirus type 6 (HHV-6) in their peripheral mononuclear cells (for example, lymphocytes, monocytes, macrophages) that are 1000 to 10,000 times higher than the average person carries. (Latent HHV-6 infections are widespread in the general population.) Subsequent studies revealed that these high loads of HHV-6 were often present in multiple members of a family and that in these families, HHV-6 genomes were inherited in the germ line integrated in certain chromosomes, specifically in telomeric DNA. Thus, a mechanism that HHV-6 usually employs in order to enter into a latent state (that is, by integrating its genome into one or another chromosome; see Section 3.6) occasionally results in germ-line transmission. The clinical consequences of inherited HHV-6 genomes remain unclear. 19
  22. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.2 Boveri and Hansemann independently hypothesized genetic abnormality as the cause of cancer cells’ malignant behavior The discovery that cancers arise as a consequence of aberrations in genetic material is widely attributed to the work of Theodor Boveri (1862–1915), a dominant figure in early twentieth-century German biology. This story, like many accounts of scientific history, is actually a bit more complicated and more interesting than conventional accounts depict. The roots of this notion actually reach back to an earlier set of observations by Boveri’s contemporary David Paul von Hansemann (1858– 1920) (Figure S4.2A). In 1890, Hansemann, a pathologist, first reported his observations that the mitotic figures (for example, see Figure 12.38) of cancer cells contained a variety of abnormal chromosomal configurations, including multipolar mitotic spindles— unusual arrays of chromosomes at anaphase, and asymmetric mitoses. He also proposed that the cells within tumors underwent partial dedifferentiation, another theme that anticipated twentiethcentury histopathological studies of tumors. Hansemann continued to report his observations in the decade that followed, but at the time had little insight into the cell-biological consequences of abnormal chromosome number and configuration. In 1900, the work of Mendel, long forgotten, was rediscovered by three geneticists. They popularized the idea that cells are essentially diploid, with pairs of genetic determinants being separated from one another during meiosis. In the several years that followed, the behaviors of Mendel’s genetic determinants were realized to parallel that of chromosomes, leading many to conclude that the chromosomes were the seat of heredity. Hence, cells with abnormal (A) © 2014 Garland Science numbers of chromosomes were perceived as being genetically mutant, though the term “mutant” had not yet come into currency. Unlike Hansemann, Boveri (see Figure S4.2B) was a zoologist and worked largely with early sea urchin embryos. He hardly acknowledged the work of Mendel and minimally that of Hansemann. Instead, his own work in manipulating metaphase figures in early sea urchin embryos led him to propose that the karyotypic aberrations observed occasionally in otherwise normal cells represented genetic abnormalities, that each chromosome carries a distinct subset of the genetic information present in the genome as a whole, and that the karyotypic abnormalities within cancer cells were responsible, in some fashion, for the aberrant behavior of cancer cells. The corrupted information content present in one cell was, he proposed, heritable from one cell generation to the next. Boveri’s posthumously published 1916 monograph, in which he laid out these ideas, was followed 13 years later by an English translation, carried out by his doting widow, the former Marcella O’Grady of Boston, Massachusetts. Hansemann lacked a loyal widow and translator, and so awareness of his pioneering contributions in connecting an abnormal genetic constitution of cells with the development of tumors receded. Boveri’s personality also may have helped impress on the scientific community his unique role in many of these advances; an otherwise uncritical colleague described him as “vehement, inflexible, and [a] relentless assailant.” Today, few remember that Hansemann began the charge that led to the now widely embraced notion that cancer cells are genetically abnormal, and that these cells’ chromosomes are responsible for their malignant behavior. (B) Figure S4.2 The first clues to the genetic origins of human tumor development (A) David Paul von Hansemann described the abnormal mitotic figures and chromosomes in human cancer cells without knowing their significance. (B) Theodor Boveri drew on Hansemann’s observations and his own to propose that abnormal chromosomes somehow were responsible for the abnormal behaviors of cancer cells. (A, courtesy of Wolfgang von Hansemann. B, courtesy of the Biocenter, University of Wüzburg.) TBoC2 s4.02 20
  23. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.3 Southern and Northern blotting Southern blotting was invented in 1975 by the British biologist Edwin Southern as a means of detecting specific DNA fragments that are generated by restriction enzyme cleavage of DNA; these fragments may be present amid a millionfold excess of other concomitantly generated DNA fragments, revealing the sensitivity of this procedure (Figure S4.3). Within several years, the procedure was adapted to detect specific mRNA molecules, once again present amid a vast excess of unrelated molecules, in this case other mRNA species; for want of a better name, this (A) s resi pho tro elec labeled RNA or DNA of known sizes as size markers unlabeled RNA or DNA © 2014 Garland Science newer procedure was dubbed Northern blotting. Thereafter, a further adaptation of the procedure was developed in order to detect individual proteins amid a vast excess of unrelated proteins and termed the Western blotting procedure (not shown here). While the Southern and Northern blots use radiolabeled nucleic acid probes, the Western blotting procedure uses specific antibodies, usually monoclonal antibodies, to detect proteins that have been adsorbed to filters and for this reason is also termed immunoblotting. stack of absorptive paper towels (B) REMOVE NITROCELLULOSE PAPER WITH TIGHTLY BOUND NUCLEIC ACIDS (C) gel nitrocellulose paper agarose gel NUCLEIC ACIDS SEPARATED ACCORDING TO SIZE BY AGAROSE GEL ELECTROPHORESIS sponge buffer SEPARATION OF NUCLEIC ACIDS BLOTTED ONTO NITROCELLULOSE PAPER BY SUCTION OF BUFFER THROUGH GEL AND PAPER Figure S4.3 Southern and Northern blotting procedures Use of these blotting procedures makes possible the detection of specific fragments of the cell genome (or specific RNA transcripts) if an appropriate radiolabeled DNA probe is available. (A) Either DNA fragments that have been generated by restriction enzyme cleavage of genomic DNA (in a Southern blot) or a mixture of cellular RNAs (in a Northern blot) are resolved by gel electrophoresis. (B) The gel slab is then placed beneath a nitrocellulose filter, and paper towels (or other absorptive material) are used to wick fluid through the gel, allowing the DNA (or RNA) molecules to adsorb to the filter paper, creating a replica of their previous positions in the gel. (C) The filter paper is peeled away from the gel and (D) placed in a plastic bag together with a solution of radiolabeled probe (pink). (E) Hybridization of the probe with complementary DNA (or RNA) molecules adsorbed on the filter and subsequent autoradiography with a photographic emulsion allow the detection of DNA fragments (or RNA molecules) that were present in the initial DNA (or RNA) preparation; these are manifested by bands of silver grains on the film. (From B. Alberts et al., Molecular Biology of the Cell, 5th ed. New York: Garland Science, 2008.) (D) LABELED PROBE HYBRIDIZED TO ADSORBED DNA OR RNA sealed plastic bag labeled probe in buffer LABELED PROBE HYBRIDIZED TO COMPLEMENTARY DNA OR RNA; BANDS VISUALIZED BY AUTORADIOGRAPHY (E) positions of labeled markers labeled bands TBoC2 b4.04/s4.03 21
  24. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 4.4 Genes undergo amplification for a variety of reasons The process of gene amplification is defined as a preferential increase in the copy number of a discrete chromosomal region (an amplicon), rather than increases in the ploidy of an entire genome, as may occur in certain specialized mammalian cells. In mammalian cells, gene amplification seems to occur only as a pathological process accompanying and driving tumor promotion (Figure S4.4). In other phyla, however, it may be part of a normal developmental program that responds to the need to express large amounts of a certain protein or group of proteins; this is achieved by directed, localized gene amplification, which results in proportional increases of the corresponding mRNA and protein. For example, in Drosophila, genes specifying chorion proteins undergo amplification in order to enable the synthesis of massive amounts of these proteins during oogenesis. ss nick or DSB (A) Alu elements MYB MYB (B) strand invasion and homologous recombination with upstream repeat on sister chromatid MYB MYB (C) MYB MYB (D) MYB MYB MYB © 2014 Garland Science Figure S4.4 Mechanism of generating tandemly duplicated genes A number of molecular mechanisms have been proposed to explain the generation of tandemly duplicated genes, such as those that form the homogeneously staining regions (HSRs) that create oncogene amplification. In the example presented here, the mechanism of amplification of the MYB oncogene via tandem duplication was studied in a human T-cell acute lymphoblastic leukemia. The amplification mechanism was traced, with high likelihood, to the presence of Alu elements, which are present in about one million copies scattered throughout the human genome (see Figure 4.7). In this case, the normal human MYB gene is flanked by two Alu repeat sequences. (A) When the MYB gene underwent replication during the S phase of the cell cycle, a single-strand (ss) nick or doublestrand break (DSB) in the newly synthesized upper sister chromatid allowed the resulting free end (which contains an Alu sequence, empty rectangle) to invade the doublestrand helix of the lower sister chromatid and anneal to another Alu sequence (dark red rectangle) that lies upstream of the MYB gene on the lower sister chromatid (B). The 3ʹ end of the newly synthesized, annealed strand could then be elongated using as a template this lower sister chromatid (light blue). (C). Subsequently, this elongating strand could jump back to the upper sister chromatid (dark green) and continue elongation, generating two head-to-tail tandem copies (D) of the MYB gene. This process might then be repeated, leading to an exponential expansion of this chromosomal region. (From J. O’Neil et al., J. Exp. Med. 204:3059–3066, 2007.) TBoC2 s4.04 22
  25. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 5.1 Making anti-Src antibodies presented a major challenge Thorough biochemical analyses of thousands of cellular proteins have depended upon the ability to detect and isolate a protein of interest (for example, Src) amid the complex soup of proteins present in cells. This detection frequently relies on the use of antibodies that specifically recognize and bind a protein such as Src while ignoring all other cellular proteins around it. The production of such antibodies involves the injection of an intact protein (for example, the entire Src protein) or a chemically synthesized oligopeptide (for example, a short protein whose amino acid sequence reflects the sequence of a small segment of the Src protein) into an animal, usually a mouse, rabbit, or goat. The hope is that the injected material will be immunogenic (will elicit an immune response) in the animal, causing it to form antibodies that specifically bind and precipitate this protein from a cell lysate (a preparation of disrupted cells). The immune systems of mammals often react vigorously to a foreign antigen, that is, a protein carrying peptide sequences that are unfamiliar. Conversely, the immune system often mounts no immune response at all (and thus fails to make antibody) against an injected protein that closely resembles one of the proteins normally present in the animal. This lack of immunoreactivity against familiar proteins is termed immunological tolerance, a phenomenon that we will return to in Chapter 15. Those who first produced anti-Src antibodies used a strain of RSV that can infect and transform mammalian cells and can therefore induce tumors in mammals. They anticipated that the resulting tumors would express the chicken Src protein, and that dying tumor cells would expose this protein to the immune systems of the tumor-bearing mammalian hosts and thus provoke an immune response. In fact, the Src protein proved to be poorly immunogenic in most animals. The reason for this was discovered much later: the structure of the Src protein is highly conserved evolutionarily. Accordingly, the chicken Src protein is almost identical to the corresponding protein of most mammalian species and is therefore not readily recognized as a foreign protein by these species’ immune systems. In the end, these investigators succeeded (for unknown reasons) in producing anti-Src antiserum only from rabbits bearing RSVinduced tumors. The factors that determine the immunogenicity of a protein such as Src in one or another species are complex and often obscure. Once in hand, the anti-Src antiserum could be used to precipitate (that is, immunoprecipitate) Src protein from lysates of a wide variety of RSV-transformed cells and from normal, untransformed cells (Figure S5.1). As in many such analyses, these lysates were prepared from cells that had © 2014 Garland Science been incubated in the presence of radiolabeled amino acids, resulting in the incorporation of radiolabel in the cells’ proteins. Following immunoprecipitation, the resulting proteins were resolved by gel electrophoresis, and the presence of radiolabeled proteins was detected by autoradiography. In the present case, the long-sought Src protein was detected as a polypeptide migrating as a 60-kD protein. 150,000 88,000 Pr76 Pr66 60,000 Pr53 35,000 p27 (daltons mol. wt.) p19 T7 1 2 3 4 5 6 7 8 9 p12, 15 Figure S5.1 Immunoprecipate of Src protein Initially, the only means of studying the Src protein depended on first immunoprecipitating it from the mixture of tens of thousands of other cellular proteins. Many attempts at making an anti-Src serum finally resulted in an antibody preparation that could be used to immunoprecipitate this protein from cells. Molecular weight markers are in the leftmost channel T7; channels 1–3: lysate of normal, uninfected cells; channels 4–6: lysate of cells infected with an RSV mutant with a deleted src gene; channels TBoC2 b5.06/s5.01 7–9: lysate of cells infected with wild-type RSV. Channel 7: normal rabbit serum; channel 8: tumor-bearing rabbit serum; channel 9: same as channel 8 except pre-incubated with lysate of RSV particles. The band at 60 kD in channels 8 and 9 (arrow) represented the first detection of the RSV oncoprotein. (p12, p15, p19, p27, Pr53, Pr66, and Pr76 are RSV virion proteins.) (From A.F. Purchio et al., Proc. Natl. Acad. Sci. USA 75:1567–1571, 1978.) 23
  26. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 5.2 The protozoan roots of metazoan signaling In the Cambrian era, ~540 million years ago, a diverse array of complex multicellular organisms evolved; many of these were the ancestors of contemporary metazoan phyla and already had recognizable features of modern organisms. Examination of contemporary animal phyla leads to a key insight into our origins: a metazoan organization plan was invented only once, and all modern metazoa descend from a common pre-metazoan ancestor that somehow solved the problem of cell–cell communication and the organization of complex tissues. This organization depended in significant part on the evolution of intercellular signaling systems, including the growth factors, receptors, and cell–cell adhesion molecules that are found in all modern metazoa. The fossil record indicates that during the earlier Vendian/Ediacaran era, which spanned from 650 to 540 million years ago, a variety of soft-bodied metazoa were already present, pushing the evolutionary origins of metazoa back another ~100 million years. The nature of the common pre-metazoan ancestor has been obscure until recently. However, accumulating evidence now implicates choanoflagellates as being the closest living relatives of metazoans. Each single-cell form of a choanoflagellate has a (A) © 2014 Garland Science single flagellum (which provides propulsion) that is surrounded by a large collar (assembled from 30–40 microvilli) that enables these protozoans to trap the bacteria that they consume for food (Figure S5.2). Large multicellular choanoflagellate colonies contain flagellated cells on the outside and possibly other flagellum-free cell types on the inside. The evolution of these multicellular colonies presumably depended on the development of cell–cell signaling and adhesion molecules. Importantly, sequencing of a choanoflagellate genome indicates that these protozoans make proteins that are found in higher metazoa, including receptor tyrosine kinases, EGFlike proteins, G-protein–coupled receptors, a TNF receptor-like molecule (Chapter 9), SH2 groups (Chapter 6), and cadherins (which bind metazoan epithelial cells together in two-dimensional sheets; Chapter 13). Once evolved to enable the assembly of these primitive multicellular colonies, these proteins could then be adapted in various ways to permit the formation and explosive diversification of far more complex multicellular animals during the early Cambrian period. At the same time, the signaling systems that were assembled from these proteins laid the foundations for the disease that, half a billion years later, we call cancer. (B) 5 µm Figure S5.2 Origins of cell–cell communication The tyrosine kinase receptors, along with a series of other important signaling molecules, were clearly developed long before the Cambrian explosion generated the ancestors of the various modern metazoan phyla. The choanoflagellate genome encodes many of these critical metazoan signaling molecules. (A) The choanoflagellates received their name from the distinctive collar that surrounds their single flagellum and is composed of an array of actin-rich microvilli (red). The single flagella, like the microtubules of the cytoplasm, are revealed here by an anti-tubulin antibody (green), while the nuclei are stained blue. (B) This scanning electron micrograph reveals that each solitary cell extends a single long flagellum with a surrounding collar that, in this case, has been disrupted into its individual component microvilli. Bacteria, which are trapped by these microvilli and used as food by the choanoflagellates, are seen nearby. (A, courtesy of Melissa TBoC2 n5.108,9/s5.02 Mott; from N. King, Curr. Biol. 16:R113–114, 2004. B, courtesy of M.J. Dayel, K. McDonald, and N. King.) 24
  27. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 5.3 Lateral interactions of cell surface receptors While GF receptors, such as those responding to EGF or PDGF, and integrins have been presented here as independently acting signaling proteins, it is becoming increasingly apparent that these cell surface receptors often form lateral complexes with one another and regulate each other’s signaling. In addition, integrin:RTK complexes are formed through association of these proteins with components of the ECM (Figure S5.3). For example, after binding its extracellular matrix (ECM) ligands, the αvβ1 integrin can cause phosphorylation of the EGF receptor even in the absence of EGF or EGFrelated ligands. This cross-phosphorylation appears to require physical association between the integrin and the EGF-R and depends, as well, on the complex of signaling molecules that the integrin attracts to its C-terminal tail in the cytoplasm. The Src tyrosine kinase, an important component of these integrin signaling complexes (see Figure 6.24) is activated following αvβ1 integrin binding to the ECM and is required for the phosphorylation of tyrosine residues in the C-terminal tail of the closely apposed EGF-R. This phosphorylation occurs independently of the EGF-R’s binding its normal ligands and results in a different spectrum of C-terminal phosphotyrosines than when the EGF-R binds its ligands. In addition, the amount of EGF-R present on the cell surface increases after the αvβ1 integrin binds ECM ligands. Indeed, efficient EGF-R signaling may require integrin association and integrin-mediated signaling. This example is only one of many. Thus, integrin-mediated modulation of tyrosine kinase receptor (RTK) signaling has been described for the platelet-derived growth factor-β (PDGF-β) receptor; a vascular endothelial growth factor (VEGF) receptor; the hepatocyte growth factor (HGF) receptor, termed Met, and the Met-related Ron RTK, which binds HGFL (HGF-like) ligand; the insulin receptor (IR); and the EGF-R–related ErbB2/HER2 receptor. And not unexpectedly, interactions between integrins and RTKs can be reciprocal: activation of RTKs by their respective ligands can stimulate signaling by the proteins associated with the C-terminal tails of integrins. Such reciprocal signaling suggests that RTKs signal in part by activating integrin-associated signaling proteins, while integrins signal in part by activating RTK-associated proteins. As receptor biochemistry is studied in increasing detail, it is also apparent that cross-phosphorylation between receptors occurs commonly and may represent a critical mechanism for controlling cell type–specific signaling. Within the EGF-R family of receptors, for example, HER2 can form heterodimeric complexes with HER3 and phosphorylate the C-terminal tail of the latter; in fact, HER3 itself lacks a catalytically functional kinase. Perhaps more unexpected are the signals exchanged between unrelated receptors. Thus, the mutant truncated EGF-R present in glioblastomas (Section 5.4) can drive phosphorylation of the activation loop of the tyrosine kinase domain of the HGF-R (termed Met); such phosphorylation causes the activation loop to swing out of the way of the catalytic cleft of this kinase, permitting its active signaling. In lung carcinomas that have become resistant to a low–molecular-weight inhibitor of the EGF-R tyrosine kinase, a new signaling channel is erected in which the Met receptor can drive HER3 tyrosine phosphorylation—a biochemical function that is usually mediated by the HER2 receptor–associated kinase. The formation of physical complexes between these various cross-talking receptors has yet to be directly demonstrated. fibrillin LTBP © 2014 Garland Science fibronectin LAP syndecan TGF-β HGF VEGF Met αvβ6 integrin P P P P α5β1 integrin α4β1 integrin P II I TGFβR VEGFR2 P P P P P P P P Figure S5.3 Association of integrins and growth factor receptors in the form of immobilized ligands. In addition, TGF-β receptors with extracellular matrix components and with is synthesized in an inactive form and is activated in association one another Many growth factors do not function as diffusible with LAP (dark blue), LTBP (yellow), and fibrillin (purple) , and signaling molecules. Instead, they are frequently attached to αvβ6 integrin. Moreover, the αvβ6, α5β1, and α4β1 integrins components of the extracellular matrix (ECM) following initial provide physical anchorage to the ECM via their association with synthesis and secretion by a signal-emitting cell and are later fibronectin and syndecan, a third ECM protein that binds, via a mobilized from the ECM and activated by proteases produced by specialized domain (heparan sulfate, purple, blue), to fibronectin. TBoC2 n5.111/s5.03 a second, signal-receiving cell or presented as solid-phase ligands These various associations with common ECM components serve attached to the matrix. Fibronectin is a key component of the also to nucleate multi-protein complexes containing both integrins ECM (green and pink). The HGF, TGF-β, VEGF growth factors are and RTKs. (Adapted from R.O. Hynes, Science 326:1216–1219, bound to the ECM and are apparently presented to their cognate 2009.) 25
  28. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 6.1 Systematic surveys of phosphotyrosine and SH2 interactions As illustrated in Figure 6.9, different tyrosine kinase receptors (RTKs) display distinct sets of phosphotyrosine residues following ligand-mediated binding and transphosphorylation. These phosphotyrosines serve as binding sites for a variety of SH2-domain–containing proteins. The identity of each phosphotyrosine recognized and bound by an SH2 is defined by its sequence context, more specifically, by the sequence of the three amino acid residues that flank the phosphotyrosine (pY) on its C-terminal side. In fact, there is a second distinct type of protein domain, termed PTB (phosphotyrosinebinding), that also functions to recognize and bind pYs; in contrast to SH2 domains, the recognition of a pY by a PTB domain is determined by the amino acid residues that flank the pY on its N-terminal side. All told, the human genome encodes more than 140 proteins that possess either an SH2 domain, a PTB domain, or both domains. While the RTKs possess tyrosine kinase catalytic domains that are very similar to one another, these receptors induce very distinct types of biochemical and thus cell-biological changes following receptor activation by growth factor ligands. These differences in responses to RTK activation are likely due to the fact that the various RTKs bind and thereby signal through distinct but overlapping sets of SH2- and PTB-containing partner proteins. In order to study in greater detail the interactions of a single receptor with its signaling partners, the binding affinities between a receptor’s array of pYs and the cell’s complement of SH2 and PTB domains have been measured. In the case of the EGF, FGF, and IGF1 RTKs, there are a large number of binding partners (39 are indicated in Figure S6.1) that are shared in common; many of these are involved in activating the signaling pathways, such as the Ras → Raf → MAPK pathway, that are described in detail in this chapter. Other interactions with binding partners are unique to individual receptors. For example, there are 11 SH2- or PTBcontaining binding partners that associate only with the EGF-R but not with the other two RTKs. These types of analyses should reveal why different RTKs evoke different responses from cells. The details of these interactions for a given RTK—in this case the EGF-R—are illustrated in (see Figure S6.1B). This figure indicates that the binding affinities of the individual SH2 and PTB domains with the pY-containing phosphopeptides of the EGF-R vary greatly, there being as much as 20-fold differences in binding constants. These differences in binding are likely to affect the strength of the signaling that results following binding of various SH2- or PTB-containing signaling proteins to the receptor. These binding interactions have also been surveyed for a number of other RTKs. The complexity of these interactions illustrates the difficulty that confronts biochemists in understanding with precision how ligand-activated receptors affect the cells into which they are releasing signals. (B) FGF-R1 IGF1-R IGF1-R & FGF-R1 891 992 974 1068 1045 1101 PLCG2 GRB2 N C GRB7 GRB10 GRB14 GRAP2 CRK CRKL NCK1 NCK2 APS SH3BP2 LNK SH2B C 1148 1114 1086 PLCG1 N C N 1173 C N C C N C N C N C SH2 domain PTB domain Phosphopeptide <100 nM 500 nM PTPN6 PTPN11 E18941 HSH2D CTEN E109111 E138606 E169291 E105251 SHB N CHN2 SH2D3A RIN1 BLNK LCP2 BRDG1 EAT2 SH2D1A TENS1 TENC1 TNS EGF-R & IGF1-R 954 N RASA1 3 845 C RABGAP1L CBL STAT2 STAT1 JAK3 JAK2 SHC3 SHC1 VAV3 VAV2 VAV1 SH2D3C SH2D2A BCAR3 INPPL1 4 2 N APBB1 39 APBB2 11 C SYK N C ZAP70 N C APBB3 7 IGF1-R, FGF-R1 & EGF-R EB-1 CCM2 EPS8L2 GULP1 APPL ANKS1 RABGAP1 FRS3 IRS4 IRS1 DOK5L DOK5 DOK4 DOK2 DOK1 E129946 NUMB NUMBL DAB2 DAB1 APBA3 APBA2 APBA1 BMX PI3KR3 N 11 DAPP1 ABL1 ABL2 FGR SRC SLA2 EGFR PI3KR2 EGF-R & FGF-R1 PI3KR1 EGF-R TEC LYN BLK ITK BTK TXK FER HCK PTK6 LCK FES YES1 MATK (A) © 2014 Garland Science 1 µM KD 1.5 µM 2 µM stronger weaker binding 26
  29. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Figure S6.1 Phosphotyrosine-binding signal-transducing proteins (A) Three widely studied RTKs—the EGF, IGF1, and FGF receptors—induce quite different cellular responses because they bind distinct sets of SH2- and PTB-containing signaling proteins. In the work illustrated here, the oligopeptides of the SH2 and PTB domains of more than 100 proteins thought to bind to the phosphotyrosines (pYs) of ligand-activated RTKs were synthesized, along with the pY-containing oligopeptides of these three receptors. The binding affinities of the phosphopeptides to the individual SH2 and PTB domains were then studied in vitro. Those binding affinities above a certain threshold are represented in the Venn diagram shown here. They reveal which of the SH2 or PTB domains are able to bind phosphopeptides that are present in the cytoplasmic tails of one, two, or all three receptors of these RTKs. As illustrated, a group of 39 SH2 and PTB domains is able to bind to pYs displayed by all three receptors, 11 domains (yellow) are able to bind pYs generated by activation of the EGF and FGF RTKs (but unable to bind to a pY on the IGF1-R), and 4 domains (light blue) are able to bind pYs generated by activation of the IGF1 and FGF RTKs (but unable to bind to a pY on the EGF-R). Finally, 11 domains (red) bind only to the EGF-R, 3 exclusively to the IGF-R (dark blue), and 7 to the FGF-R1 (green). (B) The EGF-R has 12 pY sites (only 7 of which are illustrated in Figure 6.9). © 2014 Garland Science Each of the associated phosphopeptides (red circles) is identified here by the residue number of its tyrosine in the overall EGF-R amino acid sequence. Individual SH2-containing domains (green circles) and PTB domains (blue circles) are arrayed around the edge of the rectangle and are identified according to the protein that carries them. Each binding interaction is indicated by a line connecting a given SH2- or PTB-containing domain with a specific phosphopeptide. For example, the GRB2 (= Grb2) protein (top right) has a single SH2 domain whose affinities for each of the 12 pY-containing phosphopeptides of the EGF-R were measured separately. The strength of binding is indicated by the color of the line, with strong interactions indicated by red lines and much weaker interactions indicated by blue lines. In the case of GRB2, it associates significantly with only 2 of the 12 phosphopeptides of the EGF-R. It has a relatively strong (red) interaction with the phosphopeptide-containing pY 1068 and a second, slightly weaker (orange) binding to pY 1114. Conversely, the phosphopeptidecontaining pY 1173 can bind both SHC1 (= Shc1) and ABL2, both with slightly weaker binding affinities. (A, from A. Kaushansky et al., Mol. Biosyst. 4:643–653, 2008. B, from R.B. Jones et al., Nature 439:168–174, 2006; A. Kaushansky et al., Chem. Biol. 15:808– 817, 2008; and courtesy of G. MacBeath.) 27
  30. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 6.2 The complexities of understanding RTK signaling One goal of research into the biochemistry of signal transduction is to generate mathematical models that explain why and how cells respond to signals from specific ligand-activated growth factor receptors (RTKs), such as the EGF and PDGF receptors. Such models must, by necessity, take into account the behavior of individual signal-transducing proteins that lie downstream of these receptors and are responsible for processing receptor-initiated signals and mediating eventual cell-biological responses. This goal is far from being realized because of the large number of biochemical parameters that affect responses to ligand-activated receptors. Included among these are 1. The diverse array of receptor tyrosine kinases (58 in all) that are displayed in various combinations on the surfaces of various cell types 2. The concentrations of each of these receptors on the surface of a given cell type 3. The functional interactions of these RTKs in fostering signaling by one another and in competing for limiting amounts of downstream signal-transducing proteins 4. The actual physiologic concentrations of various growth factor ligands in the extracellular spaces 5. The influences on receptor function of other cell-surface proteins (for example, integrins) that potentiate or limit receptor activation 6. The intensity of tyrosine phosphorylation on various phosphotyrosine residues following ligand binding by receptors 7. The expression in differentiated cell types of various combinations of the 103 signaltransducing cytoplasmic partner proteins possessing one or more SH2 domains and 42 with one or more PTB-containing domains (see Supplementary Sidebar 6.1), these proteins binding to the phosphotyrosines (pYs) created in the cytoplasmic tails of ligand-activated receptors 8. The functional interactions between these partner proteins in enhancing or blocking each other’s binding to ligand-activated receptors 9. The binding affinities of the various SH2- and PTB-containing partner proteins to the various receptor-associated phosphotyrosines (see Figure S6.1) 10. The lifetimes of these phosphotyrosines (that is, the period between the initial formation of each pY and its destruction by a phosphotyrosine phosphatase) 11. Various other negative-feedback controls on individual receptors, such as ligandinduced endocytosis, that are designed to delimit their signaling lifetimes (for example, see Figure 6.31) 12. Parameters governing the signal-transducing efficiencies of downstream signaltransducing partner proteins that govern their cross-communicating interactions with one another Together, these parameters interact in myriad ways to create a complexity that is vastly beyond our current abilities to analyze and to exploit in order to generate truly predictive models of cell-biological responses to growth factor stimulation. 28
  31. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 6.3 The rationale for multi-kinase signaling cascades A number of mechanisms have been invoked to explain why eukaryotic cells have assembled multikinase cascades, some involving three and even four kinases (A) in succession, rather than single signal-emitting kinases. A number of distinct functional advantages have been ascribed to the organization of multi-kinase cascades (Figure S6.2) A B C D D B C D A (B) 1 2 B C D © 2014 Garland Science D 1 2 1 2 (C) B 3 7 C 3 7 D 3 4 7 A (D) A 5 6 B B 5 6 C C 5 6 D D Figure S6.2 Advantages of multi-kinase cascades A number of distinct functional advantages for these multi-component kinase cascades have been proposed. (A) These cascades offer the advantage of great signal amplification, so that a relatively small signal that is used to activate the first kinase in a cascade can be amplified by each of the intervening kinases (since a single upstream kinase molecule can activate multiple downstream kinase molecules). This can result in an extremely strong signal released by the TBoC2 s6.02 lowest kinases in the cascade. (B) Each of the multiple kinases in a cascade may have its own constituency of substrates in addition to the next kinase in the cascade. Accordingly, these offer the ability to organize a group of radiating pathways that can affect a diverse array of intra-cellular regulators and thus cell-biological processes. (C) The multiple kinases offer multiple opportunities for other signaling pathways to intervene in the kinase cascade and modulate the flux of signals passing through this pathway. (D) Negative-feedback controls (black loops) may be imposed at several distinct steps of these cascades. 29
  32. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 6.4 Non-canonical Wnt signaling A subset of Wnt ligands signals via an alternative set of Frizzled receptors to evoke downstream responses in cells. Because these pathways were discovered after the initially characterized Wnt– β-catenin pathway (see Figure 6.26), they are termed collectively “non-canonical Wnt” signaling pathways. (In fact, some of the non-canonical Wnt ligands can also activate to some extent the canonical Frizzled receptors.) In addition to activating a subset of Frizzled receptors, some of these non-canonical Wnts (A) Frizzled also bind and activate a tyrosine kinase receptor (RTK) termed Ror2. These various ligand-receptor interactions elicit a number of downstream signaling responses (Figure S6.3) that are quite different from those described previously in the Wnt–β-catenin pathway (Figure 6.26). Among these are losses in cell polarity and acquisition of motility and invasiveness; non-canonical Wnt signaling may also contribute to activation of the epithelial–mesenchymal transition (EMT) program and entrance into the epithelial stem cell state (see Figure 14.20). (B) Wnt © 2014 Garland Science (C) Ror2 CYTOPLASM Dishevelled nuclear membrane Ca++ aPKC Par FLNA JNK filopodia PKC Calpain FLNA FL NA Tcf/Lef cell polarity & migration cell migration & invasion formation of focal adhesions arising from the binding by integrins Figure S6.3 Non-canonical Wnt signaling pathways of components of the extracellular matrix (ECM; see Figure 6.24). A number of signaling pathways that are activated by nonThe focal adhesions are critical, in turn, for cell locomotion. In canonical Wnt ligands evoke quite different biochemical responses addition, these signaling complexes influence the apico-basal than those triggered by the canonical Wnt–β-catenin pathway. polarity of epithelial cells (see Figure 2.4), doing so with the aid (A) The Ror2 tyrosine kinase receptor (RTK) contains a ligandof two other signaling proteins, atypical protein kinase C (aPKC) binding ectodomain that resembles the Wnt-binding domain of and a Par protein. The latter cooperate in a still-unclear fashion the Frizzled receptors. The binding of non-canonical Wnt ligands to activate the Jun N-terminal kinase (JNK), thereby aiding in (for example, Wnt5a) to the Ror2 receptor enables the latter TBoC2 s6.03 the establishment of cell polarity. (C) Yet another downstream to interact in an unknown fashion with non-canonical Frizzled consequence of non-canonical Wnt signaling is the mobilization receptors. The resulting downstream signaling, mediated by of intracellular calcium, which results in turn in the activation of still-unknown proteins, is transduced into the nucleus, where it a protein kinase C (PKC) and the calpain protease. By cleaving functions to antagonize the β-catenin–mediated transcription FLNA, this enzyme works with the PKC to enable certain aspects complexes that have been activated by the canonical Wnt of migration and invasiveness. Yet other pathways that operate signaling pathway. (B) The non-canonical Wnt ligand–activated downstream of non-canonical Wnt signals have been proposed Ror2 receptor has been proposed to activate Filamin A (FLNA), but are not illustrated here. (Adapted in part from M. Nishita et an actin-binding protein that transduces mechanical signals al., Trends Cell Biol. 20:346–364, 2010.) affecting the actin cytoskeleton and aids in the formation of filopodia (see Figure 14.37); the latter operate to enable the 30
  33. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 6.5 The Hippo pathway and control of stem cell proliferation The Hippo–Yap pathway was originally characterized in Drosophila, as genes encoding a number of its components, when mutated, were found to affect the size of organs. Only later were the mammalian homologs of these genes isolated and characterized. In both arthropods and mammals, this pathway controls organ size, which in turn is affected by the number of stem cells in developing organs. At the top of this pathway (Figure S6.4), afferent signals, influenced by various interactions with their extracellular environment, are fed into this pathway. These interactions include cell–cell contacts (including molecules responsible for regulating contact inhibition; see Section 3.2) and associations with the extracellular matrix (ECM), including notably the stiffness of this matrix. CD44 plasma membrane ? ? © 2014 Garland Science The connections between this pathway and cancer development are still being forged. Here are some examples. The YAP and TAZ transcription factors (TFs) are overexpressed in a variety of human cancers, sometimes in concert with the nuclear localization of these TFs, and experimental overexpression of YAP in cultured cells can cause their transformation. Loss of the pathway components NF2 (also known as Merlin), Sav, and the pair Mst1 and Mst2 results in liver enlargement and eventual carcinomas; indeed, the Merlin/NF2 tumor suppressor protein depends on YAP to regulate cell proliferation. In addition, knockdown of the transcriptional co-factor that operates with YAP/TAZ to drive expression of apoptosis-suppressing and proliferation-inducing genes, results in loss of the ability of YAP to induce cell transformation and the epithelial–mesenchymal transition (EMT; see Section 14.3). Nonetheless, the connections between these signaling proteins and cancer development are still poorly characterized, a situation that will surely change over the coming years. ? FRMD1/6? RASSFs NF2 Mst1/2 KIBRA Sav1 RASSF6 Mats P Ajuba P Lats1/2 Lats1/2 nuclear membrane P YAP/TAZ YAP/TAZ TEAD anti-apoptosis pro-proliferation genes Figure S6.4 The Hippo–Yap pathway This pathway was first uncovered by genetic studies of Drosophila development and only later extended, through the discovery of highly conserved homologs in mammalian cells. The mammalian orthologs of Hippo in flies are the Mst1 and Mst2 Ser/Thr kinases, seen here at the center of this pathway. (This pathway, even when studied in mammalian cells, is still named after its components operating in Drosophila cells.) Upstream of Mst1/2 are cell surface proteins, one of which is the CD44 protein, which appears to play a key role in regulating the stem cell state in certain normal and neoplastic epithelial cells. Yet other cell surface receptors, still not identified, are involved in sensing the extracellular environment and feeding signals into the Hippo–YAP pathway. NF2, often termed Merlin, is involved in acquiring signals from cell surface receptors and interacting with the actin cytoskeleton, which enables it to control the process of contact inhibition. The Lats1 and Lats2 serine/threonine kinases (the mammalian orthologs of the fly Hippo protein) function to phosphorylate the YAP/TAZ transcription factors, inactivating them and causing their export from the nucleus to the cytoplasm, where they are degraded. (Adapted from D. Pan, Dev. Cell 19:491–505, 2010.) TBoC2 s6.04 31
  34. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 7.1 Heterozygosity in the human gene pool The human gene pool carries millions of small variations in DNA sequence, which ensure that at many genetic loci, the gene copy we receive from one parent is subtly different from the gene copy we inherit from the other. This heterogeneity is often called genetic polymorphism (see Section 1.2, Figure 1.6). Recall that this term refers to a specific type of genetic variability in DNA sequence that has no effect on the functioning of a gene or its encoded protein product and therefore has no effect on cell or organismic phenotype. In the case of two nonidentical gene copies, these sequence polymorphisms will create, in a molecular-biological sense, two distinct allelic variants of a gene. (For a molecular biologist, a gene cannot be considered homozygous unless its two copies have identical nucleotide sequences.) Because such sequence polymorphisms do not affect phenotype, their behavior cannot be tracked by the classical procedures of Mendelian genetics. In the intergenic regions on a chromosome, there are often polymorphisms that distinguish the paternal from the maternal copy of a sequence. Though these sequence copies cannot be considered to be alleles of a gene (since they are far removed from any gene on the chromosome), they nonetheless behave just like Mendelian alleles, that is, they are transmitted in a Mendelian fashion from parent to offspring. Hence, © 2014 Garland Science the sequence AAGCC in one chromosome and its counterpart AAGGC in the paired homologous chromosome will be transmitted like Mendelian alleles from generation to generation. Because these sequence variants cannot affect phenotype, their existence and patterns of transmission must be gauged through biochemical analyses of DNA, such as DNA sequencing. These sequences, because they have no affiliation with known genes, are sometimes termed “anonymous” sequence markers. We humans descend from a small population (fewer than several thousand) of founding ancestors who lived 100,000 to 150,000 years ago. We might imagine that during the 6000 to 10,000 generations that have intervened between then and now, a variety of additional mutations, often deriving from DNA copying errors, have accumulated in human genomes, resulting in a progressive increase in the amount of polymorphism that can be detected in the now-large human gene pool. In truth, on an evolutionary time scale, this time period has been too short for large numbers of additional polymorphisms to accumulate in human genomes, and so we basically own the same genes and DNA sequences that our early ancestors carried around during the founding of our species. Hence, the great bulk of polymorphisms in the contemporary human population were already present in the gene pool of this founder population. 32
  35. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 7.2 Which is more likely—LOH or secondary, independent mutations? The logic of LOH would seem to indicate that this mechanism, rather than a mutational event striking the second, still-intact copy of a tumor suppressor gene, is invariably responsible for the loss of the surviving wild-type allele during tumor development. In truth, it is very difficult to predict the relative probabilities of these two alternative mechanisms, which vary from one gene to another. For example, the probability of a tumor suppressor gene suffering LOH through mitotic recombination (see Figure 7.7) is roughly proportional to the distance of this tumor suppressor gene from the centromere of a chromosome. Hence, tumor suppressor genes mapping close to centromeres lose heterozygosity only infrequently through mitotic recombination. Moreover, the likelihood that this recombination will occur is affected by the intranuclear environment, which varies from one cell type to another and seems to influence whether the two homologous chromosomes will be in constant, close proximity to one another within the nucleus and therefore poised to recombine. The chances of losing the second, wild-type gene copy through independent mutation are also affected by other variables. Large genes encompassing many tens of kilobases of DNA are more vulnerable to being struck and inactivated by a © 2014 Garland Science mutation than are small, compact genes. Genes that have large numbers of CpG sequences (where the cytidine is often methylated) are also more vulnerable, since the methyl-CpG is susceptible to a spontaneous chemical conversion of cytidine to uridine, resulting after DNA replication in the replacement of a CpG sequence by a TpG (see Section 12.5). Yet other factors, including whether or not a gene is being actively transcribed in a particular cell type, will also affect the likelihood of its accumulating mutations. Together, these complex factors make it very difficult to predict precisely how much LOH is favored over genetic mutation as the mechanism for eliminating the second, still-intact copies of tumor suppressor genes. To cite a specific example, in one study of 158 retinoblastomas, 101 (64%) were found to have undergone LOH at the Rb locus; the remaining 57 tumors presumably suffered independent mutations that struck the second Rb gene copy. Of those that underwent LOH, 94 showed true homozygosity in the chromosomal region surrounding the Rb gene, and 7 showed hemizygosity due to total deletion of the chromosomal region that included the second Rb gene copy. Consequently, these 7 tumors also suffered independent, secondary mutations—in these cases massive deletions of an entire chromosomal region. 33
  36. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 7.3 The polymerase chain reaction makes it possible to genetically map tumor suppressor genes rapidly In recent years, the use of RFLPs (see Figure 7.13) has been replaced by markers that can be detected using the polymerase chain reaction (PCR) to amplify specific chromosomal segments. In a simple form of this LOH analysis, one of the two DNA primers in a PCR reaction may be complementary to one allelic version of a DNA sequence that is polymorphic in the human gene pool; the other primer binds to a sequence on the complementary strand that is invariant in the human gene pool. The ability of the first primer to bind to one allelic version © 2014 Garland Science of the sequence and not to another can be used to determine whether or not the particular allele is present in a DNA preparation (Figure S7.1). The ability to perform multiple PCRs simultaneously enables geneticists to survey the configuration of many polymorphic markers at the same time; this has facilitated the discovery of thousands of these markers scattered throughout the genome. The resulting vastly increased density of markers along each chromosomal arm makes it possible to localize tumor suppressor genes far more precisely than the use of the very rough mapping strategy illustrated in Figure 7.13. (A) first primer TCGATGA AGCTACT allele A first primer anneals, elongation proceeds TCGATGA AGCTACT etc. second primer (B) first primer TCGATG A AGCTACG allele B primer fails to bind completely, no elongation no product Figure S7.1 Use of the polymerase chain reaction to determine sequence polymorphisms The polymerase chain reaction (PCR) can be used to determine the presence or absence of a single-nucleotide polymorphism (SNP)—a polymorphism where the two alleles differ by only a single nucleotide. (A) In one version of this procedure, the first primer (blue) is perfectly complementary to one allelic sequence (red strand), and therefore the rightward-moving primer extension reaction (blue line) proceeds normally, thereby generating a template for the leftward-moving elongation from the second primer (brown dashed line). (B) However, if the first primer is not perfectly complementary to the sequence present in the other allele (light green), all rightward-moving primer extensions will fail. Hence, the presence or absence of a SNP can be ascertained by the presence or absence of the resulting PCR products, which will accumulate to millions of copies in one set of reactions and will be absent in the other. The arrowheads indicate the 5ʹ to 3ʹ polarity of the DNA strands. TBoC2 s7.01 34
  37. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 7.4 The MSP reaction makes it possible to gauge methylation status of promoters The methylation-specific polymerase chain reaction (MSP) entails treatment of genomic DNA with sodium bisulfite under alkaline conditions (Figure S7.2). This treatment converts unmethylated cytosines (C’s) to uracils (U’s) while leaving methylated cytosines untouched. Segments of interest in this treated DNA (for example, promoter regions of critical genes) are then amplified, in parallel with segments of untreated DNA as control, using the polymerase chain reaction (PCR). Some of the primers used in the PCR reaction are designed so that they will anneal to the U-containing DNA (U’s being recognized as T’s) and will © 2014 Garland Science not anneal to the sequence present in untreated DNA. Yet other primers will anneal to the sequence present in the untreated DNA and not to the sequence present after bisulfite treatment. The result is the preferential amplification in each PCR of DNA fragments whose presence indicates methylation or the absence thereof. The readout of this reaction is a series of PCR products. Alternatively, following the bisulfite treatment, a DNA segment of interest can be amplified using PCR and then sequenced. To date, this technique has been used to study only a tiny proportion of the 30 million or so methylated C’s present in the haploid genome. GATCCTGATTGC Me bisulfite treatment GATUUTGATTGC Me add methylated DNA-specific primer CTAAAACTAACG GATUUTGATTGC add nonmethylated DNA-specific primer CTAAAACTAAC A GATUUTGATTGC Me Me begin PCR reaction begin PCR reaction CTAAAACTAACG GATUUTGATTGC primer fails to anneal properly, no PCR products; therefore CpG was methylated primer anneals, elongation proceeds, generate many DNA fragments via PCR amplification; therefore CpG was methylated Figure S7.2 Methylation-specific PCR The methylation-specific polymerase chain reaction (PCR) depends on the fact that the treatment of cellular DNA with sodium bisulfite causes the conversion of all regular cytosine residues to uracil residues (which behave like thymidine residues in the subsequent reactions). In contrast, the methylcytosines, which are frequently present in TBoC2 s7.02 CpG sequences, are resistant to this modification. Hence, DNAs can be treated with bisulfite and PCR primers can be designed that will hybridize with DNA molecules that were initially either methylated or unmethylated at critical CpGs. The annealing of these primers to the DNA (as in Figure S7.1) will determine, in turn, whether or not certain DNA fragments will be produced in the subsequent PCR. The second-strand PCR elongation product (lower left) is not shown. (Adapted from J.G. Herman et al., Proc. Natl. Acad. Sci. USA 93:9821–9826, 1996.) 35
  38. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Table S7.1 Advantages of ubiquitin-regulated proteolysis Supplementary Sidebar 7.5 Ubiquitylation tags cellular proteins for destruction in proteasomes The concentrations of many cellular proteins must be tightly controlled in response to a variety of physiologic signals. Much of this control is achieved through the selective degradation of these proteins. Thus, under certain conditions, a protein may be long-lived and is therefore permitted to accumulate within a cell, while under other circumstances it is rapidly degraded at a rate that is regulated by certain physiologic signals. This degradation is accomplished, almost always, by the ubiquitin–proteasome system (Table S7.1). To cite an example, many proteins are phosphorylated at critical amino acids by a kinase once their degradation is called for. The resulting phosphoamino acid, in the context of neighboring amino acid residues, attracts a complex of enzymes that covalently attaches a ubiquitin molecule to the protein—the process of ubiquitylation (often called ubiquitination). As an alternative mechanism, the exposure by a protein of one of its normally hidden subdomains may attract the attentions of a ubiquitylating complex even without the specific marking created by phosphorylation. Ubiquitin is a small (76-residue) protein whose sequence is highly conserved between single-cell eukaryotes and mammalian cells; only 3 of 76 amino acid residues differ between the yeast and human versions of this protein. (Its ubiquitous presence in the biosphere inspired its name.) One ubiquitin molecule is initially linked via its C-terminal glycine to the ε-amino side chain of a lysine present in a target protein; a second ubiquitin molecule may then be linked to lysine-48 of the first ubiquitin, and the process is repeated a number of times, yielding a polyubiquitin chain. This tagging and polyubiquitylation are achieved by a complex of E1, E2, and E3 proteins (Figure S7.3). A protein molecule tagged in this fashion makes Ubi Ubi ubiquitin ligase complex Ubi (E1 + E2 + E3) Ubi Ubi Ubi targeted protein (A) Ubi Ubi A. Unidirectional—unlike phosphorylation and other post-translational modifications of proteins, degradation cannot be rapidly reversed B. Rapid—a large number of protein molecules can be eliminated in a regulated fashion in a matter of minutes C. Fine-tuning—provides another way to finely adjust the levels of critical regulatory proteins D. Localized—can be confined to specific subcellular compartments E. Specific—a small set of proteins can be degraded without any effect on all other proteins Courtesy of M. Pagano. its way to a proteasome in the nucleus or cytoplasm, in which it is degraded. (An alternative version of the polyubiquitin chain, in which ubiquitin–ubiquitin linkages are formed through the lysine-63 residues of ubiquitin monomers, appears to be involved in the functional activation of tagged proteins rather than their degradation.) The widespread importance of this protein-degradation machinery is evident from sequence analysis indicating that at least 630 of the approximately 19,000 genes in the human genome appear to encode E3 ubiquitin ligases—the proteins responsible for initially recognizing substrate proteins that are to be targeted for ubiquitylation and destruction; 33 genes encode E2 enzymes, and as many as 110 candidate de-ubiquitylating enzymes (DUBs) have been identified. The latter proteases, by reversing ubiquitylation, often reverse the work of the ubiquitin ligase complexes. Ubi Ubi Ubi ATP © 2014 Garland Science N′ oligopeptides, amino acids hydrophobic globular core Ubi site of attachment of next ubiquitin ATP ADP polyubiquitylation 26S proteasome antigen presentation (B) Figure S7.3 Ubiquitylation and proteasomes Much of protein degradation in the mammalian cell is carried out by the ubiquitin– proteasome pathway. (A) A complex of three proteins (E1, E2, and E3), which together constitute a ubiquitin ligase, recognizes a protein destined for degradation and tags this protein with a chain of ubiquitins (Ubi). Following polyubiquitylation, the protein is conveyed to a proteasome (see Figure S7.4), where it is de-ubiquitylated and degraded into oligopeptides that are C′ point of attachment to lysine side chains of proteins either degraded further into amino acids or used for antigen presentation by the immune system; see Section 15.3. (In fact, a specialized proteasome is used to process proteins for antigen presentation.) (B) Ubiquitin itself is a relatively short protein of 76 amino acid residues whose amino acid sequence and structure are almost totally conserved among all eukaryotic cells. Its structure is indicated here in this ribbon diagram determined by X-ray crystallography. 36
  39. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Proteasomes are cellular workshops dedicated to the degradation of proteins that are presented to them. The proteasome is a large, hollow, cylindrical complex of multiple protein subunits, which uses ATP to unfold a protein prior to degrading this protein in the cylinder’s interior (Figure S7.4). Polyubiquitylated proteins that are introduced into the proteasome are first deubiquitylated and then digested into short peptide fragments ranging in size from 3 to 25 amino acid residues. The latter are then degraded by cytosolic proteases or, in certain instances, imported into the endoplasmic reticulum, where they become bound by MHC class I molecules and presented on the cell surface, a topic that is discussed at length in Section 15.3. Polyubiquitylation is known to be responsible for the degradation of many short-lived growth-regulating proteins such as Myc, p53, Jun, and certain cyclins (described in Chapter 8). Altogether, more than 80% of the proteins in mammalian cells, some far more long-lived, are degraded in proteasomes. While polyubiquitylation marks proteins for destruction, the function of mono-ubiquitylation (where only a single ubiquitin is attached) is complex and poorly understood. There are clear indications that mono-ubiquitylation is used in certain cellular contexts for regulating endocytosis, protein sorting, trafficking of proteins within the nucleus, and even regulating gene expression. For example, mono-ubiquitylation of the p53 tumor suppressor protein tags it for export from the nucleus; subsequent polyubiquitylation can then mark p53 for destruction in proteasomes. Mono-ubiquitylated growth factor receptors are marked for endocytosis. There are several additional dimensions of complexity in the post-translational modification of proteins. To begin, ubiquitin contains seven internal lysines. Of these, linkage via Lys48 residues is used to generate the polyubiquitin chains that are described above, which tag proteins for destruction in proteasomes. An alternative linkage can be forged via Lys63 residues of ubiquitin, and the resulting polyubiquitin chains thereby confer certain positive effects on the modified protein rather than marking them for destruction. End-to-end linkage of ubiquitin © 2014 Garland Science molecules also occurs with yet other consequences for the modified protein Yet another dimension of complexity derives from the fact that ubiquitin is only one of a group of as many as 10 distinct marker proteins (for example, the ubiquitin-like Sumo and NEDD8 proteins) that are used to tag various proteins, marking them for a variety of metabolic fates other than proteolysis. (A) (B) Figure S7.4 The proteasome The proteasome is a complex of about 2.5 megadaltons assembled from more than 30 distinct protein species. (A) This reconstructed image of a proteasome, determined by X-ray crystallography, indicates two “cap” regions (purple), where ubiquitylated proteins are bound, de-ubiquitylated, and then introduced into the centrally located barrel region (yellow), in which they are processed by proteolytic degradation into oligopeptides or amino acid residues. (B) A schematic diagram of the barrel region in cross section indicates the region (red dots) where the actual proteolysis takes place. (A, from W. Baumeister et al., Cell 92:367–380, 1998.) TBoC2 s7.04 37
  40. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 7.6 Krebs cycle enzymes and cancer development A class of mutations affecting enzymes that play important rules in tricarboxylic acid cycle have been found in a variety of human tumors. At first glance, these mutations might be thought to affect the overall energy balance of the cell, perhaps contributing in some fashion to the Warburg effect (see Section 2.6). Instead, these mutant enzymes seem to favor tumor development via far more subtle and interesting mechanisms. Somatic mutations in the genes encoding isocitrate dehydrogenase 1 (IDH1) occur in more than 70% of human gliomas and derived glioblastomas; tumors of these classes that lack IDH1 mutant alleles often carry comparable mutations in the IDH2 gene. Such mutations have also been found, albeit less frequently, in the genomes of colon and prostate carcinomas as well as acute myelogenous leukemias. Normally, IDH1 is a cytosolic enzyme, (also present in peroxisomes) which is involved in the conversion of isocitrate into α-ketoglutarate (Figure S7.5A); the same reaction is catalyzed by IDH2, albeit within mitochondria. Provocatively, these mutations are all point mutations altering an arginine residue (R132) in the catalytic site of IDH1 or the point mutation in the corresponding arginine residue 172 of IDH2. Like mutations affecting the ras gene, these point mutations are confined to altering a small number of codons (see Figure 4.10), suggesting that they potentiate function rather than simply inactivating it. These amino acid substitutions confer on IDH1/2 the novel ability to reduce α-ketoglutarate—the initial product of isocitrate oxidation—to 2-hydroxyglutarate (2-HG; see Figure S7.5A); because these mutations confer a novel function on the enzymes rather than inactivating them, they are termed neomorphic mutations. Moreover, because the mutant alleles do not undergo loss of heterozygosity (LOH), they appear to function in a dominant fashion and, accordingly, IDH1/2 are not considered tumor suppressor genes. On the contrary, as implied above, these mutations act like those affecting the ras protooncogenes to impart a function rather than to eliminate it. In this sense, the IDH1 and IDH2 genes can be considered proto-oncogenes and their derived mutant alleles oncogenes. Thus, when a mutant form of IDH1 is expressed in human glioblastoma cells that otherwise have very low levels of the 2HG oncometabolite, its levels increase dramatically (see Figure S7.5B), often accumulating to intracellular concentrations as high as 10 or 20 mM, an extraordinarily high level for any metabolite. The R132 mutant of IDH1, when expressed in wild-type cells, has been found by some researchers to cause a marked increase in the levels of both HIF-1α and its downstream effector, VEGF. This suggested that alterations in the levels of α-ketoglutarate and closely related metabolites, specifically 2HG, affect the regulation of proline hydroxylase, a dioxygenase enzyme that © 2014 Garland Science is responsible for oxidizing HIF-1α and HIF-2α, thereby tagging them for ubiquitylation and destruction (see Figure 7.28). However, other research groups have failed to replicate this observation. In fact, the 2HG that accumulates in very high concentrations in R132 mutant cells can function as a competitive inhibitor of dioxygenase enzymes that normally use α-ketoglutarate as their electron acceptor (see Figure S7.5C). This suggests, in turn, that increases in 2-HG can have wide-ranging effects on cells by affecting a diverse array of dioxygenase enzymes. Included among these are the proline hydroxylase (PHD) that is responsible for the HIF-1/2α proline oxidation cited above (see Figure S7.5D), the TET enzymes that are responsible for oxidizing and ultimately demethylating methyl-CpG dinucleotides in DNA (see Figure S7.5E,F), and the Jumonji domain-containing demethylases that are responsible for demethylating lysine residues of histone H3, thereby affecting chromatin structure and regulation of transcription (see Figure S7.5G). In normal cells, demethylation of CpGs instigated by the actions of TET enzymes might serve two purposes: Certain genes whose CpG islands were previously hypermethylated may, in response to physiologic signals, become demethylated through the actions of TETs; since methylated CpG islands usually repress gene expression, this might enable the de-repression of such genes. Alternatively, the TET enzymes may continuously cruise the genome and oxidize methyl groups on CpGs that have, for one reason or another, become inappropriately methylated. Accordingly, during the course of tumorigenesis, if active TET enzyme function is lacking, inappropriately methylated CpG dinucleotides may accumulate progressively. Yet other mechanisms may need to be invoked to rationalize the mutations found in genes encoding other enzymes involved in carbohydrate metabolism. Thus, mutant loss-of-function alleles of the genes encoding two other enzymes playing key roles in the tricarboxylic/Krebs cycle have been discovered in human tumors. These involve fumarate hydratase (FH) and succinate dehydrogenase (SDH). These mutations are likewise correlated with increases in HIF-1/2α levels. Indeed, the increased levels of succinate that result from mutations in the SDH gene have been reported to interfere with proline hydroxylase function, once again leading to increases in HIF-1α levels. Some mutations in SDH genes are acquired somatically while others are inherited in the germ line; the latter have been associated with susceptibility to develop pheochromocytomas (tumors of the adrenal glands) and paragangliomas (tumors of various neuroendocrine components of the sympathetic nervous system). In a subset of these cases, there are indications of an involvement of HIF1/2 in tumor pathogenesis. 38
  41. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) oxidation COO– CH2 C HO decarboxylation NADPH + H+ CH2 COO– C H NADP+ COO– H O H isocitrate CO2 COO– C C – COO reduction COO– CH2 COO– NADPH + H+ NADP+ CH2 COO– COO– C HO α-ketoglutarate oxalosuccinate CH2 CH2 (partial reverse reaction) mutant IDH COO– C O H+ © 2014 Garland Science 2-hydroxyglutarate (2-HG) (forward reactions) wild-type IDH (C) 120 100 80 60 40 20 0 (D) α-ketoglutarate 2-hydroxyglutarate H2C α-ketoglutarate (2-oxoglutarate) H2C c my 132 H proline of HIF-1α HO 2-hydroxyglutarate C N hydroxyproline H 1 (R Me IDH CpG (E) Me GpC 2nd round CpG replication Me C N H H 2 C C H2C H)- 1-m yc al ent IDH CO2, succinate H2 C proline hydroxylase Fe2+, ascorbate par 2-HG conc. (fold change) (B) GpC CpG 1st round GpC newly synthesized daughter strands replication newly synthesized daughter strands failure to methylate CpG GpC MeOH CpG Me CpG GpC MeOH Me 2nd round CpG Me MeOH CpG CpG replication GpC 1st round GpC Me α-ketoglutarate number of differently methylated CpG Loci MeOH (G) H3C 200 hypermethylated hypomethylated 150 GpC GpC succinate 2-hydroxyglutarate (F) CpG CpG MeOH TET2 newly synthesized daughter strands newly synthesized daughter strands replication GpC GpC H3C R2 CH2OH CH3 N+ α-keto- succinate glutarate + CO2 R1 R2 R1 N+ N+ Jumonji C 200 N 50 0 IDH mutant IDH wild-type H O (6-N,6-N,6-N) trimethyllysine O N 2-hydroxyglutarate H O HCH N H O 39
  42. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Figure S7.5 IDH mutations and their effects on multiple signaling pathways The mutant forms of isocitrate dehydrogenase (IDH) that are found in gliomas and, to a lesser extent, in other types of tumors, operate in complex ways to perturb cell signaling and enable tumor formation. (A) IDH is found in several isoforms in cells. IDH1 operates within the cytosol and peroxisomes, whereas IDH2 functions within mitochondria; they both normally catalyze the same reaction. In both cases, the metabolism of isocitrate results in a coupled oxidation and decarboxylation, producing α-ketoglutarate (α-KG). In the presence of mutant forms of the two IDH isoforms, this normal “forward reaction” proceeds into a “partial reverse reaction,” in which α-KG is reduced to 2-hydroxyglutarate (2-HG). (B) The actions of the mutant IDH1/2 enzymes results in a dramatic increase in 2-HG production, which often accumulates in concentrations within the cell exceeding 10 mM. In this experiment, the concentration of 2-HG was measured in parental human glioblastoma cells, used here as control, relative to the concentrations found in these cells following transfection of a plasmid expressing either the wild-type form of the enzyme (IDH1-myc) or a mutant form of the enzyme [IDH1 (R132H)-myc] often found in human gliomas and glioblastomas. (C) The 2-HG that is formed appears to act as a competitive inhibitor of a group of enzymes, termed dioxygenases, that use α-KG as their electron acceptors. Here, as one example of this class of enzymes, we see the interactions between the catalytic cleft of a histone demethylase enzyme (the JmjC domain of CeKDM7A) and its normal substrate (α-KG, above) or alternatively between this catalytic cleft and 2-HG (below). As is apparent, 2-HG (here seen in its D isomeric form) binds to the same site in the catalytic cleft of this enzyme as is normally bound by α-KG. The extremely high concentrations of 2-HG that accumulate in IDH1/2-mutant cells, which may be more than two orders-of-magnitude greater than that of α-KG, ensure strong inhibition of this dioxygenase via the mechanism of competitive inhibition; the effects of 2-HG on other dioxygenase enzymes of this class are presumed to be similar. (D) A key dioxygenase that is inhibited by high levels of 2-HG is the proline hydroxyase (PHD) enzyme responsible for oxidizing prolines of HIF-1α (as well as HIF-2α, not shown) (see Figure 7.27A). Normally, under conditions of hypoxia, HIF-1/2α escape oxidization and the HIF-1/2 transcription factors accumulate, driving the expression of a number of genes responsible for regulating angiogenesis, cell survival, and carbohydrate metabolism. However, in the presence of high levels of 2-HG, the PHD enzyme is inhibited, HIF-1/2 activity rises, and many of the responses usually orchestrated by these transcription factors under conditions of hypoxia are now expressed constitutively. (E) A second group of dioxygenases play important roles in regulating the methylation of CpG dinucleotides in DNA. As seen in Figure 1.19, maintenance DNA methylases can methylate © 2014 Garland Science the CpG residues of hemi-methylated DNA shortly after DNA replication, thereby ensuring the heritability of meCpG modifications. The countervailing mechanisms by which DNA is demethylated are less clear. One simple mechanism, sometimes termed “demethylation by neglect,” generates demethylated CpGs simply by failing to undertake maintenance methylation following one round of DNA replication (above). Following a subsequent round of demethylation, one of the daughter helices deriving from the purple strand will be fully demethylated at the indicated CpG dinucleotide. The alternative mechanism (below) involves the active oxidation of methyl groups by the TET1, TET2, and TET3 dioxygenases, which results in the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC); of these, only TET2 has been implicated in cancer pathogenesis. The resulting 5hmC is not recognized by the maintenance DNA methylases responsible for ensuring heritable transmission of 5mC groups from one cell generation to the next. Indeed, cancer cells with compromised TET2 function have been shown to accumulate hypermethylated DNA (see Panel F), indicating that the mechanism of removing 5mC from DNA is likely to play a major role in holding down the levels of this modified base in the DNA. Moreover, as seen in panel (B), the 2HG produced by mutant IDH1/2 alleles is a competitive inhibitor of the α-KG that is the normal substrate of TET enzymes. In fact, in acute myeloid leukemias (AML), the presence of TET2 mutant alleles is mutually exclusive with the presence of IDH1/2 mutant alleles in leukemia cell genomes. (F) The genomes of 131 gliomas (belonging to a variety of subtypes of this disease) were analyzed for the mutation status of their IDH1/2 alleles and for the methylation status of CpG loci that underwent statistically significant hyper- or hypomethylation. These data demonstrate the profound effect that IDH1/2 function had on the methylation status of these CpG loci in these tumors, (G) An important modification of histones that affects chromatin structure and transcription involves the methylation (including mono-, di- and trimethylation) of lysine residues, such as those in positions 4, 9, and 27 of histone H3, which have opposing effects on transcription (see Figure 1.20). Enzymes of the Jumonji C (JmjC) class are able to oxidize one, two, or three methyl groups from the 6-N position of lysine, using once again α-KG as an electron acceptor. The reaction reaches completion when the initially formed hydroxymethyl group is further oxidized (not shown) and then released as a formaldehyde molecule. These enzymes also appear to be inhibited by the 2-HG that accumulates in IDH1/2mutant cells, potentially generating extremely widespread and complex changes in gene expression. (B, from L. Dang, D.W. White, S. Gross et al., Nature 462:739–744, 2009. C, from W. Xu, H. Yang, Y. Liu et al., Cancer Cell 19:17–30, 2011. F, from B.C. Christensen, A.A. Smith, S.Zheng et al., J. Natl. Canc. Inst. 103:143–153.) 40
  43. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 7.7 Homologous recombination allows restructuring of the mouse germ line The natural process of homologous recombination can be exploited to introduce well-defined genetic changes into the mouse germ line. A cloned gene fragment that is introduced by electroporation or micro-injection into a somatic cell or an embryonic stem (ES; see Supplementary Sidebar 8.1) cell is able, with a low but significant frequency, to recombine with homologous DNA sequences residing in the chromosomal DNA of this cell. Use of appropriate selection markers, in this case neor (for neomycin resistance gene) and tkHSV (herpesvirus thymidine kinase gene, which renders cells sensitive to killing by the drug ganciclovir), can be exploited to select for the rare cells in which homologous recombination has taken place (Figure S7.6A). Thus, application of neomycin selects for cells that have stably integrated the donor DNA into their chromosomes, while ganciclovir causes (A) formation of ES cells carrying a knockout mutation r HOMOLOGOUS RECOMBINATION disrupted gene X insert into ES cells nonrecombinant cells recombinants with gene-targeted insertion other genes GENE-TARGETED INSERTION mutation in gene X NONHOMOLOGOUS RECOMBINATION recombinants with random insertion TREAT WITH NEOMYCIN (POSITIVE SELECTION) ES cell DNA gene X (B) positive and negative selection of recombinant ES cells tkHSV neo gene replacement vector © 2014 Garland Science RANDOM INSERTION TREAT WITH GANCICLOVIR (NEGATIVE SELECTION) no mutation in gene X recombinant cells cells are resistant to neomycin and ganciclovir (C) female mouse cells are resistant to neomycin but sensitive to ganciclovir INJECT ES CELLS INTO BLASTOCOEL MATE AND WAIT 3 DAYS surviving cells ES cells with targeted disruption in gene X EARLY EMBRYO PARTLY FORMED FROM ES CELLS INTRODUCE EARLY EMBRYO INTO PSEUDOPREGNANT MOUSE BIRTH isolated blastocyst Figure S7.6 Homologous recombination of mouse germ-line genes (A) A cloned fragment of a mouse gene is linked using recombinant DNA procedures with the neor and tkHSV drug selection markers. The neor marker selects in the presence of neomycin for cells that have stably acquired the cloned DNA segment, while the tkHSV marker selects in the presence of ganciclovir against cells that have acquired the cloned DNA fragment through nonhomologous recombination. This cloned DNA fragment can then be introduced via electroporation or transfection into a mouse embryonic stem (ES) cell. (B) Drug selection can then be performed to select for those ES cells that have stably acquired the cloned DNA fragment and have done so via homologous recombination. The configuration of the introduced DNA in the mouse genome can then be verified by Southern blotting analysis (not shown). (C) ES cells carrying properly integrated DNA segments are then injected into mouse blastocysts, whereupon their progeny may become incorporated into the tissues of the resulting embryo. Newborn mice may carry the introduced DNA fragment in their germ cells, thus enabling the germ-line transmission of this DNA fragment to their descendants. (A and B, adapted from H. Lodish et al., Molecular Cell Biology, 5th ed. New York: Freeman, 2004. C, adapted from B. Alberts et al., Molecular Biology of the Cell, 5th ed. New York: Garland Science, 2008.) CELLS OF OFFSPRING TESTED FOR PRESENCE OF ALTERED GENE; THOSE WITH ALTERED GENE ARE BRED TO DETERMINE WHETHER THEY CAN TRANSMIT IT TO PROGENY TRANSGENIC MOUSE WITH ONE COPY OF TARGET GENE REPLACED BY ALTERED GENE IN GERM LINE 41
  44. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg killing of cells that have retained tkHSV and therefore have integrated the donor DNA through nonhomologous recombination with the chromosomal DNA of the embryonic stem (ES) cell (see Figure S7.6B). ES cells that have acquired the cloned DNA via homologous recombination can then be introduced via micro-injection into the blastocoel cavity of a mouse blastocyst (an early embryo), and the embryo can then be introduced into a pseudopregnant female (see Figure S7.6C). Because ES cells are pluripotent (able to differentiate into all cell types in the body), the injected cells may then insert themselves into (chimerize) the developing tissues of the resulting embryo, creating a genetic mosaic in which some of the cells and tissues are descendants of the injected ES cells while others derive from the host blastocyst embryo. In the event that these genetically altered ES cells © 2014 Garland Science generate descendants that succeed in chimerizing the developing gonads, the germ cells in the gonads of resulting adults may then transmit the experimentally altered allele to descendant organisms. The originally used donor DNA fragment can contain sequences that, following recombination with the chromosomal gene, disrupt the chromosomal gene’s function, in which case this procedure is commonly termed a “gene knockout.” Alternatively, other types of alterations can be introduced into this cloned donor DNA fragment prior to micro-injection. Following homologous recombination, the targeted gene may retain some function and express, for example, a mutant form of the protein that it normally expresses or even a foreign protein; the introduction of such new sequences into the resident gene is often termed “gene knock-in.” 42
  45. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 8.1 The origins of embryonic stem cells As indicated in Figure S8.1, a variety of cells from early mouse embryos can be extracted and placed in culture. Depending on the stage of embryonic development, the resulting cells can yield variously embryonic stem cells (ESCs, or ES cells), embryonic carcinoma cells (ECCs, or EC cells), or embryonic germ cells (EGCs, or EG cells). Mouse ES cells, in particular, were first produced in the early 1980s through the culture in vitro of cells derived from early mouse embryos and have proven very useful for mouse genetics, including experiments in which specific genetic alterations can be introduced into the mouse germ line (see Supplementary Sidebar 7.7). Careful manipulation of these cells has yielded cell lines that appear to be genetically wild type and retain the capability, upon implantation into the blastocysts of mouse embryos, of integrating into such embryos and participating in the formation of all the embryonic tissues of the mouse; accordingly, such cells are termed pluripotent, in EO 1 0.5 1.5 2 3.5 5 7 8 ESC 9 10 11 that they are able to generate all of the tissues of the developing embryo with the exception of the extraembryonic tissues forming the placenta. A mouse resulting from such manipulation may have chimeric tissues, in that some of the differentiated cells in one of its tissues derive from the original blastocyst into which ES cells were injected and other phenotypically identical cells in the same tissue are descended from the injected ES cells. Genetic modification of the ES cells prior to injection into the host blastocyst (see Supplementary Sidebar 7.7) can generate a chimeric tissue in which the host-derived and ES-derived cells are genetically distinct. In the event that the injected ES cells are able to colonize the germ tissues, their descendants may form either sperm or egg, resulting in the transmission of the ES-derived genome through the germ line of the mouse that arises following development of the embryo arising from the injected blastocyst. 13 PO EGC ECC blastocyst ICM transfer ICM cells to extra-uterine sites cultivation embryonic stem cells feeder cells teratocarcinoma © 2014 Garland Science PGC cultivation cultivation embryonic carcinoma cells embryonic germ cells Figure S8.1 Derivation of pluripotent embryonic stem cell lines of the mouse As indicated here, at least three types of embryonic cells and derived cell lines can be derived by explanting cells from early embryos. The scheme depicts the derivation of mouse embryonic stem cells (ESCs), embryonic carcinoma cells (ECCs), and embryonic germ cells (EGCs) from different embryonic stages and compartments of the mouse embryos. The ESCs, hereafter referred to as ES cells, can be derived by explanting cells from the inner cell mass (ICM) of blastocysts. In vitro propagation of the resulting cells initially depended on culturing these cells above a monolayer of “feeder cells” that secreted factors enabling the ES cells to be propagated essentially for unlimited periods of time in vitro. Alternatively, the ES cells could be propagated in the presence of medium that had been conditioned by embryonic carcinoma cells (ECCs); such conditioning implies that the cells in question (the ECCs) release factors into the supernatant culture medium that can then be transferred to other cultures in which they benefit the survival and proliferation of other types of cells. As indicated also here, the ECCs are prepared from malignant teratocarcinomas that originate when embryonic cells, including ES cells, are implanted in a number of anatomical sites in the adult mouse, whereupon they generate tumors that are often composed of sectors that exhibit a number of distinct tissue-specific differentiation programs (see Figure 2.11). A third type of cell depicted here—EGCs—can be prepared by culturing primordial germ cells (PGCs) from the early sites of gonad development, termed genital ridges, that are apparent between days 9 and 12.5 of mouse embryonic development. (From A.M. Wobus and K.R. Boheler, Physiol. Rev. 85:635–678, 2005; photos from K.R. Boheler et al., Circ. Res. 91:189–201, 2002.) 43
  46. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 8.2 Plasticity of the cell cycle clock While the portrayal in Section 8.3 of the normal workings of the cell cycle clock is reasonably accurate, the functions of the clock and its various cyclin–CDK complexes can be reconfigured in major ways, as indicated by experiments in which the genes encoding various component parts are inactivated, that is, knocked out, in the mouse germ-line; see Supplementary Sidebar 7.7. For example, when the genes encoding CDK4 and CDK6 are knocked out, D-type cyclins can associate with and activate CDK2, allowing cultured cells to proliferate; mouse embryos lacking these two CDKs are viable through mid-gestation. Mice lacking either CDK 4, 6, or 2 are viable. Mice lacking CDK2 survive for two years—an essentially normal life span—but are sterile, while those lacking both CDK2 and CDK4 complete embryonic development but die at full term. Experiments like these indicate great functional redundancy and plasticity in the cell cycle machinery. Moreover, they suggest that in certain cell types and at certain stages of embryonic development, the organization of this machinery may be quite different from that depicted in this chapter. 44
  47. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 8.3 Chromatin immunoprecipitation (ChIP) In the ChIP procedure, the molecules within chromatin (containing both DNA and associated proteins) are crosslinked, usually by brief formaldehyde treatment (Figure S8.2). The treated chromatin is then mechanically sheared into small fragments, usually by sonication, yielding fragments 300–1000 nucleotides in length; alternatively, it may be fragmented by micrococcal nuclease treatment. Following brief centrifugation to eliminate cellular debris, the chromatin fragments are incubated with an antibody that reacts specifically with a particular transcription factor (TF). (To expedite isolation of the immunoprecipitates, the antibody molecules are cross-linked to beads prior to being used.) The resulting immunoprecipitates are then treated with a protease, such as proteinase K, after which the DNA molecules are extracted and amplified by the PCR (polymerase chain reaction). The amplified DNA can then be subjected directly to DNA sequencing (sometimes termed ChIP-seq), or it can be incubated with a microarray that contains a large number of specific genomic segments; successful annealing of the PCR-amplified DNA fragments with specific sequence probes in the microarray—the procedure of ChIP-on-chip—is then registered, once again yielding information on the genomic regions with which the immunoprecipitated protein was associated prior to cell lysis and cross-linking. In one alternative to this procedure, the antibody that is used reacts with a specific modified histone (see Section 1.8), enabling surveys across the genome of various histone modifications. The resulting sequences are then matched, using bioinformatics, with the sequences of all of the known genes present in the human or mouse genome, resulting in the identification of genes that are bound by a particular TF or associated with a particular modified histone. Application of this ChIP technique to a wide variety of TFs promises to reveal the identities of the cohorts of genes that are targets of transcriptional induction or repression by, for example, each of the known oncogenic TFs. Importantly, the demonstrated binding of a TF to the promoter of a gene does not, on its own, prove that transcription of the gene is actually regulated by the TF; instead, a rigorous demonstration of such regulation depends on functional tests of the gene in the presence or absence of the TF. Figure S8.2 Chromatin immunoprecipitation The procedure of chromatin immunoprecipitation requires the treatment of cells with an agent that cross-links genomic DNA with the associated chromatin proteins. Following fragmentation of the resulting cross-linked chromatin, the fragments are incubated with an antibody that binds one or another chromatin-associated protein, such as a transcription factor (TF; green spheres) or modified histone. In this case, the TF molecules are bound to DNA segments a, b and c, which might be parts of the promoters of three distinct genes. The resulting immunoprecipitates are then purified, and the associated DNA is isolated and, following PCR-based amplification, analyzed, usually either by direct sequencing or by annealing to a DNA microarray. © 2014 Garland Science treatment of cell with cross-linking agent cell lysis b a c sonication or treatment with micrococcal nuclease b c a incubation with antibody-coated beads b a c bead-antibody protein-target DNA complex protein digestion, DNA purification c a b genomic DNA fragments PCR amplification a b c a c c a b analysis by sequencing or annealing to microarrays identification of genes associated with immunoprecipitated proteins TBoC2 s8.02 45
  48. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 8.4 Some tumors increase Id concentrations by de-ubiquitylating them The Id proteins are increasingly perceived as critical regulators of processes such as differentiation and senescence, both of which they block when present in high concentrations. This is achieved largely, and perhaps entirely, via their ability to associate with and inhibit various bHLH proteins, some of which are important master regulators of certain differentiation programs. The Id1, Id2, and Id3 proteins are usually turned over rapidly, with half-lives estimated to be between 2 and 20 minutes, while the turnover of Id4 is slower, resulting in a half-life of more than an hour. Of note, the concentrations of the Ids are high in rapidly dividing tissues, and the concentrations of several of these (for example, Id2) are markedly elevated in a broad range of malignancies. The modulation in steady-state levels of certain Ids suggests a finely tuned regulatory apparatus that governs the synthesis and degradation of the first three Id proteins. Indeed, soon after being synthesized, they are usually rapidly ubiquitylated by a complex involving an E3 ubiquitin ligase identified as APC/Cdh1 (the latter representing the anaphase-promoter complex and its Cdh1 activator protein; see Supplementary Sidebar 7.5). Equally important are deubiquitylases (DUBs), which remove the polyubiquitin chains before ubiquitylated proteins, including the Ids, are transported to proteasomes for degradation; removal of the polyubiquitin chains by DUBs spares the ubiquitylated © 2014 Garland Science proteins, allowing their accumulation and higher steady-state levels. These dynamics suggest that the elevated levels of Id proteins found in many tumors may be consequences of a suppression of the ubiquitylase enzymes or an overexpression of the responsible DUB, which has been identified as USP1. In fact, in a number of tumors, the expression of both Id2 and the USP1 DUB have been found to be coordinately up-regulated. This correlation supports the notion that the elevated levels of USP1 are responsible for the increased accumulation of Id2, a notion that has been supported by direct experimentation. This suggests that an important means of deregulating the Rb circuitry in human tumors depends on increasing levels of this DUB, which results in higher concentrations of Ids that can, in turn, antagonize Rb function. The important role of Id proteins in blocking differentiation has been directly tested in an osteosarcoma cell line in which Id2 is overexpressed. Reducing the concentrations of the USP1 DUB (achieved through an introduced shRNA directed against its mRNA) resulted in marked reduction in Id2 levels, cessation of cell proliferation, and the osteogenic differentiation of the osteosarcoma cells into osteoblasts. This experiment reveals how critical this Id protein is in blocking differentiation, thereby holding the osteosarcoma cells in an actively proliferating, undifferentiated state. 46
  49. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 8.5 Specific targeting of cell cycle regulators by E3 ubiquitin ligases Much of the regulation of protein function during cell cycle progression is dependent on controlling the levels of key regulatory proteins achieved by controlling their levels post-translationally; this is achieved, in turn, by modulating the levels and activities of specific ubiquitin ligase complexes (see Supplementary Sidebar 7.5). The specific recognition of the protein substrate that is to be ubiquitylated is achieved by the E3 subunits of the multi-subunit ligase complex. In the case of cyclin E, its ubiquitylation and resulting degradation in proteasomes occurs at the G1/S boundary and the beginning of S phase (see Figure 8.10). At the R point, the formation of small numbers of cyclin E–CDK2 complexes allows the phosphorylation of, among other substrates, p27Kip1. This phosphorylation results in the recognition of the phosphorylated p27Kip1 by a ubiquitin ligase by Skp2 and Cks1 (above; see also Figure 8.37B). A very similar multi-subunit complex operates later at the G1/S boundary (Figure S8.3). In this case, however, the E3 subunit is involved in the recognition of the cyclin E substrate by an Fbw7 subunit. Figure S8.3 An alternative mechanism of protein degradation A very similar multiprotein complex to the one that is assembled at the R point in order to ubiquitylate p27Kip1 (thereby tagging it for destruction in proteasomes; see Figure 8.37B) operates soon thereafter at the G1/S transition to trigger the degradation of cyclin E. As seen here (below), the F-box protein Fbw7 (red) recognizes and binds a domain of cyclin E (aquamarine), driving its ubiquitylation and destruction. (The phosphorylated form of cyclin E that arises at the G1/S boundary, not shown, is bound by this ubiquitylation complex.) The remainder of the Fbw7-associated complex (not shown) is composed of Skp1, Cul1, and Rbx1—creating precisely the same multi-subunit assembly that forms the complex driving p27Kip1 ubiquitylation and degradation, shown above and in Figure 8.37B. The complexes differ in their respective subunits involved in substrate recognition: the complex driving p27Kip1 degradation contains Skp2 instead of the Fbw7 seen here. (Top, from B. Schulman et al., Nature 408:381–386, 2000; bottom, from B. Hao et al., Mol. Cell 26:131–143, 2007.) © 2014 Garland Science p27Kip1 C′ N′ N′ N′ N′ cyclin E Fbw7 Skp1 TBoC2 s8.03 47
  50. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 8.6 The major puzzle surrounding the RB gene: retinoblastomas While pRb clearly plays a role in governing cell cycle progression in a wide array of cell types throughout the body, its loss in cells early in life leads, peculiarly, to retinal tumors. The retina (see Figure 7.3A) is a relatively tiny organ compared with other tissues throughout the body. This only heightens the mystery, since the absolute numbers of cells in an organ should provide some indication of its intrinsic vulnerability to spawn tumors: tissues with large numbers of cells should be more susceptible to develop cancers, since they represent a far larger target size for the accidental triggering of a tumorigenic event. Some clues to the resolution of this puzzle have come to light in recent years, although a coherent mechanistic explanation is still lacking. To begin, cyclin D1 is expressed at extraordinarily high levels in the developing mouse retina, as much as two orders of magnitude higher than in other organs at this stage of development (Figure S8.4). While not yet documented, it is presumed that comparable levels of high expression are present in other mammalian embryos, including those of humans. Cyclin D1 operates as a major antagonist to drive partial phosphorylation and functional inactivation of pRb (see Figure 8.22) and sequestration of the CDK2 antagonist p27Kip1 (see Figure 8.17B); this would seem to make developing retinal cells especially dependent on the functioning of pRb in order to constrain their own proliferation. A second contributing factor appears to derive from the responses of many cells to loss of pRb function. The immediate effect is hyperactivity of the E2F1 transcription factor, which © 2014 Garland Science induces, in turn, expression of p14ARF (p19ARF in mice); the latter sequesters and thereby inhibits Mdm2, thus permitting high levels of p53 to accumulate (to be described in Chapter 9). Hence, loss of pRb results, through this chain of events, in increases in the pro-apoptotic p53 protein and the associated triggering of apoptosis. This seems to represent a naturally implanted safeguard against cancer development, since it causes the elimination by apoptosis of cells that have, for one reason or another, lost pRb function (Figure S8.5). This safeguard mechanism is thwarted in retinal cells, specifically, cone precursor cells, in which Mdm2 levels are naturally very high. These Mdm2 proteins apparently succeed in suppressing p53 function, even when pRb is lost in these cells. Accordingly, such cone precursor cells, which appear to be closely related to the cells from which retinoblastomas arise, are particularly tolerant of pRb loss, since the apoptotic elimination process that operates in many cell types elsewhere in the body is rendered inoperative by the high Mdm2 levels. According to this logic, an Rb+/– heterozygous child may lose the remaining wildtype Rb gene copy in many cells throughout his or her body, but those cells will never spawn tumors because they are eliminated by apoptosis. Only nullizygous (Rb–/–) retinal cells will therefore have a chance of surviving and generating tumors. (One clear exception to this rule is the bone-forming osteoblasts, which spawn the osteosarcomas that occur at greatly elevated rates in Rb+/– heterozygous children.) Figure S8.4 Expression of cyclin D1 in the developing retina Use of in situ hybridization reveals that cyclin D1 mRNA (red) is expressed in the developing retina of a 14.5-day mouse embryo at levels that are as much as 100-fold higher than in most other organs of the embryo. (A series of in situ hybridizations of sections of the same embryo have been joined together to reveal the expression of the mRNA in the entire embryo.) While not yet demonstrated directly, comparable elevated levels of expression are likely to be present in the developing retinas of other mammals, including humans. This high-level expression may, in ways that remain unclear, contribute to the special susceptibility of the retina to tumor formation in the absence of pRb function. (Courtesy of P. Sicinski and S. Elledge; from P. Sicinski et al., Cell 82:621–630, 1995.) 48
  51. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg most cell types pRb © 2014 Garland Science all Mdm2 sequestered by ARF; no p53 degradation Md m2 E2F1 most cell types p53 p14ARF E2F1 Mdm2 apoptosis m2 Md ARF ARF retinal precursor cells pRb retinal precursor cells Md m2 ARF m2 Mdm2 E2F1 Md p14ARF E2F1 p53 Mdm2 Md m2 Mdm2 RXRγ Mdm2 Mdm2 Md m2 mdm2 Mdm2 Md m2 Mdm2 excess Mdm2; p53 degradation no apoptosis Figure S8.5 Origin of retinoblastomas Retinoblastomas appear p14ARF then sequesters Mdm2, allowing p53 to escape Mdm2to arise from a cell type that is closely related to the precursors driven degradation and to accumulate to high levels and trigger of the cone cells in the eye that give us color vision. The reason apoptosis. In cone cell precursors, the first steps of this process Rb gene inactivation leads specifically to retinal tumors in young proceed identically, i.e., Rb inactivation leads, once again, to children has been elusive, since this gene and its encoded protein production of p14ARF. However, in these cells, a relative of the are responsible for regulating cell cycle progression in many cell retinoic acid receptor, another nuclear receptor (see Section 5.8) types throughout the body. One important contributing factor termed RXRγ, drives high levels of Mdm2 transcription, leading derives from the responses that many cell types throughout TBoC2 s8.05 to high concentrations of Mdm2 protein. These high levels of the body mount following inactivation of the Rb gene or its Mdm2 overwhelm p14ARF and thereby succeed in triggering product, pRb. As described in Sections 9.7 and 9.8, the resulting p53 degradation, allowing these retinal cells to evade p53deregulated activation of the E2F transcription factors (E2F1 is initiated apoptosis. As a consequence, Rb nullizygous retinal cells shown here) often leads to expression of p14ARF (p19ARF in mice). apparently can survive and proliferate, leading to retinal tumors. 49
  52. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg pro-apoptotic genes P P P P P P P P P P P P P active δNp63α P inactive δNp63α P activated p53 degradation E6 P P HIPK2 p53 U bi Chk2 ATM ATR E6 U bi U bi bi U bi U U bi U bi U bi U U bi bi U bi U bi bi U bi U bi DNA damage E6 E6 bi U OR E6 U Supplementary Sidebar 9.1 UV-B radiation, HPV, and cutaneous squamous cell carcinomas Squamous cell carcinomas (SCCs) of the skin occur with a relatively low frequency in the general population, often in light-skinned individuals, and with a greatly increased frequency in organ transplant recipients, who are immunosuppressed in order to prevent organ graft rejection (see Figure 15.18). These tumors are more threatening than the far more common basal cell carcinomas of the skin, which rarely if ever metastasize. Several human papillomavirus (HPV) types have been found in these lesions, although the etiologic role of these has not yet been firmly established. Recent evidence provides some insights into the mechanisms by which HPV, acting through its E6 protein, might act synergistically with UV-B radiation from the sun to enable the survival of heavily irradiated skin cells, thereby enabling any UV-mutated cells to persist in the skin and serve, over the long term, as the progenitors of SSC skin tumors. In effect, HPV, acting through its encoded E6 oncoprotein, is able to neutralize p53, thereby creating a phenocopy of the state seen in keratinocytes in which the chromosomal p53 gene has been mutated and rendered inactive. Normally, in response to DNA damage, HIPK2 (homeodomain-interacting protein kinase 2) phosphorylates p53 on its serine 46 residue, thereby placing p53 in an apoptosis-promoting configuration (Figure S9.1). (In fact, this phosphorylation may prove to be a key determinant of whether p53, acting as a transcription factor, induces expression of proapoptotic genes rather than, for example, genes that lead to cell senescence.) Such HIPK2-mediated phosphorylation of p53 occurs not only in response to UV-induced DNA damage, but also following genotoxic damage inflicted by a variety of other agents, including cisplatin and doxorubicin, two commonly used chemotherapeutic drugs. In addition, HIPK2 phosphorylates and inactivates δNp63α, a truncated form of p63, the cousin of p53. δNp63α usually acts as a dominant-negative inhibitor of p53, preventing it from triggering apoptosis. (Its high expression in rapidly proliferating epithelial cells helps to ensure the survival of these cells.) The E6 oncoprotein made by HPV23—a strain of this virus that is © 2014 Garland Science cytoplasmic proteasome Figure S9.1 HIPK2, HPV E6, and the inactivation of p53 pro-apoptotic functions The HIPK2 kinase is activated in response to severe DNA damage. Once activated, it phosphorylates p53 on its serine 46, thereby converting p53 into a pro-apoptotic transcription factor. [This site of p53 phosphorylation is distinct from the sites of ATM phosphorylation (serine 15) and of p53 phosphorylation by Chk2 (serine 20) that are described in Figure 9.13. Each modification has a distinct effect on p53 function in TBoC2 s9.01 response to DNA damage.] In addition, the E6 oncoprotein of human papillomavirus (HPV) is deployed by this class of DNA tumor viruses in order to tag p53 for ubiquitylation and destruction in proteasomes (see Table 9.1). Moreover, HPV E6 operates to block the interaction of HIPK2 with p53, thereby preventing p53’s conversion, via phosphorylation by HIPK2, into a pro-apoptotic transcription factor. Thus, HPV E6 blocks p53 function via at least two distinct mechanisms. This may provide a mechanistic explanation of how UV radiation and cutaneous HPV types function synergistically to trigger the outgrowth of cutaneous SCCs in humans. 50
  53. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg commonly associated with skin cancers—is able to bind HIPK2 and prevent this kinase from phosphorylating p53, thereby blocking the apoptosis-activating signal released by HIPK2. Moreover, by inactivating HIPK2, HPV E6 protects δNp63α from phosphorylation and resulting degradation; δNp63α can then continue to inhibit the pro-apoptotic functions of p53. In addition, E6 blocks p53 function directly; acting together with E6AP, a ubiquitin ligase, E6 tags p53 with polyubiquitin (see Supplementary Sidebar 7.5), thereby marking p53 for rapid destruction in proteasomes. This means that the inactivation of the p53 gene resulting from UV-B–induced mutations in © 2014 Garland Science keratinocytes can be functionally replaced by an HPV23 infection, which acts via two mechanisms to neutralize p53 protein function. Indeed, since the HPV23 E6 protein is able, in principle, to inactivate all of the HIPK2 in a cell, viral infection may be a far more effective way to prevent p53-induced apoptosis than UV-induced somatic mutations of the p53 gene. This protection of a virus-infected keratinocyte allows these skin cells to survive and accumulate additional UV-induced somatic mutations in other genes; the resulting multiply altered cells (carrying both the viral E6 oncogene and mutated cell genes) may then spawn a squamous cell carcinoma of the skin. 51
  54. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 9.2 The TUNEL assay The TUNEL assay provides a highly specific test of whether a cell is in the apoptotic state. It is in frequent use because it is reproducible and relatively simple to execute experimentally (Figure S9.2). apoptosis add TdT & BrdUTP add anti-BrdU monoclonal antibody Figure S9.2 The TUNEL procedure Apoptotic cells can be detected because their chromosomal DNA has become fragmented (see Figure 9.18C), exposing 3ʹ-OH DNA ends. The latter can be extended by the terminal deoxyribonucleotide transferase (TdT) enzyme, which acts processively to generate long tails from these ends, in this case doing so using bromodeoxyuridine triphosphate (BrdUTP) as substrate. The resulting BrdU-incorporated oligonucleotide tails can be detected with an anti-BrdU monoclonal antibody that has TBoC2 s9.02 been coupled to a dye molecule (yellow green). 52
  55. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 9.3 Dominant-negative functions of mutant p53 alleles: functional interactions between p53 and its p63 and p73 cousins Evidence is rapidly accumulating that p53 strongly influences the functioning of p63 and p73, almost certainly by forming mixed tetramers or mixed oligomers with the two paralogous proteins. Perhaps the most graphic evidence comes from experiments with a p53 mutant allele that carries a mutation in the sequence encoding the DNAbinding domain of p53. The resulting amino acid substitution not only causes a loss of p53’s ability to bind its usual target sequences in chromosomal DNA, but also causes p53 to undergo partial denaturation, leading to the formation of aggregates that accumulate in the cytoplasm, that is, away from p53’s normal site of functioning in the nucleus. The resulting complexes also involve p63 and p73 proteins, which form mixed aggregates with p53 in the cytoplasm, ostensibly compromising their ability to function as transcription factors because of this cytoplasmic sequestration (Figure S9.3). These aggregates testify to the ability of mutant alleles of the p53 gene to operate in a dominant-negative fashion to compromise the functions not only of wild-type p53 alleles but also of wild-type p63 and p73 alleles. DAPI p53 p63 © 2014 Garland Science p53 p63 Figure S9.3 Sequestration of p63 in cytoplasmic complexes with mutant p53 The products of certain mutant alleles of p53, such as the R110P allele studied here, undergo partial denaturation within cells—a consequence of amino acid substitutions in their DNATBoC2 s9.03 binding domains. The mutant p53 proteins then form aggregates in the cytoplasm that include both p63 and p73; only effects on p63 are shown here. These aggregates provide evidence that p53 has a natural tendency to associate with its p63 and p73 cousins, ostensibly forming mixed tetramers and possibly higher-order complexes in normal cells. These images are 3-dimensional computer-generated reconstructions of confocal micrograph serial sections that image an individual cell stained with an anti-p53 antibody (red), an anti-p63 antibody (green), and DAPI stain for DNA (blue). Any overlap of red and green (right panels) staining yields computer-generated yellow regions (left panel). (From J. Xu et al., Nat. Chem. Biol. 7:285–295, 2011.) 53
  56. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 9.4 Some mutant p53 alleles cause highly specific tumors Some mutant germ-line alleles of p53 have effects that are quite different from those observed typically in Li–Fraumeni families. One such unusual example has been reported in southern Brazil, where pediatric adrenal cortical carcinomas (affecting the cortex of the adrenal glands, which sit above the kidneys) are encountered at rates that are 10 to 15 times higher than elsewhere in the world. Remarkably, of 36 patients from this region who were examined, 35 showed an identical germ-line mutation in the p53 gene, which caused an arginine-to-histidine substitution in amino acid residue 337. This finding is difficult to reconcile with the current notion that inherited p53 alleles should affect a variety of tissues throughout the body. However, recent efforts to characterize the effects of this amino acid substitution on p53 structure have shown that the tetramerization domain of the mutant protein (see Figure 9.6) is less stable than that of wild-type p53 and is sensitive to disruption at acidic pH—precisely the environment encountered within the adrenal gland. This may account for the peculiar, tissue-specific effects of this mutant germ-line allele. (As an aside, the fact that 35 patients living in a confined geographic area carried the same rare germ-line mutations testifies to their descent from a common ancestor in which this mutation occurred—a genetic phenomenon known as a founder effect.) 54
  57. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 9.5 Autophagy is critical to postfertilization development About 4 hours after fertilization, widespread autophagy is initiated in the mouse oocyte and can be observed by visualizing the hundreds of autophagosomes that are formed (Figure S9.4). The formation of these autophagosomes is triggered by the process of fertilization and is essential for further development. Thus, if an oocyte is © 2014 Garland Science deprived of a critical autophagy protein, then development halts between the 4- and 8-cell stage of the subsequently formed embryo. Similarly, shortly after birth, widespread autophagy occurs in order to generate amino acids for the vigorous protein synthesis occurring in the cells of the neonate; these recycled amino acids are critical since the neonate no longer is supplied with amino acids via the placental circulation. Figure S9.4 Formation of autophagosomes in fertilized mouse eggs In these eggs, LC3 protein, an autophagosome-associated protein, has been labeled by fusing it with green fluorescent protein (GFP). The resulting GFP-LC3 protein, when concentrated in autophagosomes, generates visible fluorescent spots. An unfertilized oocyte (left) has only a few fluorescing autophagosomes, with most of the fluorescent label being distributed diffusely throughout the oocyte. However, shortly after fertilization (right), large numbers of concentrated autophagosome foci suddenly appear. (From N. Mizushima and B. Levine, Nat. Cell Biol. 12:823–830, 2010.) TBoC2 s9.04 55
  58. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 10.1 The use of the TRAP assay and adaptations thereof permits the rapid and quantitative assessment of the levels of the catalytic activity of telomerase enzyme in eukaryotic cells In adult human cells, with the exception of the hTERT catalytic subunit, the remaining multiple subunits of the telomerase holoenzyme appear to be present at levels that are consistently adequate for robust telomerase activity. Consequently, enzyme activity is governed by changes in the levels of the hTERT catalytic subunit, which may vary dramatically from one cell type to another (Figure S10.1). © 2014 Garland Science hexanucleotide sequence of G-rich strand nontelomeric sequence 5′ 3′ + telomerase + Taq DNA polymerase + Figure S10.1 Detecting telomerase activity The telomeric repeat amplification protocol (TRAP) assay permits detection of minute levels of telomerase activity in cell lysates by relying on the polymerase chain reaction (PCR) to amplify the products of the telomerase enzyme. A primer consisting of nontelomeric sequences (dark pink) and telomeric hexanucleotide sequences (from the G-rich strand; light green) is added to a cell lysate in the presence of deoxyribonucleotide triphosphates. This primer is extended (light orange) by any telomerase that may be present in a cell lysate. The thermostable Taq polymerase is then added together with a primer from the C-rich strand (dark green), and the second strand is elongated (light blue). These two DNA strands are then denatured and recopied repeatedly by the PCR in the presence of appropriate primers. hexanucleotide sequence of C-rich strand denature + further amplification by polymerase chain reaction (PCR) TBoC2 b10.21a/s10.01 56
  59. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 11.1 Monoclonal antibodies and fluorescence-activated cell sorting (FACS) The procedures for analyzing and fractionating cell populations by sorting them according to their display of cell surface proteins depend on the technologies for producing monoclonal antibodies (MoAbs; Figure S11.1A) and the instruments of fluorescence-activated cell sorting (FACS; see Figure S11.1B). The motivation for producing a monoclonal antibody (MoAb) is to generate a pure and homogeneous solution of antibody molecules, all of which are identical to one another and therefore recognize the same antigen with identical avidity. Such a MoAb contrasts with the antibodies that are usually produced by the immune system of an animal in response to immunization with an antigen of interest, usually a protein; these antibodies constitute a complex mixture of antibody species that bind to different epitopes (antigen sites) on the immunizing protein, with differing avidities (binding strengths), and with differing © 2014 Garland Science specificities, in that some antibodies may be totally specific for the antigen of interest, while others may recognize and bind unrelated proteins and are therefore termed cross-reactive. The serum of an immunized animal may be used to recognize the antigen of interest even though it contains a diverse array of other functionally unrelated antibody molecules; alternatively, the antibody molecules of interest may be greatly enriched through their ability to bind to the antigen of interest followed by their elution from this antigen. In both cases, this complex mixture is considered to be a polyclonal antibody. The production of a monoclonal antibody begins with the immunization of a mammal (usually a mouse, but occasionally a rat or rabbit) with an antigen of interest, such as a protein (see Figure S11.1A). The procedure of fluorescence-activated cell sorting (FACS) begins by treating cells with a monoclonal antibody, as described in Figure S11.1B. (A) myeloma cells (immortal) lacking antibody secretion and the enzyme HGPRT mix and fuse cells with PEG transfer to HAT medium immortal hybridomas proliferate; mortal spleen cells and unfused HGPRT– myeloma cells die select the hybridoma that makes antibody specific for antigen A selected hybridoma clone produces monoclonal antibodies spleen cells producing antibody from mouse immunized with antigen A add antibody coupled to fluorescent dye pass cells in front of laser that excites dye to cells photoelectric cell reads intensity of fluorescent excitation as each cell passes by separate cells on the basis of intensity of light emission distribution of light intensity plotted no. of cells having a certain intensity of light emission (B) intensity of fluorescent emissions 57
  60. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Figure S11.1 Monoclonal antibodies and fluorescence-activated cell sorting (FACS) (A) A mammal is immunized (usually repeatedly) to expand the number of B cells in its spleen that produce antibodies that specifically recognize the immunizing antigen. (Each of these B cells is known to produce a discrete and specific antibody species.) A mixture of B cells (lower left) is then prepared from the spleen of this immunized animal and each of these cells is fused using polyethylene glycol (PEG) with a myeloma cell belonging with a myeloma cell belonging to a special myeloma cell line (upper left) whose cells contain dominantly acting oncogenes but lack the ability to make both their own antibodies and the HGPRT enzyme. As a consequence, each of the resulting hybrid cells (left middle) contains a genome that (from the B-cell parent) specifies the production of a specific antibody and (from the myeloma parent) confers the ability to proliferate indefinitely in culture. These cells are transferred to a special medium termed HAT (hypoxanthine-aminopterin-thymidine), which selects for the outgrowth of cells that contain both parental genomes. The surviving hybrid cells are then introduced into microtiter plates at such high dilutions that each well of the plate contains at most a single cell. Each founding cell within a well should proliferate and its progeny should secrete antibody into the supernatant culture medium in the well. The medium from each of the wells is screened for the presence of antibody molecules that bind the original immunizing antigen (middle right). The cells from a well producing an antibody of interest are retrieved and expanded and constitute a hybridoma, i.e., a neoplastic hybrid cell line that produces a single, discrete antibody species, ideally with the desired specificity and antigen-binding affinity termed avidity (far right). The antibody produced by this hybridoma cell population is termed a monoclonal antibody. (B) Cells are treated with a monoclonal antibody that recognizes and binds a cell surface antigen displayed by some but not all of the cells in a population; alternatively, as shown, all of the cells may display this antigen but to differing extents. This antibody is usually coupled with a fluorescent dye, resulting in staining of the antigen-positive cells. Hence, among the cells being analyzed, some express the antigen at higher levels and therefore are stained more strongly by the antibody (darker red), while others are stained weakly (pink) or not at all (gray). This mixture of cells, placed in suspension, is then passed in single file past a laser beam that excites the dye (and causes it to emit light) and a photoelectric cell that measures the intensity of fluorescence emission by the dye molecules staining each cell. These measurements may then be integrated and plotted as a distribution of signals, in which case this procedure is often termed “flow cytometry” (below). Alternatively, if the labeling procedure has not damaged these cells, they can be separated from one another on the basis of their fluorescence intensity (e.g., through use of an electrical field), which allows the biological properties of these cells to be gauged through other tests (right). Often, staining by two different antibodies, each bearing a distinct fluorescent dye, allows the simultaneous measurement of intensities of two different antigens, in which case a different graphing convention is used, as shown in Figure 11.16A. Measurements of fluorescence intensity obtained in this way are usually plotted on a logarithmic scale. More recent innovations of FACS have allowed more than two antigens to be monitored simultaneously (not shown here) and the permeabilization of cells to allow access of antibody molecules to intracellular antigens. (A, from K. Murphy et al., Immunobiology, 8th ed. New York: Garland Science, 2011.) 58
  61. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 11.2 How does multi-step tumor progression actually take place? The scheme of multi-step tumor progression presented in Figure 11.15 indicates that a normal cell sustains an initiating mutation, which in turn spawns by clonal expansion a new cell population in which all cells carry this initiating mutation. One of these descendant cells will thereafter acquire a second mutation, resulting in a second clonal expansion, and so forth. However, with the discovery of the hierarchical organization of neoplastic tissues, this scheme must be revised to include cancer stem cells and transit-amplifying/progenitor cells. Because normal epithelial tissues are organized in a hierarchical fashion (involving stem cells, transit-amplifying/ progenitor cells, and more differentiated cells) and because the cells within derived fully neoplastic carcinomas appear to be organized in a very similar fashion, it is highly likely that each of the cell populations present in the intermediate stages of multistep tumor progression is also organized in a hierarchical fashion. Accordingly, in the simplest version of the revised scheme involving cancer stem cells, an initiating mutation strikes a normal stem cell and the resulting singly mutated stem cells subsequently sustain a second mutation, generating doubly mutated stem cells, which in turn develop into triply mutated stem cells and so forth. Each of these stem cells generates its own cohort of transit-amplifying and more differentiated descendants. In essence, one stem cell population evolves directly into another, as depicted in Figure S11.2A. While this scheme is logical, it holds several inherent difficulties that cannot be readily reconciled with real-world biology. To begin, it implies that the targets of mutation are, at each stage of tumor progression, individual cells within the stem cell subpopulation. Experience to date indicates that, in general, stem cells represent only a small proportion of the overall cells within a tissue, normal or neoplastic. Since mutations occur at a low rate per cell generation (for example, 1 per 106 cell divisions), this means that tumor-promoting mutations are mathematically relatively unlikely to strike this cell population because the absolute size of this cell population is small. (In the colonic crypt of the mouse, ~16 stem cells usually coexist with more than 500 transit-amplifying/progenitor cells.) © 2014 Garland Science A second major difficulty stems from the process of mutation. Mutations generally occur in actively dividing cells and in part are associated with mistakes in DNA replication of one sort or another, as will be discussed in Chapter 12; in rapidly proliferating cells yet other mutations arise from the replication of still-unrepaired DNA. If stem cells divide relatively infrequently (see Section 11.6), this also decreases the likelihood that they represent the targets of mutation. Taken together, these two factors argue against stem cells as the being the direct targets of the mutations that drive multi-step tumor progression. A solution to this problem derives from the discovery that the hierarchical scheme depicted in Figure 11.18B requires revision: non-stem cells can, with a certain frequency, dedifferentiate into stem cells. Accordingly, this hierarchical scheme now needs to be redrawn (see Figure S11.2B). This dedifferentiation can occur in both immortalized epithelial cell populations and in their transformed derivatives, both in vitro and in vivo; it may also occur in fully normal epithelial cell populations. The altered scheme depicted in Figure S11.2B now makes it attractive to embrace a model of how multi-step tumor progression actually proceeds (see Figure S11.2C). In particular, mutations strike a cell in the transit-amplifying/progenitor cell populations, which are both far larger than the stem cell populations and are actively dividing, making the occurrence of a mutation far more probable (relative to a mutation striking a member of the stem cell population). Thus, as the scheme of Figure S11.2C indicates, once a mutation hits a transit-amplifying cell, the dedifferentiation of this cell into a stem cell ensures that the new mutation can now be introduced into the stem cell pool; moreover, the mutant cell with this new mutation gradually displaces the other stem cells in this population, and these now generate transit-amplifying and more-differentiated cells carrying this new mutation. A second mutation that may trigger a subsequent round of clonal expansion will, as before, strike a cell in the newly formed pool of transit-amplifying cells and once again will be introduced into the stem cell pool via dedifferentiation. Experimental validation of this scheme is still required. Figure S11.2 Cancer stem cells, spontaneous dedifferentiation, and the actual course of multi-step tumor progression Cancer stem cells (CSCs) must be incorporated into the schemes of multi-step tumor pathogenesis described in this chapter. (A) The simplest scheme depicts fully normal stem cells (SCs) evolving directly into initiated SCs that have acquired an initiating mutation (red sector). The latter SCs now proliferate and acquire a second lesion (brown sector) and evolve directly into a third SC population, and so forth. The more-differentiated derivatives of each of these SC populations, including transit-amplifying and more-differentiated cells, are not depicted here. (B) Certain types of transit-amplifying/progenitor cells have been found to spontaneously dedifferentiate into stem cells (red arrows) under certain conditions of culture in vitro and in vivo as well, forcing revision of the existing, widely accepted scheme by which SCs differentiate unidirectionally into non-SCs. (C) The scheme shown in A, while attractive because of its simplicity, is complicated by two important considerations: (1) There are generally far fewer SCs than transit-amplifying/progenitor cells in a tissue; since the likelihood of a mutation occurring is proportional to the number of target cells in a population, this implies a far smaller likelihood of cancer-causing mutations being acquired by SCs relative to those arising in the transit-amplifying population. (2) Mutations occur far more commonly in actively proliferating cells, such as those in the transit-amplifying compartment; this contrasts with cells in SC compartments, which proliferate only infrequently. Taken together, these two considerations make the transit-amplifying cells into far more attractive candidates as the cells that acquire novel mutant oncogenic alleles. The dedifferentiation of non-SCs into SCs, as described in B, provides an interesting solution to this quandary. Thus, a transit-amplifying cell at one stage of tumor progression may acquire a mutation (red sector) and thereafter dedifferentiate into stem cells of the next stage of malignant progression. The resulting SCs may then spawn transit-amplifying cells, in one of which a second mutation (brown sector) strikes; this cell and its descendants may dedifferentiate into a successor SC population, and so forth. Such back-and-forth alternation between SC and transit-amplifying states appears to address the problems arising from the scheme depicted in A. 59
  62. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) STEP OF MULTI-STAGE PROGRESSION STEM CELLS MORE DIFFERENTIATED CELLS TRANSITAMPLIFYING CELLS © 2014 Garland Science (B) self-renewing stem cell I transitamplifying cells etc. II second mutation III third mutation IV etc. (C) STEP OF MULTI-STAGE PROGRESSION STEM CELLS TRANSITAMPLIFYING CELLS MORE DIFFERENTIATED CELLS I 1st mutation II 2nd mutation III 3rd mutation IV etc. 4th mutation n11.119,120,b11.16/s11.02 60
  63. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 11.3 Symbiosis between distinct subpopulations within a tumor The phenotypic heterogeneity of cells within tumors raises the question of whether in certain circumstances two subpopulations of cells cooperate with one another in some type of symbiosis rather than actively competing with one another. In one set of experiments, the detailed characterization of cells within certain tumors has revealed two subpopulations that coexist and do indeed appear to support one another’s survival and energy metabolism. One subpopulation resides more distantly from the microvasculature and is therefore more hypoxic (see Section 7.12). The cells in this subpopulation appear to utilize the “aerobic glycolysis” scheme of glucose metabolism, taking up the glucose that diffuses from microvessels through multiple cell layers to reach these cells; their mode of Warburg-like energy metabolism was described in Section 2.6. In accord with Warburg’s hypothesis, these hypoxic cells release lactic acid into the surrounding microenvironment. (A) pimonidazole © 2014 Garland Science A second, phenotypically distinct subpopulation of cells resides closer to microvessels and is therefore less hypoxic. This second subpopulation of cells takes up the lactic acid released by the first subpopulation and metabolizes this lactic acid through the citric acid/Krebs cycle, using the oxygen provided by the nearby microvessels. Hence, these cells behave much like conventional cells that normally have access to adequate oxygen, except that they use lactic acid instead of glucose to fuel their energy metabolism (Figure S11.3). Importantly, because the second class of cells do not use glucose as their carbon source, they permit the glucose diffusing from microvessels to reach the first, hypoxic subpopulation, that is, they do not intercept this glucose before it reaches the first subpopulation. Hence, the two subpopulations exist symbiotically: the hypoxic subpopulation provides lactic acid to the second subpopulation; and the second, by not intercepting the glucose leaving the microvessels, allows this glucose to reach the first subpopulation that is located more distantly from the microvessels. (B) MCT1 MCT1 Figure S11.3 Certain tumors harbor symbiotic subpopulations with distinct metabolisms When SiHa human cervical squamous cell carcinoma cells are grown as xenografts, the resulting tumors develop sectors that exhibit distinct metabolisms. (A) Cells that are hypoxic, as indicated by pink staining of the pimonidazole dye (outside of white dotted line), exhibit a metabolism that reflects the aerobic glycolysis that was first described by Warburg (see Section 2.6). These cells take up large amounts of glucose, much of which they process into lactate, which they secrete. (B) A second population of cells in the same tumor, which are less hypoxic because of n11.118/s11.03 better access to the tumor vasculature, express relatively high levels of monocarboxylate transporter 1 (MCT1; pink, inside of white dotted line), which they employ to take up the lactate that is released by their more hypoxic neighbors. These cells then feed the imported lactate into the Krebs/citric acid cycle and, because they have greater access to oxygen, are able to use this lactate in almost the same way as normal cells use glucose. Moreover, these MCT1-positive cells are less able to use glucose for this purpose. Together, this results in a symbiosis between the two populations: The cells with more direct access to the vasculature, and thus to both glucose and oxygen, do not utilize the glucose but instead allow it to diffuse to the more distantly located, hypoxic cells; the latter metabolize this glucose to lactate, which is then taken up by the MCT1-positive, more oxygenated cells and used as their prime carbon source for generating ATP in the Krebs/citric acid cycle. (From P. Sonveaux et al., J. Clin. Invest. 118:3930–3942, 2008.) 61
  64. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 11.4 Comparative genomic hybridization The techniques associated with the technology of comparative genomic hybridization (CGH) have been designed to determine whether a given genomic region has suffered either amplification or deletion during the course of multi-step tumor progression (Figure S11.4). These techniques are useful for localizing chromosomal regions that are likely to harbor oncogenes (in the case of amplification) or tumor suppressor genes (in the case of deletions). Since (1) both types of genetic changes are likely to involve chromosomal regions that are far larger than individual genes and (2) the precise locations of beginnings and ends of affected DNA segments are generally scattered randomly within a chromosomal region, it is usually necessary to perform CGH analyses on the genomes of a number of independently arising tumors in order to find regions that are recurrently altered in one or the other way; this narrows down the location of the specific gene whose alteration was functionally advantageous to the tumor cells. probes made from normal DNA and tumor DNA mix probes and anneal to normal DNA clones chromosomes green:red ratio copy number normal DNA anneals, tumor DNA does not, therefore red signal both normal DNA and tumor DNA anneal, therefore yellow signal normal DNA anneals, tumor DNA anneals more, therefore green signal amplification deletion distance along genome Figure S11.4 Outline of the experimental strategies of CGH CGH enables an investigator to determine whether the genome of a tumor cell contains segments that are present in greater or lesser copy numbers than exist in the normal diploid genome. As shown, fragments of normal DNA (red) and tumor DNA (green), each labeled with distinctly colored dye molecules, can be hybridized to DNA segments derived from defined chromosomal regions throughout the normal cell genome. Those chromosomal segments that anneal with only normal DNA will register as red, indicating that the corresponding DNA is missing in the b11.20/s11.04 tumor DNA. Conversely, those segments that are labeled in green indicate that an elevated copy number of the segment (e.g., as a consequence of gene amplification) is present in the tumor DNA. DNA segments that are present in equal number in normal and tumor DNA will register as yellow. (Courtesy of J.W. Gray.) 62
  65. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg agents) generally have nonlinear dose response curves (Figure S11.5). If actual human exposures to a compound being tested involve effective doses that are thousands of times lower than the MTD of this agent, and if the carcinogenic effect being scored in rodents derives largely from tumor promotion (because of toxicity at the MTD), then the outcomes of such high-dose rodent tests may have no implications whatsoever for human health risks. Ames’s critique of MTD carcinogen testing remains hotly debated, and the issue is largely unresolved. biological effect Supplementary Sidebar 11.5 Are rodent carcinogen tests reliable indicators of danger to humans? Bruce Ames, whose Salmonella test has had such a profound effect on our testing of potential human carcinogens (see Section 2.10), has questioned the meaningfulness of many of the laboratory tests currently performed to assess the potential carcinogenicity of various chemical compounds. These tests involve exposing rats and mice to high doses of a test compound for an extended period of time, followed by histopathological surveys to detect tumors that may have arisen in the exposed animals. Many of these rodent tests are conducted at doses that are close to or at the maximum tolerated doses (MTDs) of these compounds; above these doses, the test compounds begin to induce obvious damage in various organ systems of the exposed animals and therefore generate conspicuous effects on the animals’ health. (These MTD tests usually involve exposure to concentrations of agents that are orders of magnitude higher than those experienced by human populations. For example, human exposure to heterocyclic amines, a product of cooking meat at high temperature—see Section 12.6—has been estimated to be under 1 μg per kg body weight per day, although most laboratory animal studies have been conducted at doses in excess of 10 mg/kg body weight per day—more than 10,000 times higher. The motive for testing at these high dose levels is a desire to detect biological effects in a population of several hundred laboratory animals that might normally only be observed in several individuals among a human population size of one million.) Ames argues that a compound tested at the MTD may well be exerting its effect by killing cells in specific tissues, thereby provoking compensatory proliferation of the surviving cells in those tissues. Hence, a compound introduced into a rodent at concentrations near the MTD may be acting as a tissue-specific mitogen and thus as a tumor promoter, whether or not it also has mutagenic powers (see Section 11.13). Alternatively, certain chemicals tested at the MTD may favor inflammation, once again fostering tumor progression. The toxicity and secondary mitogenic effects of compounds, both natural and synthetic, are usually apparent only above a certain threshold dose, below which they are not detectable. Thus, unlike mutagens, whose powers for creating mutations in the genome are likely to be a linear function of dosage, compounds that act like tumor promoters (including cytotoxic © 2014 Garland Science dose-dependent effect of cytotoxic agent or tumor promoter dose-dependent effect of mutagen increasing dose Figure S11.5 Dose response curves of genotoxic and nongenotoxic agents Numerous studies have shown that the mutational burden inflicted by mutagens is, up to a certain concentration, linearly proportional to the cumulative dose of b11.42/s11.05 administered mutagen (red line). The risk of cancer, in turn, is likely to be linearly proportional to the burden of inflicted mutations. For example, 1% of a full dose of a mutagenic carcinogen is likely to yield 1% as much risk of carcinogenesis. In contrast, many tumor-promoting agents, including cytotoxic agents, operate differently (blue curve). Because their biological effects depend on their binding affinities to protein targets within cells, promoters usually have a sigmoid curve of the sort indicated. In this type of dose response, once the agent falls significantly below a certain threshold, it has virtually no biological effect. Conversely, once it rises above a certain threshold, its biological effects are maximal and cannot increase further. Accordingly, 1% of the dose of such a promoting agent may elicit far less than 1% of a biological effect. 63
  66. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 11.6 Does saccharin cause cancer? A controversy has roiled the scientific community for four decades. In 1970, researchers discovered that saccharin pellets implanted in the bladders of male rats resulted in a markedly increased rate of bladder cancer. This led to a widespread fear that the millions of obese and diabetic individuals who were using this artificial sweetener were being exposed to a carcinogen in their daily diet. As it turned out, the evidence pointing to saccharin’s carcinogenicity was most peculiar. It never registered as a mutagen in the Ames test. Indeed, since the saccharin ion present in solution is negatively charged, it has little affinity for DNA. Moreover, the carcinogenic effects of the implanted pellets were seen long after the saccharin had been leached out, leaving behind the vehicle used to construct the pellets—cholesterol. This suggested that mechanical irritation of the wall of the bladder created by these pellets contributed to the observed tumorigenesis. Tumors were observed only when saccharin constituted 2.5% or 5.0% of the total diet of male rats (a dose equivalent to a human drinking 750 cups of coffee daily, each with a saccharin pill in it). Bladder cancer was not observed in any other mammalian species exposed to saccharin, including monkeys who consumed substantial doses for more than 20 years. Mice exposed to a diet of 7.5% saccharin showed no sign of bladder cancer. Male (but not female) rats excrete into their urine large amounts of a protein called “major urinary protein” (MUP); the concentration of MUP in rat urine has been estimated to be 100 to 1000 times higher than in human urine. MUP, acting together with saccharin ions, triggered the formation of co-precipitates and co-crystals, which accumulated in high concentrations in the male rat bladder. (These crystals were also formed by salts of other ionized acids, which were also effective bladder tumor promoters in male rats.) These precipitates irritated and inflamed cells of the male rat urothelium— the specialized epithelium lining the bladder—yielding a tumor-promoting effect. Experiments like these illustrate the perils of carcinogen testing in rodent models. It is a widespread but still not universal consensus in the cancer research community that saccharin is totally harmless. Forty years after this controversy began, there was still no epidemiologic evidence that the vast numbers of diabetics experiencing chronic exposure to saccharin over many years’ time suffered any increased risk of bladder carcinomas. (The sweetener remains banned in Canada and California and in the minds of many, the perceived carcinogenicity of saccharin implies that other, chemically unrelated artifiical sweeteners are also carcinogenic!) 64
  67. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 11.7 How does diet affect colon cancer incidence? Colon cancer rates vary by as much as twentyfold between countries. These dramatic differences are due to environmental differences rather than differing degrees of genetic susceptibility, as indicated by the cancer incidence rates of migrants (see Figure 2.23). The “environment” in this case is, without doubt, foodstuffs in the diet. In the case of colon cancer, a striking influence of diet has been reported in a study of patients whose primary colon carcinomas were removed surgically followed by adjuvant chemotherapy. These patients were followed up for a period of ~5 years with special attention paid to their diet during this period. In particular, these patients were stratified in quintiles according to their consumption during this period of a “Western diet” (involving high consumption of red meat, high caloric burden, refined grains, and fried foods) at one end of the scale, in contrast to a “prudent diet” (which involved the consumption of fruits, vegetables, fish, poultry, and whole grains) at the other end. The highest- and lowest-quintile cohorts exhibited a stunning difference in disease recurrence, with those on the Western diet exhibiting a >3-fold higher relapse rate within the ~5-yearlong postoperative period. This schedule of rapid disease reoccurrence should be contrasted with the overall timetable of spontaneous colon cancer pathogenesis, which is assumed to usually involve periods of three or four decades if not longer (for example, see Figures 11.1 and 11.8); this extended period presumably reflects the difficulties that evolving pre-neoplastic cells encounter in acquiring critical genetic alleles that arise with low probability per cell generation. These contrasting schedules can only mean that the effects of the Western diet derived from its ability to stimulate the outgrowth of latent but already-neoplastic metastatic growths that had escaped detection and excision during initial surgery. Stated differently, the rapidity of these relapses can only mean that the diet acted as a tumor promoter on cells that, at the time of this surgery, had already acquired most if not all of the mutations required for full-fledged neoplastic behavior. The identities of components of the Western diet that were responsible for promoting the eruption of these previously latent growths were not revealed by this clinical study. However, epidemiology points to the most likely culprit—red meat consumption—which has been repeatedly correlated with increased incidence of colorectal, prostate, pancreatic liver, esophageal and even lung, carcinomas. But how might red meat (from beef, lamb, and pork) function as a tumor promoter that stimulates the outgrowth of nests of latent colorectal carcinoma cells? One novel hypothesis has derived from the observation that red meat contains a form of sialic acid that is not present naturally in the human body. (Sialic acid is used as one component of the complex post-translational modifications of proteins that yield glycoproteins; virtually all secreted and cell-surface proteins are glycosylated during their biogenesis.) Most mammals © 2014 Garland Science synthesize two forms of sialic acid, N-acetylneuraminic acid (Neu5Ac) and the hydroxylated form N-glycolylneuraminic acid (Neu5Gc) (Figure S11.6A). However, 2 to 3 million years ago one of our hominid ancestors lost the gene encoding enzyme required for Neu5Gc synthesis; as a consequence, the glycoproteins that our cells synthesize include only the Neu5Ac isoform of sialic acid on their carbohydrate side chains. This has an effect on the development of our immune system, which becomes tolerant to Neu5Ac early in the development of its lymphocytes (see Section 15.5); conversely, tolerance is not developed toward Neu5Gc, which may be recognized as a foreign antigen in the event that our immune system encounters it, provoking some type of immunologic attack. In humans, confrontation with Neu5Gc occurs through the consumption of muscle from species that do indeed synthesize this sialic acid isoform and incorporate it into their glycoproteins. It is expressed at particularly high levels in the red meats prepared from beef, lamb, and pork, while the meat of other vertebrates (chicken, fish) expresses relatively low levels of Neu5Gc. The dietary Neu5Gc is metabolized in the gut, perhaps by commensal bacteria, and some of it enters into the circulation and is eventually incorporated into the glycoproteins of our normal and even neoplastic tissues, with preferential incorporation into secretory epithelia and blood vessels (see Figure S11.6B). This lack of tolerance toward Neu5Gc can lead, in turn, to the production of anti-Neu5Gc antibodies by the immune system and chronic, low-level inflammation as cells of our immune system attempt to attack and eliminate cells that express this “foreign” antigen. (Indeed, antibodies reactive with Neu5Gc are common in human sera.) Because processing of the carbohydrate side chains is altered in many types of human tumor cells, the Neu5Gc of dietary origin may actually be incorporated at higher levels into the glycoproteins of tumor cells than normal cells. As a consequence, because of antibody-mediated binding to the surfaces of normal and neoplastic cells, attacks on these cells mediated either by macrophages or by complement fixation may generate chronic low-level local inflammation in both normal tissues and tumors (for example, see Section 15.16). (Higher levels of immune reactivity against tumor cells might in principle lead to attack of the tumors and their elimination by various arms of the immune system; it is unclear whether such immune rejection of tumors displaying Neu5Gc actually occurs in human cancer patients.) These complex dynamics represent an attractive but still-unproven mechanism that connects read meat consumption with increased cancer risk. Of note, a very large epidemiologic study reported in 2012 that the hazard ratio for the development of a variety of human cancers was 1.10 for consumption of unprocessed red meat and 1.16 for the consumption of processed red meat, in both cases involving one serving per day. Given the absolute number of cancer cases diagnosed yearly, this represents a large number of diagnosed tumors and, quite often, subsequent deaths. 65
  68. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) O OH HO OR O HO H N O– HO O made by most mammalian species N-acetylneuraminic acid (Neu5Ac) HO not made by humans HO O OH O HO H N O O– OR HO N-glycolylneuraminic acid (Neu5Gc) control IgY lung carcinoma normal lung (B) anti-Neu5Gc IgY © 2014 Garland Science Figure S11.6 Incorporation of Neu5Gc in human tissues The consumption of red meat and dairy products has been shown to lead to the introduction into human tissues of N-glycolylneuraminic acid (Neu5Gc), which is generated from the digestion products of proteins from beef, lamb, and pork. Thereafter, this form of sialic acid may be introduced via the circulation into a variety of tissues, into which it is incorporated during the course of the biosynthesis of native human glycoproteins. (A) Most mammals make two forms of sialic acid, which becomes incorporated into a vast array of glycoproteins. The inability of humans to synthesize Neu5Gc means that this molecule is not experienced during the development of immune tolerance and may thereafter be perceived as a foreign antigen by several arms of the adaptive immune system (see Section 15.5). (B) Neu5Gc is not naturally synthesized in human cells and therefore cannot be used by the glycosylation machinery that undertakes the post-translational modification of proteins by assembling the complex carbohydrate sidechains and attaching them to the primary products of translation to construct glycoproteins. However, Neu5Gc can be detected in both human epithelial tissues such as the one analyzed here as well as in the endothelial lining of human blood vessels (not shown). This Neu5Gc derives from dietary sources, largely red meat and dairy products. Here a monospecific polyclonal chicken IgY antibody has been used (right column) to immunostain both normal (upper row) and neoplastic (lower row) lung tissue for the presence of Neu5Gc. The results of staining both tissues with a control chicken IgY antibody are seen in the left column. The presence of Neu5Gc is indicated by the brown staining. (B, from N.M. Varki et al., Annu. Rev. Pathol. Mech. Dis. 6:365–393, 2011.) TBoC2 n11.122,n11.123/S11.06 66
  69. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 12.1 Hematopoiesis as a model for the organization of many kinds of tissues Our understanding of the organization of a variety of self-renewing tissues derives in large part from the study of the organization of hematopoiesis (Figure S12.1). Experiments performed in the 1960s and 1970s led to a number of concepts that remain deeply embedded in our thinking to this day. Among them are the following: © 2014 Garland Science 1. A primitive, relatively undifferentiated cell type can spawn progeny that ultimately generate multiple distinct differentiated cell types. 2. These primitive cell types reside long-term within a tissue and, upon dividing, ensure that at least one of their daughters remains a stem cell, while the other may exit the stem cell state and begin a process of differentiation. The generation NK cell THYMUS T cell common lymphoid progenitor B cell dendritic cell dendritic cell multipotent hematopoietic stem cell multipotent hematopoietic progenitor macrophage monocyte osteoclast neutrophil eosinophil common myeloid progenitor basophil platelets megakaryocyte erythrocyte STEM CELL COMMITTED PROGENITORS DIFFERENTIATED CELLS a tissue; instead, in certain tissues such as this one, “committed Figure S12.1 Hematopoietic differentiation Our current progenitors” (i.e., the lymphoid and myeloid stem cells shown understanding of hematopoietic cell differentiation teaches a here) as well as some of their descendants have self-renewal number of lessons. (1) It indicates that a single cell type—the capability. The fact that a patient suffering from CML (chronic multipotent hematopoietic stem cell (HSC; left)—is capable of myelogenous leukemia) often exhibits several distinct differentiated generating virtually all of the cell types in the blood and in the lymphoid and myeloid cell types carrying the Ph1 chromosome immune system. (2) It shows that a single stem cell type can spawn multiple types of “committed” stem progenitor cells, in this (and a BCR-ABL translocation) provides strong indication that this case, the two stem cell types that are committed to generating abnormal chromosome was initially formed in some multipotent lymphoid and myeloid cell types. (3) It shows that self-renewal HSC or progenitor. (From B. Alberts et al., Molecular Biology of the ability (curved arrows) is not confined to a single stem cell type in Cell, b12.04/s12.01 5th ed. New York: Garland Science, 2008.) 67
  70. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg of two phenotypically distinct daughter cells is termed asymmetric division. 3. This generation of a daughter cell that is indistinguishable from its mother is the process of self-renewal, guaranteeing that the pool of stem cells in a tissue is not depleted by such cell divisions. 4. The non-stem cell daughter that arises following division of a stem cell may not immediately undertake to differentiate, but instead may participate in a succession of symmetric cell divisions, generating more cells like itself. Such cells, which are often termed either transit-amplifying or progenitor cells, may increase exponentially, ensuring that a single cell division of the original mother cell—the stem cell—yields dozens if not hundreds of progenitor cell progeny. This means that the stem cell need only occasionally pass through a cycle of growth and division, minimizing the likelihood that the genome of this cell and its stem cell descendants will be susceptible to the © 2014 Garland Science genetic accidents that accompany active cell division. 5. After a certain number of divisions, the progenitor cells may then generate fully differentiated progeny that undertake the work of the tissue. Since these differentiated cells often have entered into a post-mitotic state, this means that the great bulk of the mitotic activity in a tissue is often associated with the transit-amplifying/progenitor cell population. The model presented in Figure S12.1was strongly supported by experiments in which a mouse that had been lethally irradiated, thereby depleting its hematopoietic cells, could be rescued from otherwise-inevitable death by implanting hematopoietic stem cells. These experiments demonstrated, among other things, that all the cells in the hematopoietic tissues of such a rescued mouse might derive from a single engrafted stem cell, proving that this stem cell was multipotent, in that its progeny could enter into multiple distinct states of differentiation and thereby generate multiple differentiated cell types. 68
  71. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 12.2 Stem cell pools may explain the protective effects of pregnancy A clear explanation for the complex epidemiology of human breast cancer is still to be produced. As is apparent from Figure S12.2A, the age of first parity has a profound effect on breast cancer risk: women who give birth before the age of 18 have one-third the breast cancer rate of those who postpone childbearing until their mid-30s. Premenopausal women who are nulliparous (having never given birth) experience as much as twice the lifelong risk of breast cancer of women who are parous (having borne one or more children) early in life. And women who postpone parity until later in life have an even greater risk of breast cancer than nulliparous women. This complex behavior may be explained by the following three mechanisms. 1. During a full-term pregnancy, a portion of the relatively undifferentiated stem cells in the mammary epithelium are recruited to form at least three of the differentiated cell types that are involved in the formation of ducts and of the alveoli in which milk production occurs. Consequently, repeated pregnancies progressively deplete the less differentiated cells in the mammary gland, and this depletion is correlated with the progressive decrease in the population of cells that are susceptible to mitogenic stimulation and to malignant transformation (see Figure S12.2B and C). 2. The pulse of hormones that a woman experiences during each menstrual cycle (see Figure 11.34) provides a potent mitogenic stimulus to the mammary epithelium and therefore has a tumor-promoting effect; indeed, breast cancer risk grows progressively with increased numbers of menstrual cycles (see Section 11.14). Hence a nulliparous woman, whose menstrual cycling has not been suppressed by repeated periods of pregnancy and lactation, experiences © 2014 Garland Science many more tumor-promoting menstrual cycles carrying a large pool of susceptible, undifferentiated stem cells that has not been depleted by pregnancies. 3. The large hormonal exposure experienced during pregnancy also promotes breast cancer development. If a woman has children early, the tumor-promoting effect of pregnancy and of subsequent menstrual cycles will be countered by the elimination of most of the pool of susceptible stem cells in her breasts. However, if she postpones parity until later in life (for example, age 40), she will have forgone virtually all of the benefit of pregnancy removing cells from this stem cell pool before the bulk of her menstrual cycles (>300) have taken place. At the same time, the hormones of pregnancy will, like those of menstruation, act as potent tumor promoters of cells that may have become initiated during the previous quarter century, making her even more susceptible to breast cancer than a nulliparous woman. The striking effects of pregnancy on the breast can be modeled in laboratory animals. For example, dimethylbenz[a]anthracene (DMBA) has been found to be a potent mammary carcinogen if introduced into virgin rats, but it loses its carcinogenic powers if introduced into female rats after they have undergone a pregnancy. The hormone human chorionic gonadotropin (hCG) can be introduced into virgin rats and can mimic many of the effects of pregnancy, including the induction of steroid hormones and the differentiation of stem cells in the mammary epithelium. If hCG is administered to virgin rats together with DMBA, then the number of resulting mammary tumors is drastically reduced (see Figure S12.2D). Experiments like these provide strong support for the notion (see Figure S12.2E and F) that the size of undifferentiated or less differentiated cell compartments is critical to the rate of tumor formation. 69
  72. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (B) 1.4 1.0 childless women 0.6 0.2 15 25 35 age at which woman has first child (C) Lob1 18 yr. old nulliparous Lob2 24 yr. old nulliparous Lob3 35 yr. old parous 25 (D) 100 20 80 CIS-DMBA 15 60 10 40 CIS-DMBA + hCG 20 CIS/gland % lobular structure woman's relative chance of developing breast cancer (A) © 2014 Garland Science 5 0 hCG treatment 0 1 2 3 1 2 3 nulliparous parous pre-menopausal lobule type 60 70 80 90 100 110 age in days 120 130 (E) PAROUS FEMALE breast cancer pregnancy + menstruation pregnancy + menstruation pregnancy undifferentiated, stem-cell-like, susceptible pregnancy pregnancy pregnancy differentiated, not susceptible (F) NULLIPAROUS FEMALE breast cancer menstruation menstruation undifferentiated, stem-cell-like, susceptible 70 n12.109,n12.114/s12.02
  73. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Figure S12.2 Parity and breast cancer susceptibility (A) The age at which a woman first bears children and whether she has children profoundly affect her risk of developing breast cancer. Studies have estimated that every year that first-birth childbearing is delayed results in a 3.5% increase in lifetime breast cancer risk. The risk of breast cancer of a nulliparous woman (horizontal dashed line) has been set here arbitrarily as 1.0. (B) This risk appears to be attributable, in part, to the changing histology of a woman’s breast following pregnancy. The lobular structures in the human breast can be placed into three histological categories, sometimes termed Lob1, Lob2, and Lob3, showing (in that order) increasing degrees of ductal branching and differentiation, as well as decreasing percentages of mitotically active cells. (For example, Lob2 has a mean of 47 ductules per lobular unit whereas Lob3 has a mean of 81. Note that the clear delineation of these distinct histomorphologies in the human breast and their presence in women of one or another parity status remain a matter of controversy among breast pathologists.) (C) Among pre-menopausal nulliparous women, the breast tissue shows a high degree of type 1 lobules (least differentiated, pink bars), while among pre-menopausal parous women, the type 3 lobules predominate (most differentiated, blue bars). The type 2 lobules, having an intermediate degree of branching and differentiation, are shown in green bars. (D) The hormone hCG (human chorionic gonadotropin) is active during pregnancy, when it induces, among other responses, differentiation of a portion of the epithelial cells in the breast. As seen here, treatment of female rats with the potent carcinogen DMBA induces a significant number of mammary carcinomas in situ (CISs) by 4 months of age (red curve). However, if the female rats are also injected with hCG, the subsequent development of these lesions is almost totally prevented (blue line). (E) This model helps to explain the effects of pregnancy on breast cancer risk in human females. Each time a woman undergoes a pregnancy, a portion of the undifferentiated stem cell-like cells (brown) are induced to differentiate into cells (blue) that are no longer susceptible to neoplastic transformation. While both pregnancy and menstrual cycling contribute to the risk of transforming the undifferentiated cells to a neoplastic state (red cells), the probability of this occurring (width of orange arrows) decreases with each pregnancy because of the progressive depletion of the pool of undifferentiated, susceptible cells. (F) In a nulliparous woman, however, because of the absence of pregnancy, the size of the population of undifferentiated cells that are susceptible to transformation (brown) remains undiminished, and the cumulative risk of breast cancer increases steadily until menopause. (A, from J. Cairns, Cancer: Science and Society. San Francisco: W.H. Freeman, 1978. Reproduced from B. MacMahon, P. Cole and J. Brown, Bull. World Health Organ. 43:209–221, 1970. B, from J. Russo et al., J. Natl. Cancer Inst. Monogr. 27:17–37, 2000. C, from J. Russo and I.H. Russo, Endocr. Relat. Cancer 4:7–21, 1997. D, from J. Russo and I.H. Russo, in S.P. Ethier, ed., Endocrine Oncology. Totowa, NJ: Humana Press, 2000, pp. 121–136.) 71
  74. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 12.3 The conserved-strand mechanism and protection of the stem cell genome The rationale behind “the conserved-strand” strategy, first proposed in 1975, derives from the molecular details of the DNA replication occurring in stem cells. When stem cells divide, the division is usually asymmetric, in that one daughter cell remains a stem cell and the other enters into a differentiation pathway by producing transit-amplifying cells (see Figure 12.1). Ideally, the genome that is donated to the daughter that remains a stem cell should be afforded more protection than the genome that is passed on to the daughter destined for differentiation, because descendants of the latter are destined to be discarded sooner or later. As illustrated in Figure S12.3, the asymmetric allocation of DNA strands can help to accomplish this aim. The idea here is based on the fact that DNA replication is inherently error-prone. By some estimates, each time a cell passes through S phase and replicates its DNA, several nucleotide substitutions occur per cell genome because DNA polymerases make mistakes that escape subsequent detection and repair (see Section 12.4). (In truth, this number may greatly underestimate the errors in DNA replication.) Consequently, DNA strands that were not synthesized during the most recent cycle of DNA replication—the “conserved” strands—are more likely to retain wildtype sequences than are those “nonconserved” strands that are indeed the products of this DNA synthesis. This suggests that in a well-designed tissue, the DNA strands that have not been created by recent DNA replication should be retained by the daughter cell that remains in the stem cell compartment, while those DNA strands that are the products of DNA replication should be allocated to the daughter cell whose descendants are destined to differentiate and eventually die. As Figure S12.3A makes clear, one DNA strand (the conserved, “immortal” strand) can, in principle, be transmitted indefinitely through a lineage of stem cells by such asymmetric segregation of DNA molecules. Stated differently, stem cells may carry DNA strands that have repeatedly served as templates for DNA replication but are only infrequently synthesized as products of replication (at least since the adult tissue was first formed). This model of asymmetric strand allocation can be tested experimentally. In fact, we have already seen one manifestation of this behavior in the experiment illustrated in Figure 12.2C. In that case, stem cells were allowed to incorporate 3H-thymidine for a brief period of time. The radioactive precursor became incorporated into the DNA strands being replicated during this short period, after which further incorporation ceased. (Such an experimental protocol is often termed “pulse-chase” labeling.) The fate of the radiolabeled DNA molecules was then followed using autoradiography, a technique in which a photographic emulsion is placed on a slice of tissue and yields a readily visualized dark grain whenever a radioactive atom such as a tritium Figure S12.3 Conserved DNA strands and the stem cell genome (A) The “immortal-strand” model depicts a stem cell (blue) donating a conserved strand (yellow) of its chromosomal DNA to a daughter cell that will remain a stem cell. This DNA strand is said to be conserved because it is not the product of recent DNA replication. Conversely, the “nonconserved strand” (red) that is indeed the product of recent DNA replication will be allocated preferentially to the daughter cell that spawns transit-amplifying cells (green) and therefore exits the stem cell compartment; a new round of DNA replication adds a new red strand to the nonconserved parental red strand. This model predicts that one DNA strand can persist indefinitely within the stem cell compartment. (B) This prediction is fulfilled in the mouse mammary gland. Mice can be exposed to a brief pulse of 3H-thymidine at a time during puberty when the gland is still growing and the number of mammary epithelial stem cells is continuously increasing, necessitating symmetrical divisions in which both daughters of a stem cell become stem cells (see Figure 12.3C) and therefore, hypothetically, both strands of DNA are retained as conserved strands. Hence, 3H-thymidine label incorporated into DNA during this period would be retained indefinitely in the conserved DNA strands and would be detected by autoradiography. Five weeks after initial exposure to 3 H-thymidine, only about 2% of the mammary epithelial cells (left) are seen to contain the radiolabel (dark grains). If, at that time, the mice are exposed to a pulse of bromodeoxyuridine (BrdU), a thymidine analog whose incorporation into DNA is revealed by a specific antibody (red-staining nucleus, middle), BrdU can be detected in the majority of the cells that retained radiolabel from the exposure to 3H-thymidine 5 weeks earlier (right panel). This shows that these cells are still actively proliferating and yet they retain a DNA strand that was synthesized 5 weeks earlier—a conserved strand that was not lost from these cells during the repeated rounds of growth and division. (C) When a stem cell is lost (top right) in an adult (in which the size of the stem cell pool should be constant), a surviving stem cell will divide symmetrically, so that both of its daughters will remain as stem cells, thereby reconstituting the population of stem cells in the pool (see Figure 12.3B). In this daughter (right), a DNA strand that was previously nonconserved (red) will be retained in the stem cell compartment and become an immortal, conserved strand (yellow). Hence, the killing of stem cells in an adult should make it possible for label incorporated into DNA during synthesis to be retained indefinitely in the stem cell compartment. (D) This is borne out by mouse duodenum cells. The enterocytes are normally replenished by the continual division of the stem cells near the bottom of the crypts (see Figure 12.2) and all of the radiolabeled DNA that is initially synthesized in the crypts is lost after several days as the differentiating transit-amplifying cells and their enterocyte progeny undergo apoptosis after leaving the crypts. However, if the duodenum is exposed to 8 Gy of X-irradiation (which kills some of the stem cells) before radiolabeling, cells in the crypts can be found to retain label even 8 days after a brief pulse of radioactive thymidine. Four examples of these label-retaining cells (LRCs), which are found precisely in the location of stem cells in the crypts, are shown here (arrows). (E) In this mammary duct, the mammary epithelial cells (MECs) are stained for cytokeratin expression (red). An LRC that incorporated BrdU 9 weeks earlier is immunostained in green. (F) LRCs can be found in the “bulge” region of mouse hair follicles, where keratinocyte stem cells are known to reside. These cells, which were briefly induced to express a stable form of green fluorescent protein (GFP, green) at 4 weeks of age, continue to express GFP 4 weeks later. The epithelial cells are labeled here in red. (B, from G.H. Smith, Development 132:681–687, 2005. D, courtesy of C.S. Potten. E, courtesy of B. Welm and M.A. Goodell. F, courtesy of T. Tumbar, V. Greco and E. Fuchs.) 72
  75. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) conserved strand nonconserved strand © 2014 Garland Science (B) stem cell 3 H-thymidine labeled stem cells etc. transitamplifying cells etc. (C) conserved strand BrdU labeled 3 H-thymidine + BrdU labeled (D) etc. etc. nonconserved strand (E) stem cells symmetric division 2nd daughter is retained in stem cell compartment and becomes a stem cell asymmetric division etc. transitamplifying cell etc. one of the recently replaced DNA strands becomes a conserved strand that is thereafter retained in the stem cell compartment (F) etc. atom decays. If the allocation of DNA strands were symmetrical, then we would expect some of the radiolabel would remain behind in the stem cell compartment and some would be distributed to the differentiating cells that had left the stem cell compartment. However, in the experiment, virtually all of the radiolabeled DNA strands migrated out of the stem cell compartment with the transit-amplifying cells that had begun to differentiate. This supports the notion that the newly synthesized strands (that is, those that were synthesized during the 3 H-thymidine pulse) were preferentially donated to the daughter cells that spawned transit-amplifying cells and their more differentiated descendants that migrated out of the crypts. Conversely, we discover that it is extremely difficult to label the DNA strands that are retained in the stem cell compartment within the crypts. b12.05/s12.03 Actually, there is an alternative interpretation to this observation: the radiolabel disappears from the stem cell compartment because it is rapidly diluted by the repeated cycles of growth and division occurring there. This notion can be tested by exposing stem cells to 3H-thymidine at a time when the stem cell compartment is expanding; under these conditions, stem 73
  76. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg cells must undergo symmetric division in order to increase their number (see Figure 12.3C), and both radiolabeled DNA strands should therefore be retained in the stem cell compartment. This is just what is seen when the mammary epithelial stem cells of the mouse are allowed to incorporate 3H-thymidine during puberty, when the mammary gland is growing rapidly (see Figure S12.3B). Under these conditions, the radiolabel is retained (without visible dilution) many weeks later in the stem cell compartment, even though these “label-retaining” cells (LRCs) can be shown to be actively dividing at this later time. (The radiolabeled strand inherited from their ancestors many cell generations earlier retains its radiolabel in spite of repeated intervening cycles of cell growth and division.) Another test of the conserved-strand model comes from experiments in which some of the stem cells are killed by exposure to X-rays. The remaining cells in the stem cell compartment will attempt to replace the lost stem cells through symmetric cell divisions in which both daughters remain as stem cells (see Figure 12.3B). Consequently, a newly made DNA strand, which would normally be allocated to the differentiating daughter cell, will now be converted into a conserved strand and retained in the stem cell compartment (see Figure S12.3C). If we expose the stem cell compartment of mouse intestinal crypts to 3H-thymidine during the period of time when the lost stem cells are being replaced, we can indeed label DNA molecules © 2014 Garland Science that subsequently remain within the stem cell compartment for an indefinite period of time; that is, the labeled molecules are not “chased” out when the stem cells are exposed subsequently to non-radiolabeled thymidine precursors (see Figure S12.3D). Hence, the only time in an adult animal when we can introduce long-lived radiolabel into the stem cell compartment seems to be when we perturb this compartment by killing some of its cells. Under these conditions, both the immortal DNA strand and the recently synthesized, non-immortal strand are retained in cells that become stably ensconced in the crypts of the small intestine. Label-retaining cells are also found in other epithelial tissues (see Figure S12.3E and F). In spite of this and other evidence, most of which has been gathered in the mouse, the “immortalstrand” model remains largely a matter of speculation for most tissues and requires far more experimental validation before we can accept it as a well-established fact. If further validated, this model holds important implications for the process of carcinogenesis, as it makes predictions about how certain carcinogens work. Among the major difficulties of this model is the absence of a well-defined molecular mechanism that would allow conservation of a specific parental DNA strand in one or the other daughter of an asymmetric division—that is, how can the mitotic apparatus distinguish between the two sister chromatids that have generally been presumed to be segregated randomly at anaphase? 74
  77. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 12.4 Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular genome By recent estimates, the genomes of some human cells suffer as many as 103 oxidative hits a day, about 10-fold less than the rate at which depurination of bases occurs. The resulting oxidized bases are largely but not totally removed and replaced with the appropriate normal bases. Rat cells suffer about 10-fold more oxidative hits per cell per day in their genomes than do human cells because they have about a 7-fold greater metabolic rate (see Figure 12.11). Any unrepaired oxidative lesions will accumulate with time, especially in the genomes of cells that are not mitotically active. 8-oxo-dG is the most frequently observed nucleotide product of oxidative damage. It seems that 1 to 2% of this oxidized nucleotide fails to be removed by the DNA repair apparatus. Oxidants may oxidize the nucleotide precursor of dG prior to its incorporation into DNA and the resulting oxidized nucleoside triphosphate may then be incorporated instead of dGTP into the DNA. Alternatively, oxidants may attack the guanine base after its incorporation into DNA. The 8-oxo-dG excised from DNA is largely excreted in the urine. The importance of oxidized dGTP (that is, 8-oxo-dG triphosphate) is indicated by the fact that a specialized enzyme— MTH1 (mammalian MutT homolog 1)—is employed by mammalian cells to degrade this oxidized DNA precursor; mice lacking MTH1 develop tumors at a 3 to 4 times higher rate than their wild-type counterparts. Under standard conditions of tissue culture, early-passage human fibroblasts will usually proliferate for several dozen population doublings before entering © 2014 Garland Science senescence (see Section 10.1). However, when expression of the MTH1 enzyme was suppressed in these cells by expression of an shRNA (see Supplementary Sidebar 1.4), almost all of them entered into senescence within a single population doubling (approximately one day). This indicated that these cells were poised under these conditions of culture to enter senescence rapidly and were saved from this fate by the continuous actions of this detoxifying enzyme. Interestingly, when these cells were propagated in an environment of 3% oxygen (rather than the 21% usually present in tissue culture incubators), then this rapid entrance into senescence was avoided. This lower oxygen tension more closely approximates the conditions experienced by cells within our tissues (see also Section 10.3). Unfortunately, studies of the importance of DNA oxidation products have been subject to a number of artifacts, including the inadvertent oxidation of DNA and nucleosides in vitro. On one occasion, aliquots of one DNA preparation were sent to 21 laboratories in Europe for measurement of 8-oxo-dG content; the results yielded estimates ranging over a factor of more than 200. The estimates of the numbers of oxidized bases in cell genomes have fallen dramatically in recent years. Nonetheless, the newer, more conservative estimates place the steady-state number of 8-oxo-dG residues in the DNA isolated from an average human cell at about 3000. This steady-state level is comparable to the level of chemically altered bases that are formed in the DNA of target tissues of laboratory animals that have been exposed to high, carcinogenic doses of compounds such as aflatoxin and heterocyclic amines. 75
  78. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 12.5 How does red meat cause colon cancer? High red meat consumption has been linked in a number of epidemiological studies to increased risk of colon, pancreatic, and rectal cancer (and, to a lesser extent, to cancers of the breast, lung, and urinary bladder). However, it has not been possible to associate these risks specifically with the heterocyclic amines (HCAs) that are generated by cooking meats at high temperatures (that is, by frying or grilling). Attempts to make such association are complicated by inter-individual differences in the metabolism of HCAs, by complex metabolic conversions undertaken by bacterial flora (see below), and by the possibility that other components of meat and fat, including other compounds generated by cooking, may play important contributory roles in the observed increased cancer risk. It is clear, for example, that an HCA such as PhIP (see Figure 12.18A), which is produced in substantial amounts when cooking meats and fish at high temperatures, is capable of inducing G-to-T transversions and deletions of a single G from the sequence GGG—mutations of the sort that are found in many p53 alleles carried by human colon cancer cells. However, such a correlation hardly proves causation, and the specific cause(s) of these and other mutations in colon cancer genomes remain to be demonstrated. More recent work indicates as much as a two-fold increased risk of prostate cancer in men who consumed large amounts of grilled or barbecued red meat. Consequently, while HCAs are clearly carcinogenic, their actual contributions to human cancer incidence are still the subject of great debate. Complicating this analysis are the repeated observations that processed meats are especially carcinogenic, and are associated with even higher incidence of colorectal cancer than red meats in general. These include bacon, various types of sausage including hot dogs, and packaged sandwich meat. Common to these various processed meats is the presence of sodium nitrite, which is added by meat companies as a preservative and to provide © 2014 Garland Science a bright red (and thus fresh) appearance to these meat products. Sodium nitrite can, in turn, generate nitrosamines. Thus, NaNO2 + R2NH (cellular amines) → R2N-N=O (nitrosamine) + heat → R-N2+ (diazonium ion) + DNA bases → DNA adducts. Importantly, the nitrite concentrations used to preserve red meats in the United States have decreased over the past three decades while ascorbic acid has been added to inhibit the first of the reactions described above (termed nitrosation), clearly lessening but not eliminating the carcinogenicity of nitrite preservatives. Once again compelling evidence of a direct connection between an agent—nitrites—with human cancer incidence is lacking, even though the connection between processed meats and cancer drawn from epidemiology is clear and undeniable. Additional confounding effects that complicate our ability to establish direct connections between foodstuffs and cancer incidence derive from the fact that, by some estimates, between 500 and 1000 distinct bacterial species reside in the human colon. The mix of bacterial species varies from one individual to another, being affected by genetic background, by diet, and possibly even by immune state. Each of these microbial species has its own set of enzymes that convert chemical species in the fecal matter, including xenobiotics (compounds that are not naturally produced in the body) such as HCAs, into a vastly complex collection of metabolites. Each of these metabolites, in turn, has its own particular set of chemical reactivities. Hence, the mechanisms of mutagenesis in the colon are extraordinarily complex and contrast starkly with the mechanisms operating in the skin, where many carcinogenic mutations can be traced directly and unequivocally to the actions of ultraviolet photons and the pyrimidine dimers that they generate. Finally, it is worth citing a totally independent and unrelated hypothesized mechanism of red meat-induced cancer, involving the consumption of a sialic acid isoform that engenders low level inflammation in epithelial and endothelial tissues (see Supplementary Sidebar 11.7) 76
  79. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 12.6 A convergence of bacterial, yeast, and human genetics led to the discovery of hereditary non-polyposis colon cancer genes The route through which the genetic basis of HNPCC was discovered is quite fascinating. By following the co-inheritance of HNPCC susceptibility in a number of afflicted families and the inheritance of a set of chromosomal markers scattered throughout the human genome, geneticists discovered two distinct genetic regions that seemed to be associated with disease susceptibility. In some families, HNPCC susceptibility co-segregated with genetic markers from human Chromosome 2p16, while in a second set of families, this susceptibility seemed to be inherited together with markers from chromosomal region 3p21. At the same time, astute observation of the types of genetic aberrations found in a subset of sporadic colon carcinomas noted that stretches of A’s (for example, A7) often were expanded or compressed (that is, were replaced by A6 or A8) in the genomes of colon carcinoma cells; thereafter, more complex microsatellite sequences [for example, (CA)8] were also found to undergo expansion or shrinkage (see Section 12.4). This microsatellite instability (MIN) was found to be rampant in the cancer cells of individuals suffering from HNPCC. Yet other researchers noted that these changes in DNA sequence were strikingly similar to the alterations that accumulated rapidly in E. coli that bore mutant versions of either of two genes—mutS or mutL—involved in mismatch repair (MMR; see Section 12.4); yeast carrying mutations in the homologous genes showed identical genomewide aberrations. This similarity led some scientists to investigate whether human homologs of these or other known mismatch repair genes were affected in HNPCC patients, leading rapidly to the confirmation that, indeed, a human mutS homolog (named hMSH2) mapped to human Chromosome 2p16, while a human mutL homolog (hMLH1) mapped to 3p21. Eventually, members of one set of HNPCC families were found to transmit mutant alleles of hMSH2, while those belonging to a second set of HNPCC kindreds carried mutant alleles of hMLH1. 77
  80. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 12.7 Homology-directed repair Homology-directed repair (abbreviated HDR or simply HR) is thought to depend on the ability of a DNA helix suffering a double-strand break (DSB) to consult the related, still-intact homologous sequences present in another chromosome, doing so in order to reconstruct a repaired helix whose sequences are indistinguishable from those that existed prior to the DNA damage. In fact, the reference sequences are almost invariably carried by the other chromatid that arises following DNA replication in S phase. (This other helix has presumably not suffered damage during this period.) This dictates that HR is limited to those phases in the cell cycle—S and G2—when replication has generated such a second reference helix. Conversely, HR is thought not to occur in the G1 phase of the cell cycle, a phase when such homologous chromatids are not yet present. The machinery that underlies HR depends on a series of proteins (Figure S12.4), some of which are found in defective form (or are absent) in certain human cancers. © 2014 Garland Science This leads to the notion that defects in HR, because they leave behind unrepaired, still-damaged DNA, are highly mutagenic and that this increased mutagenicity explains the increased cancer risks of women who, for example, bear mutant forms of either the BRCA1 or BRCA2 proteins. This conclusion is not fully substantiated, since proteins like BRCA1 have multiple alternative functions, many not connected directly with DNA repair. dsDNA break end resection BRCA1 BARD1 MRN CtlP RPA Figure S12.4 Detailed roles of DNA repair proteins in homology-directed repair of double-strand DNA breaks After a double-strand break (DSB) occurs in DNA, this lesion is recognized and bound by the heterotrimeric MRN complex, composed of Mre11, Rad50, and Nbs1 (also termed nibrin because of its involvement in Nijmegen breakage syndrome) (see Figure 12.33). This MRN complex recruits CtIP (CtPBinteracting protein) and, in complex with BRCA1 and BARD1, undertakes exonuclease activity to generate two 3ʹ-overhanging single-strand (ss) DNA ends. These ssDNA ends are rapidly coated by RPA (replication protein A; green). BRCA1, acting through PALB2, then recruits BRCA2, and the latter loads Rad51 (orange) onto the ssDNA, thereby displacing the previously bound RPA protein. This Rad51 protein, which acts as a recombinase, then facilitates strand invasion into the undamaged homologous region of the sister chromatid, which depends on unwinding the double-helical DNA of the sister chromatid; Rad51 also participates in the search for binding to the proper complementary sequence in the sister chromatid (termed a “homology search”). A displacement (D-) loop is produced when the 3ʹ ssDNA forms double-helical complexes with the DNA strands of the sister chromatid. This permits DNA polymerases to perform strand elongation from the 3ʹ ends of the inserted ssDNA by using the complementary ssDNA strands of the sister chromatid as templates. The result is the formation of a complex structure, termed a Holliday junction (HJ), between the two chromatids. The two HJs can be resolved in either of two ways, resulting in either crossover or lack-of-crossover when the two, now-intact sister chromatid helices separate from one another. Other variations of this basic scheme may operate in certain circumstances. (Adapted from P.J. O’Donovan and D.M. Livingston, Carcinogenesis 31:961–967, 2010.) RAD51 loading RAD51 BRCA2 strand loading and homology search RAD51 BRCA2 D-loop DNA synthesis 2nd end capture double Holliday junction formation resolution of Holliday junction crossover non-crossover n12.115/s12.04 78
  81. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 13.1 Localization of growth factors is important for proper heterotypic interactions Paracrine signaling operates over short distances between nearby cells, rather than over long distances via the circulation, the latter being termed endocrine signaling. (Some paracrine signaling is termed juxtacrine to indicate signaling between immediately adjacent cells.) In the case of microvessel formation, highly localized concentrations of PDGF are needed to ensure that the pericytes recruited to the outer layers of capillaries become directly apposed to the endothelial cells rather than dispersed randomly some distance away (Figure S13.1). Like many other growth factors, the PDGF-B molecule—the specific form of PDGF that operates here—contains a “retention motif,” which ensures its binding to proteoglycans in the extracellular matrix (ECM). Consequently, soon after PDGF-B molecules (A) © 2014 Garland Science are secreted by endothelial cells into the space around capillaries, these molecules are trapped in the nearby ECM and therefore accumulate in high concentrations immediately next to the endothelial cells. Once liberated from this tethering by proteases, the resulting high local concentrations of activated PDGF-B molecules ensure the recruitment of pericytes to locations directly adjacent to the endothelial cells. However, in mice expressing a mutant PDGF-B that lacks this retention motif, the secreted PDGF-B molecules diffuse away from endothelial cells, causing the recruitment of pericytes to the general area of the endothelial cells but not directly apposed to them. These endothelial cells therefore lack the structural support afforded by the pericytes, as well as the VEGF supplied by the pericytes that is required for long-term endothelial cell survival. (B) endothelial tube pericyte precursors ECM wt PDGF-B pericyte precursors ECM mutant PDGF-B Figure S13.1 Role of PDGF in the recruitment of pericytes to capillaries (A) Normally, the pericytes (yellow, green) are tightly attached to capillaries (red, upper panel). However, in one strain of mice, the gene encoding PDGF-B has been altered to encode mutant PDGF-B molecules that cannot attach to the extracellular matrix and therefore diffuse away from the endothelial cells producing this growth factor. As a consequence, the pericytes are no longer attracted to the endothelial cells and lack intimate contact with the capillary tubes (arrows, lower panels). (B) This S13.01 close juxtaposition of pericytes with the capillaries depends on the localization of PDGF-B, which is secreted by endothelial cells (lighter green). Normally (above), this wild-type (wt) PDGF-B (dark green) is trapped immediately adjacent to the endothelial cells in the extracellular matrix (ECM, brown), where it serves to attract pericyte precursors (pink) that attach themselves (red) tightly to the capillary tubes formed by the endothelial cells. However (below), if PDGF-B is deprived of the amino acid sequences that normally tether it to the ECM (purple), as described in panel A, it diffuses away from the capillary tubes, and any pericytes that are recruited are scattered at some distance from the capillaries. (A, from A. Abramsson, P. Lindblom and C. Betsholtz, J. Clin. Invest. 112:1142–1151, 2003.) 79
  82. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 13.2 Ongoing heterotypic signaling in carcinomas A variety of heterotypic signaling channels have been described in carcinomas involving signaling from the (A) © 2014 Garland Science neoplastic epithelial cells to various cellular components of the stroma and in the reverse direction from stroma to the epithelial cells. Three examples are given in Figure S13.2. (B) (C) T T S S E E control PDGF-CC (ligand) T Sonic Hedgehog (ligand) T S T S T T PDGF-Rα (receptor) E E E imatinib treated Figure S13.2 Heterotypic signaling between epithelium and stroma in carcinomas A variety of heterotypic signaling axes have been documented in carcinomas in which the cells in one cell layer release a signaling ligand while the cells in an adjacent cell layer display the cognate receptor, allowing them to respond to the released ligand. (A) In a mouse model of cervical carcinoma S13.02 pathogenesis, the PDGF-CC ligand (light brown) is made by islands of carcinoma cells (T) and is absent from the surrounding stroma (S) (upper panel). Conversely, the cognate receptor—PDGF-Rα (red)—is displayed by the stromal cells (lower panel). (B) Binding of PDGF-CC by the stromal PDGF-Rα enables the stromal cells (S) to respond to PDGF (not shown) released by nearby carcinoma cells Patched (receptor) (E), doing so by secreting FGF-2 (upper panel, brown); the latter then signals back to and stimulates the epithelial carcinoma cells. When PDGF-R signaling is blocked in the stromal cells by treatment with imatinib/Gleevec (lower panel), production of FGF2 by the stromal fibroblasts is significantly attenuated. (C) In a mouse model of pancreatic carcinoma development, the Shh Hedgehog ligand (upper panel) is made by the pancreatic epithelial cells surrounding the ducts within the tumor (light brown, arrows), while cells in the stroma (lower panel) express the cognate Patched receptor (red, arrows). The ductal epithelial cells express a cytokeratin (green). (A and B, from K. Pietras et al., PLoS Med. 5:e19, 2008. C, from H. Tian et al., Proc. Natl. Acad. Sci. USA 106:4254–4259, 2009.) 80
  83. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 13.3 Certain highly advanced tumors provide exceptions to the generally observed dependence of carcinoma cells on stroma The cells in a small subset of carcinomas have evolved so far in vivo that they no longer require stromal support. Examples are provided by pleural and peritoneal effusions, that is, cancer cells that have left the primary solid tumor mass and acquired the ability to grow, essentially in suspension, in pleural fluid around the lung (Figure S13.3A) or in ascitic fluid in the abdominal cavity; the formation and accumulation of these fluids is driven in both cases by the cancer cells (Figure S13.3B). In these cases, there is no question that the cancer cells have become completely independent of direct support by other nearby cell types and instead seem to require only nutrients (and possibly soluble growth factors) for their support. Not unexpectedly, such highly progressed tumor cells adapt far more readily to tissue culture and have become (A) (B) © 2014 Garland Science the progenitors of many of the cancer cell lines used in laboratories. A prominent example here is provided by HeLa cells (see Section 10.1), which have proven to be a boon for the study of the molecular biology of human cells. These cells were readily adapted to tissue culture in 1951 because they derived from an unusually aggressive adenocarcinoma of the cervix that grew in suspension in ascites and has been propagated in culture since that time. Some evidence indicates that such highly autonomous human tumor cells produce a variety of growth factors to which they can also respond; this enables them to stimulate their own proliferation and survival through autocrine signaling loops. Similarly, in the middle of a carcinoma that has become highly malignant, one can often observe cancer cells that appear to be almost as independent, in that they have virtually no stroma near them with the exception of the tumor-associated vasculature (see Figure S13.3C). (C) malignant cancer cells, these have unusually large nuclei. (C) In Figure S13.3 Independence of cancer cells from stromal some solid tumors, such as these mouse mammary carcinomas, support (A) These cancer cells, found in the pleural fluid of one can see sectors toward the periphery (upper panel) in which a breast cancer patient, are growing essentially in suspension groups of carcinoma cells (blue nuclei) are layered between without any direct contact with a supporting tumor stroma. stromal cells, including macrophages (green) and those forming Such pleural effusions, or their abdominal counterparts, called the neovasculature (red). In the same tumors, however, there ascites, are indications of the most advanced, aggressive are sectors (lower panel) where hardly any stroma is apparent stages of many types of solid tumors. A small clump of breast besides the cells forming the neovasculature, indicating that the cancer cells is seen in the middle. (B) Ascites are often seen in cells in the center have become independent of stromal support patients with advanced tumors of abdominal organs, including except that provided by this neovasculature. (A, courtesy of carcinomas of ovary (seen here), pancreas, and colon. The tumor A. Lukacher. B, from A.T. Skarin, Atlas of Diagnostic Oncology, cells are often found growing as single cells or in small clumps 4th ed. Philadelphia: Elsevier Science Ltd., 2010. C, courtesy of in the ascitic fluid and can be best studied by concentrating E.Y. Lin and J.W. Pollard.) them by centrifugation prior to examining them under the S13.03 microscope, as was done here. Like many kinds of highly 81
  84. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 13.4 Myofibroblasts predict clinical progression of cancer A strong correlation can be observed between the predicted survival of a cancer patient and the content of myofibroblasts in the patient’s tumor. As seen in this example of patients bearing squamous cell carcinomas of the mobile portion of the tongue, an increasing content of myofibroblasts in the stroma correlates with an increase in the rapidity with which these carcinomas progress to a fatal endpoint (Figure S13.4). Another study of oral carcinomas, published in the same year, reported that the most powerful prognostic marker for rapid © 2014 Garland Science progression to fatal disease could be found not in the islands of carcinoma cells but in the tumor-associated stroma, specifically in the quantity of α-smooth muscle actin (α-SMA)–positive myofibroblasts; their presence in high numbers generated a >3-fold increased risk of death, independent of other histopathological criteria. Importantly, the data from both of these studies represent correlations, and thus do not on their own prove that myofibroblasts actually serve as causal forces driving tumor progression forward. cumulative survival 1.0 0.6 medium density 0.4 high density 0.2 0.0 (A) (B) (C) (D) low density 0.8 P = 0.002 0 50 100 150 200 250 time since diagnosis (months) (E) 100 µm Figure S13.4 Content of myofibroblasts and clinical prognosis One strategy for determining prognosis—in this case long-term survival of a cancer patient—comes from measuring the content of α-smooth muscle actin (α-SMA)–containing myofibroblasts in the tumor-associated stroma. This provides striking correlations between the content of these cells in the stroma and the rapidity of tumor progression. In these squamous cell carcinomas of the mobile portion of the tongue, different densities of α-SMA– S13.04 containing myofibroblasts can be observed: (A) low, (B and C) medium, and (D) high densities. (E) The densities of stromal myofibroblasts have been correlated with the proportion of patients surviving for various times after initial diagnosis. These data provide compelling evidence of the inverse relationship between stromal myofibroblast density and survival time of patients. (A–D, from I.O. Bello et al., Oral Oncol. 47:33–38, 2011; and courtesy of T. Salo.) 82
  85. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 13.5 A technique for separating stromal from epithelial cells The technique of laser capture microdissection (LCM) enables an investigator to separate distinct cell layers from one another in a microscopic section from a normal or neoplastic tissue (Figure S13.5). The resulting separated clusters of cells can then be subjected to various types of analysis, including those that measure differences in their genome sequences and gene expression patterns. (A) laser beam plastic cap thermoplastic transfer film affixed to cap tissue section cells of interest retrieved cells of interest transfer of selected cells glass slide (B) before LCM after LCM captured cells Figure S13.5 Laser capture microdissection (LCM) (A) The LCM procedure allows an investigator to use a laser beam to microdissect tissue sections that have been affixed to a microscope slide. It requires the positioning of a transparent film affixed to a plastic cap S13.05 above a slide to which a tissue section has been affixed. A motor-driven laser beam (orange) directed by the microscopist irradiates chosen areas (light brown), thereby melting the transfer film (formed from a thermoplastic polymer) that has been affixed to the cap; the melted film flows into the interstices between cells adhered to the glass slide and forms a stronger bond with these cells than exists between the cells and the glass slide below them. The cap with attached transfer film can then be lifted with the adherent cells, which can then be analyzed separately from the cells left behind on the slide. A sample of tissue as small as 3 microns diameter can be captured in this way. (B) As seen here, the section of a human colon carcinoma shown in the left panel contains both epithelial regions (purple) and stromal regions (pink). In this instance, the LCM procedure caused the stromal area to be left behind on the slide (middle panel) while the epithelial island was removed (right panel). Both groups of cells could then be studied by various analytical procedures, including gene expression array analyses. (A, adapted from R.F. Bonner, M. Emmert-Buck, K. Cole et al. Science 278:1481–1483, 1997. B, courtesy of M. Tangrea, M.R. Emmert-Buck and Arcturus/ Life Technologies.) 83
  86. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 13.6 Microvessel leakiness dooms many forms of anti-cancer therapy: optimizing anti-angiogenic treatments VEGF was discovered as a factor that promoted blood vessel permeability and initially was termed vascular permeability factor (VPF). When an antibody that neutralizes VEGF is introduced into the circulation of a tumor-bearing mouse, the permeability of the tumor-associated capillaries is substantially reduced, demonstrating that the deregulated production of VEGF within tumors is responsible for much of the pathological leaking of plasma-derived water into the tumor parenchyma. This leaking causes much of the elevated hydrostatic pressure within the interstitial fluids of a tumor parenchyma, that is, the fluids lying between the nonvascular cells of a tumor. In normal tissues, the fluid pressure in the interstitial spaces is 0.4 mmHg or less, but in tumors, this pressure may reach 20 mmHg and occasionally far more. This means that the steep pressure gradient that normally operates between the lumen of a blood vessel (20 to 25 mmHg) and the surrounding parenchyma (0.4 mmHg) is absent in most solid tumors. As a consequence, the introduction of low–molecular-weight drugs into tumor masses via the circulation becomes very difficult, since these molecules remain in the circulation rather than riding with surrounding water molecules down pressure gradients into the extravascular spaces. This likely explains at least some of the difficulties that have bedeviled almost all attempts at developing effective drug therapies for solid tumors; these therapies are usually stymied by the great difficulty of delivering drugs in significant concentrations to the cancer cells within the parenchyma of solid tumors. These difficulties have inspired a new form of anti-tumor therapy in which anti-angiogenic agents are given in order to normalize the tumor-associated vasculature. Shutdown of VEGF production by tumor cells, using antisense RNA directed against the VEGF mRNA, results in a dramatic decrease in capillary permeability (Figure S13.6A). In addition, when an anti-VEGF mono- clonal antibody is introduced into a tumor-bearing mouse, the poorly constructed blood vessels in the tumor vasculature are pruned back, returning the vasculature trees to a more normal configuration (Figure S13.6B). This also results in a reduction in the interstitial hypertension of the tumor parenchyma and therefore permits far more effective delivery, via the circulation, of therapeutic drugs to the tumor cells. Importantly, an even more intensive (or longer-term) anti-angiogenic therapy is more than likely counterproductive, since it results in the regression of most of the residual vasculature in a tumor, thereby making drug delivery to the tumor extremely difficult once again (see Figure S13.6B). Moreover, as discussed in Section 13.11, the local areas of hypoxia that may result from excessive reduction of the tumor microvasculature can induce an increased invasiveness in cancer cells and, quite possibly, metastatic dissemination of cancer cells. Hence, doses and dosing schedules of anti-angiogenic agents must be optimized in order for these agents to serve as effective complements to more conventional chemotherapy. These various arguments suggest that causing the normalization of the intra-tumoral vasculature rather than the regression of the vasculature may actually prove to be more productive therapeutically, and, indeed, certain clinical observations appear to support this notion. For example, intra-tumoral blood flow was measured in 30 patients suffering from glio-blastoma by using a noninvasive magnetic resonance imaging (MRI) technology. These patients were treated with an agent that inhibited the tyrosine kinase functions of all three VEGF receptor subtypes, and their tumors responded in one of three ways to the treatment: decreased blood flow (11 patients), constant blood flow (12 patients), and increased blood flow (7 patients). As is apparent from Figure S13.6C, those patients whose blood flow increased in the tumor enjoyed longer survival before they succumbed to this invariably fatal disease. This indicates that normalization of blood flow (see Figure S13.6D) may actually benefit a cancer patient. Figure S13.6 Normalization of the tumor-associated vasculature (A) The permeability of tumor vessels was gauged here through the ability of a blue dye, introduced into the circulation 2 minutes before sacrifice of a tumor-bearing mouse, to permeate into and throughout the tumor parenchyma from the circulation (left panel). Human endometrial carcinoma cells were constructed that released two angiogenic factors—FGF-2 and VEGF. When FGF-2 expression was shut down, the number of tumor-associated vessels decreased greatly, but those that remained were still highly permeable (not shown). However, when VEGF production was shut down (right panel), once again the number of tumor-associated vessels (red) decreased greatly and, in addition, the permeability of the surviving vessels was strongly reduced, as indicated by the virtual absence of light blue staining. Arrows indicate two vessels that are no longer connected with the circulation. (B) Intra-vital microscopy can be used to visualize, through an implanted glass window, the vasculature of a subcutaneously implanted tumor. Especially high resolution was obtained here by using a multiphoton laser scanning microscope. The vasculature of the same region of a subcutaneously implanted murine mammary adenocarcinoma was monitored following the introduction of anti-VEGF-R monoclonal antibody. The vasculature was highlighted through the intravenous injection of a contrast agent. In comparison with normal vasculature (far left), the vasculature of the tumor (2nd image) initially appears contorted with many large-diameter vessels. After 2 days of treatment, the vasculature has normalized substantially. However, after 5 days, the vasculature in the tumor has regressed so far that it is barely visible (far right). (C) Thirty patients suffering from glioblastoma multiforme were treated daily with cediranib (AZD2171), a drug that inhibits the tyrosine kinase domains of all three VEGF receptors. Patients in whom the intra-tumoral blood flow increased during treatment, ostensibly because of normalization of the tumor-associated microvasculature, survived longer than patients in whom the blood flow was constant or decreased. (D) Antiangiogenic treatments may elicit several alternative responses from the aberrant tumor-associated microvasculature that develops during primary tumor formation. The microvasculature may become normalized, which may facilitate drug delivery and avoid hypoxia and associated increases in tumor cell invasiveness. The microvasculature may remain unaltered, in which case continued capillary leakiness may lead to high interstitial hydrostatic pressure in the tumor parenchyma, complicating delivery of drugs from the vessel lumina to the tumor cells. Finally, the microvasculature may be almost eradicated, which may result in widespread hypoxia, tumor necrosis, and/or the emergence of tumor cells that respond to hypoxia by becoming more invasive. (A, from R. Giavazzi et al., Am. J. Pathol. 162:1913–1926, 2003. B, from R.K. Jain, Nat. Med. 9:685–693, 2003; and E.B. Brown et al., Nat. Med. 7:864–868, 2001. C, from A.G. Sorensen et al., Cancer Res.72:402–407, 2012. D, from R. Jain, Nat. Med. 7, 987–989, 2001.) 84
  87. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (C) overall survival (OS) (A) © 2014 Garland Science 1.0 0.8 increased 0.6 0.4 0.2 0 0 tumor releasing FGF and VEGF p = .019 VEGF production shut down decreased stable 100 200 300 400 500 600 time (d) (B) normal skin, striated muscle human tumor xenograft 1 day of antiVEGF-R MoAb 2 days of antiVEGF-R MoAb 5 days of antiVEGF-R MoAb “normalized”: increased flow (D) 1.0 0.0 2.0 3.0 4.0 FLOW 1.0 0.0 2.0 1.0 3.0 0.0 4.0 3.0 4.0 FLOW FLOW TREATMENT normal flow in tissue 2.0 1.0 2.0 0.0 3.0 4.0 FLOW no change: stable flow irregular, inefficient flow in tumor, eventually resulting in hypoxia, necrosis 1.0 0.0 2.0 3.0 4.0 FLOW excess pruning: decreased flow S13.06 85
  88. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 13.7 The temporary nature of vessel regression created by anti-angiogenesis therapy The highly dynamic nature of the tumor-associated neovasculature is revealed by the effects of treatment of Rip-Tag tumors by a drug, AG-013736, that targets specifically the VEGF-R expressed by the endothelial cells. After initial treatment, the endothelial cells in many locations disappear, apparently by apoptosis, and leave behind sleeves composed of both basement membrane and pericytes (Figure S13.7A and B). These sleeves appear outwardly normal (Figure S13.7C) and are frequent throughout the © 2014 Garland Science Rip-Tag tumors (Figure S13.7D). However, 2 days after the drug is withdrawn, surviving endothelial cells begin to grow into the empty sleeves, and following 7 days of drug withdrawal, the endothelial cells have regenerated a microvasculature in these tumors that is essentially indistinguishable from that present in untreated controls (Figure S13.7E). This indicates that this and likely other types of anti-angiogenic therapy must be applied continuously in order to ensure the continued regression of the tumor-associated microvasculature. Figure S13.7 Regrowth of microvessels after cessation of anti-VEGF treatment Microvessels may regenerate rather quickly after cessation of certain anti-angiogenic therapies. (A) When the anti-VEGF-R agent AG-013736 is introduced into mice bearing Rip-Tag pancreatic islet tumors for 7 days followed by its withdrawal for 2 days, immunofluorescence microscopy reveals that the microvessels in the tumor contain segments with both CD31+ endothelial cells and α-smooth muscle actin (α-SMA)–positive pericytes (yellow, left), as well as segments in which pericytes are present but endothelial cells are absent (between arrowheads). (B) Following treatment, the entire length of this microvessel contains both α-SMA–positive pericytes and collagen type IV–containing basement membrane (BM). This indicates that the segment between the two arrowheads in these two panels is an empty sleeve composed of the BM and pericytes but lacking the normally present inner core of endothelial cells. (C) This scanning electron micrograph reveals that following this antiangiogenic treatment, the microvessels (e.g., center) appear outwardly normal. (D) However, microvascular sleeves (orange, arrows), such as the one in (A), are present in abundance throughout the tumor, with microvessels of a normal constitution (yellow) being in the minority (staining as in A). (E) The reversible effects of anti-angiogenesis treatments are seen in these images of CD31+ endothelial cells (red-orange) forming microvessels in Rip-Tag tumors. An untreated control tumor (far left) exhibits extensive microvessels. However, after 7 days of treatment with AG-013736, the endothelial cells have almost completely disappeared (second panel). If this anti-VEGF-R drug is withdrawn, microvessels begin to regenerate, and by 2 days after drug withdrawal, vascular sprouts (arrows, 3rd panel) are evident, indicating new vessel branching. By 7 days after drug withdrawal, the microvasculature has almost completely re-formed (far right). (From M.R. Mancuso et al., J. Clin. Invest. 116:2611–2621, 2006.) 86
  89. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) © 2014 Garland Science (B) α-SMA (pericytes) α-SMA (pericytes) CD31 (endothelial cells) + type IV collagen (BM) (C) (D) vessel pericyte tumor cell 5 µm (E) untreated 7 day treatment 25 µm +2 day withdrawal +7 day withdrawal S13.07 87
  90. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 13.8 Kaposi’s sarcoma cells hold the record for the number of documented heterotypic signals they receive Kaposi’s sarcoma (KS) seems to derive from the cells related to those generating the endothelial linings of lymphatic vessels. The causal agent of KS—human herpesvirus-8 (HHV-8)—flourishes when immune defenses have been compromised, explaining the high frequency of this tumor in AIDS patients (see Section 3.6). Gene expression analyses from several laboratories have documented the increased expression of mRNAs encoding 15 distinct receptors in KS cells; the respective ligands for these receptors appear not to be produced by the KS cells. This suggests that the elevated expression of these receptors allows KS cells to take advantage of ligands that are supplied, via paracrine signaling, by other nearby cell types. The list of receptors includes those binding TNF-α, INF-γ, oncostatin M, bFGF, VEGF-C and -D, insulin, thromboxane A2, IL-2, IL-6, IL-10, IL-13, thrombospondin, VEGF 165, CXCL12, and CSF-1. (The dependence of KS cells on all of these signals has not yet been demonstrated experimentally.) This complexity hints that other histologically complex tumors may, like KS, rely on multiple heterotypic signals to sustain their proliferation and survival. 88
  91. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg inhibitors of the VEGF-R tyrosine kinase (Figure S13.8). Exposure to this monoclonal antibody (MoAb) can result in widespread apoptosis by the endothelial cells forming the tumor-associated microvasculature, ostensibly because VEGF-A, in addition to stimulating the formation of new capillaries, also serves as a survival factor for endothelial cells. 20 30 40 50 60 70 0 0 10 20 30 40 days post tumor implantation control (B) DC101-treated 50 60 0 m 10 100 50 /ge 0 500 101 0 + DC101 200 150 DC 500 + DC101 101 1000 300 250 DC control e control bin 1500 1000 ita 2000 1500 500 450 400 350 mc SK-RC-29 ge BxPC3 SS (C) mean pancreatic tumor 3 volume (mm ) mean tumor volume (mm3) (A) HB Supplementary Sidebar 13.9 Effects of an anti-VEGF-R monoclonal antibody on the growth of a human tumor xenograft A monoclonal antibody, termed DC101, has been produced that specifically binds and inhibits VEGF-R2, the main VEGF receptor active during tumor angiogenesis. This antibody can serve as an alternative anti-angiogenic agent to low–molecular-weight © 2014 Garland Science Detroit 562 FADU indicated by the blue lines. (B) Exposure of the tumor xenografts Figure S13.8 Use of an anti-VEGF-R2 monoclonal antibody formed by two human tumor cell lines (the Detroit 562 and as an anti-angiogenic agent An alternative to using low– FADU nasopharyngeal carcinomas lines) to DC101 results in a molecular-weight drugs to inhibit the VEGF-R–associated tyrosine profound reduction in the tumor microvessels, revealed here kinase (TK) domain is the use of an anti-VEGF-R2–specific by staining against CD31 (brown), an endothelial cell–specific monoclonal antibody (MoAb) termed DC101. Use of a MoAb S13.08 marker. Interestingly, the very large dilated microvessels present offers greater specificity in targeting the VEGF-R2, since the MoAb in the untreated tumors are the first to disappear follow DC101 is likely to affect only the VEGF-R2, unlike low–molecular-weight treatment. (C) The DC101 MoAb can act synergistically with TK inhibitors, which may additionally affect to various degrees gemcitabine, a widely used chemotherapeutic agent. In mice other cellular TKs including the other VEGF receptors. bearing orthotopically implanted HBSS human pancreatic (A) As seen here, exposure to the DC101 MoAb from the moment carcinoma cells, the combined effects of the two agents are far of implantation blocks the outgrowth of two implanted human more dramatic than when either is used alone. (A, courtesy of tumor cell lines, BxPC3 (left panel) deriving from a pancreatic ImClone, Inc., from M. Prevett et al., Cancer Res. 59:5209–5218, adenocarcinoma and SK-RC-29 from a renal cell carcinoma (right 1999. B, courtesy of ImClone, Inc. C, from C.J. Bruns et al., panel), each growing as a xenograft in immunocompromised Int. J. Cancer 102:101–108, 2002.) mouse hosts. Growth of the untreated control tumors is indicated by the red lines, while growth of the DC101-exposed tumors is 89
  92. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 13.10 Fibroblasts are heterogeneous and can change dynamically in response to signals Many of the mesenchymal cell types that are recruited into the tumor stroma, either from afar (that is, from the bone marrow) or from adjacent tissues, actually represent distinct subtypes of a single well-studied cell type (for example, keratinocytes) rather than a single, well-defined histopathological entity. Fibroblasts represent one such cell type, which under certain conditions may be induced to transdifferentiate into myofibroblasts. Fibroblasts and their derivatives exhibit great heterogeneity, depending on their tissue of origin and their association with neoplastic cells within tumors. The observations presented in Figure S13.9 reveal how internally complex this class of cells actually is. Indeed, two dimensions of heterogeneity are indicated here: the fibroblasts prepared from various normal tissues show quite distinctive gene expression patterns (Figure S13.9A), and the gene expression pattern of the stromal cells (essentially fibroblasts) from a single organ often changes as the tissue progresses increasingly toward high-grade neoplasia (Figure S13.9B). These differences in gene expression reflect underlying functional differences in fibroblast populations (Figure S13.9C). Initial surveys of macrophages prepared from tissues and involved in different physiologic responses suggest comparable complexity. Similar analyses of other stromal cell types have not yet been produced. These analyses, when taken together, indicate an array of distinct stromal cell types that vastly exceeds the ten or so that are usually invoked to explain the complexity of stromal biology. 90
  93. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (B) 24 35 19 22 15 27 14 18 23 21 1312 20 33 7 34 8 6 5 36 37 38 39 41 42 metaplasia 29 28 26 25 dysplasia 31 30 32 40 43 100 200 300 400 500 600 700 stroma genes clustered cancer 4 3 2 1 –5 .03 lower +5 32 higher (C) fetal skin fibroblasts fetal esophageal fibroblasts adult esophageal fibroblasts dysplasia (A) © 2014 Garland Science metaplasia cancer 100 200 300 400 500 600 stroma genes clustered 40 80 120 160 200 stroma genes clustered Figure S13.9 Fibroblasts represent a heterogeneous collection of cells rather than a single distinct cell type (A) Fibroblasts were prepared from normal tissues in 43 locations throughout the human body, including both skin (samples 1–35) and internal organs, specifically the lungs, liver, aorta, skeletal S13.09 muscle, and prostate gland (samples 36–43) (left). The results of gene expression analyses that were performed on each of these are presented in the heat map (right), in which the levels of expression are plotted on one axis and the origins of the analyzed sample are plotted on the other axis. The samples are arrayed horizontally and clustered hierarchically, with similar expression patterns being grouped more closely to one another, as represented in the hierarchical array above the heat map. The degree of relative expression is given by the color bar below the heat map, where the numbers above the bar represent intensity of expression on a log2 scale. This heat map reveals several dozen distinct gene expression patterns, indicating that normal fibroblasts may reside in at least this many distinct states of differentiation. (B) The stroma from three distinct stages of esophageal cancer progression was isolated by microdissection and the expression of stroma-specific genes was analyzed by microarrays. The expression patterns of the three distinct stages of progression—metaplasia (Barrett’s esophagus), dysplasia, and frank carcinoma—were compared pairwise (labels to left of heat maps). These analyses reveal quite different gene expression patterns, largely of the fibroblasts within the stroma. Gene probes are arrayed left to right, while tissue samples are arrayed vertically. High expression is indicated by red/maroon; low expression, by green/aquamarine. (C) Normal human esophageal epithelial cells were experimentally transformed through the introduction of three genes (as described in Figure 11.27). Organotypic cultures were prepared in which three distinct types of fibroblasts were embedded in extracellular matrix components in order to mimic in vitro the stroma present in the three tissues. The experimentally transformed epithelial cells were then layered above the fibroblast/ECM layer and propagated for 10 days in a series of media. The resulting cultures were sectioned and stained with hematoxylin/eosin (H&E). The fetal skin fibroblasts permitted limited invasion of the transformed keratinocytes (arrows, left panel) downward into the fibroblast/ECM layer, while fetal esophageal fibroblasts induced extensive, highly aggressive invasion by the epithelial cells. Adult esophageal fibroblasts permitted almost no invasion in the fibroblast/ECM layer. These images present a dramatic demonstration of the functional differences among different types of normal fibroblasts. (A, from J.L. Rinn et al., PloS Genet. 2:e119, 2006. B, from A. Saadi et al., Proc. Natl. Acad. Sci. USA 107:2177–2182, 2010. C, from T. Okawa et al., Genes Dev. 21:2788–2803, 2007.) 91
  94. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.1 Visualization of the dynamics of pathfinding fibroblasts followed by squamous cell carcinoma cells The role of fibroblasts in leading the invasion of a cohort of carcinoma cells can be demonstrated most dramatically by using both still and time-lapse microscopy; in the latter, the same microscopic field can be photographed repeatedly (A) following SSCs inv © 2013 Garland Science at regular time intervals. A rapid succession time-lapse microscopy, in which the same microscopic field is photographed repeatedly at regular time intervals. A rapid succession of these images can then be merged into a video film of this progression (Figure S14.1). (B) asi on leading fibroblasts upper right) in an in vitro cell culture model. Interestingly, Figure S14.1 Microscopy of fibroblasts leading squamous carcinoma cells that were more mesenchymal in their phenotype carcinoma cells (A) In this in vitro culture model, carcinomacould migrate on their own, while those that retained a more associated fibroblasts (CAFs, red) are seen migrating through epithelial phenotype depended on fibroblasts to create an invasive extracellular matrix material in a leftward direction. Following path for them and invaded as a collective cohort held together closely behind and retaining contact with these leading fibroblasts by adherens junctions. Contact between the SCC cells and the are a cohort oforal squamous cell carcinoma cells which remain TBoC2 S14.01 fibroblasts was required, and these fibroblasts contributed to associated with one another as they move in a unified cohort, SCC invasiveness through their ability to remodel the surrounding following the path cleared by the fibroblasts. (B) This succession extracellular matrix. (Courtesy of C. Gaggioli and E. Sahai, from of images, filmed at 20-minute intervals over the course of four http://www.london-research-institute.org.uk/research/101/Moviehours, shows a small group of carcinoma-associated fibroblasts Gallery and from C. Gaggioli et al., Nat. Cell Biol. 9:1392–1400, (red, lower left) clearing a path for the subsequent invasion by 2007.) SCC12 human oral squamous cell carcinoma (SCC) cells (green, 92
  95. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.2 Metastasizing cancer cells often take on hitchhikers while traveling through the blood Repeated reports over the course of the last century have documented the presence of metastasizing cancer cells within small clots, termed variously microthrombi, microemboli, or thromboemboli. These consist of multiple platelets and red blood cells adsorbed to the surface of individual tumor cells or clumps of tumor cells within blood vessels (Figure S14.2A). (The process of forming these microthrombi is sometimes termed embolization.) In fact, the direct contact of cancer cell surfaces with blood plasma tends to provoke localized clotting. In experimental mouse models of human cancer development, the formation of these microthrombi can already be observed within two minutes of the entrance of cancer cells into the circulation. (A) P-selectin+/+ © 2014 Garland Science These localized clots are likely to be triggered largely by Tissue Factor (see Figure S14.2B), a protein that is expressed on the surface of malignant carcinoma cells and much less so by benign carcinoma cells and normal epithelial cells. Tissue Factor interacts with proteins in the plasma to initiate the clotting cascade: it activates thrombin, which in turn converts fibrinogen to fibrin (which binds the cells in clots together) and activates platelets (see Figure 13.10). The importance of these microthrombi as vehicles for metastasizing cancer cells is indicated by studies using genetically altered mice that lack various components of the plateletclotting machinery, including fibrinogen and components of platelets essential for their activation. Melanoma cells were introduced via injection into the tail veins of these mice, and (B) P-selectin–/– 82 78 N C 163 157 (C) ectodomain of Tissue Factor (D) surface tumors 100 10 1 (E) P-sel +/+ Nf-E2 +/+ P-sel –/– Nf-E2 –/– Nf-E2 +/+ P-sel +/+ with heparin (F) 80 densitometry units Nf-E2 –/– p <.0001 60 40 20 0 mouse genotype L+/+ L –/ – 93
  96. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg the number of subsequently arising lung metastases was determined. Mice lacking platelets showed a metastatic tumor burden that was only 4% of that seen in comparable wild-type mice (see Figure S14.2C and D). Similarly, mutant mice carrying platelets that were unresponsive to activation by thrombin showed a metastatic burden that was only 9% of the burden seen in wildtype mice. And mice lacking the ability to form fibrin—the critical structural component of clots—exhibited lung metastases that totaled only 3% of the number counted in wild-type mice. In addition, two cell surface proteins, P- and L-selectin, seem to reinforce the adhesion of the platelets to intravasated cancer cells (see Figure S14.2E and F). Precisely how microthrombi facilitate metastasis remains a matter of conjecture. Some experiments indicate clearly that a coating of platelets protects cancer cells from various types of injury while they are traveling through the blood and are lodged in microvessels. This protection may including shielding these circulating cancer cells from attack by natural killer (NK) cells (lymphocytes that have an intrinsic ability to recognize and kill many types of cancer cells; see Chapter 15) as well as monocytes and macrophages. For example, the loss of metastatic ability resulting from the absence of fibrinogen is almost completely reversed if host mice are also unable to make NK cells. Presumably NK cells represent the major threat to metastasizing cancer cells in the circulation, and cancer cell survival Figure S14.2 Contribution of platelets to the formation of metastases The initial adhesion of platelets to intravasated cancer cells is triggered by Tissue Factor (TF), a glycoprotein that is expressed on the surfaces of most cell types outside of the circulatory system. This binding seems to be reinforced by receptors—L-selectin and P-selectin—that are expressed on the cell surface of platelets. Both selectins appear to recognize and bind mucins on the surface of cancer cells. (A) Green fluorescent protein (GFP)–labeled cells from a human colon cancer cell line were injected intravenously into mice, and 30 minutes later, frozen sections were prepared from the lungs of these mice and immunostained with an antibody that recognizes platelets (red). When this experiment was performed in mice that are wild-type at the locus for P-selectin (left), a cloud of platelets was apparent, while the same experiment performed in P-selectin–negative cells (right) failed to demonstrate platelet aggregation, indicating the key role of P-selectin in forging links between platelets and intravasated cancer cells. Similar clouds of platelets can be found at even shorter times after introduction of other cell types, such as melanoma cells, into the general circulation. (B) The attachment of platelets to cancer cells is triggered by the actions of Tissue Factor, whose ectodomain is seen here. Exposure of plasma to Tissue Factor, which occurs following wounding, results in a 105-fold up-regulation of plasma-associated FVIIa; activated FVIIa then initiates the clotting cascade that results in the conversion of fibrinogen to fibrin and the adsorption of platelets to the surfaces of cancer cells. (C) Mouse B16 melanoma cells, which express the melanin pigment (black), were injected into the tail vein of either wild-type mice or Nf-E2–/– mice; the latter have virtually no platelets. As seen here, the mutant mice (left) developed very few melanoma metastases (black spots) in their lungs, while most of the wild-type mice (right) injected with an equal number © 2014 Garland Science seems to depend on their ability to hide behind cloaks formed by adsorbed platelets. Platelets may also expedite metastasis through their ability to strongly adhere to the surfaces of injured blood vessels, specifically those in which the localized loss of the endothelial cell lining exposes the underlying vascular basement membrane and associated collagen IV (Figure S14.3). This binding may facilitate the attachment of entire microthrombi to the walls of larger vessels, and thereby enable metastasizing cancer cells within these clumps to gain their first firm foothold in a tissue. Once physically stabilized in this way, cancer cells may begin to proliferate within microthrombi, penetrate through the vessel wall (that is, extravasate), and eventually establish a new metastatic colony in the parenchyma of a tissue. In addition, as first reported in 2011, adhered platelets may help to induce components of the EMT program via the secretion of molecules like TGF-β. Indeed, this interaction, which is likely to occur after cancer cells have become trapped in distant microvessels, can perpetuate and thereby maintain expression of an EMT program that was initially activated within the confines of a primary tumor, and may thus confer the invasive traits that cancer cells require in order to extravasate by invading through the walls of these microvessels into the nearby tissue parenchyma. of melanoma cells developed large numbers of metastases in their lungs. Hence, platelets provided a critical contribution to metastasis formation. (D) The counts of metastases visible on the surfaces of the lungs in panel C vary dramatically from mouse to mouse. Note that the ordinate is a logarithmic scale. (E) In wild-type mice (left panel), a cloak of platelets (blue) is present 30 minutes after human colon carcinoma cells (green) have been introduced into the circulation. P-selectin is required for the firm attachment of platelets to cancer cells. In mice unable to make P-selectin (middle panel), the cloak of platelets is substantially reduced, and now immune cells, including macrophages and monocytes (red), are able to approach closely to the cancer cells. Similarly, when wild-type mice are injected with heparin (an anti-coagulant that prevents platelet aggregation; right panel) prior to introducing the cancer cells, the platelets are absent and immune cells are present in abundance immediately around a cancer cell. (F) L-selectin plays a similarly important role in forming these microemboli. Human colon carcinoma cells were injected intravenously via the tail vein into immunocompromised mouse hosts and the numbers of metastasized human cancer cells were determined by measuring the amount of human DNA (represented here as densitometry units) in the lungs of these animals 6 weeks later. Mice whose platelets expressed normal levels of L-selectin (L+/+) showed significant amounts of human DNA and thus metastases in their lungs, while those whose platelets lacked L-selectin (L–/–) showed few if any lung metastases. (A and E, from L. Borsig et al., Proc. Natl. Acad. Sci. USA 98:3352–3357, 2001. B, from Y.A. Muller, R.F. Kelley and A.M. de Vos, Protein Sci. 7:1106–1115, 1998. C and D, from E. Camerer et al., Blood 104:397–401, 2004. F, from L. Borsig et al., Proc. Natl. Acad. Sci. USA 99:2193–2198, 2002.) 94
  97. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) © 2014 Garland Science Figure S14.3 Adhesion of microthrombi to vessel walls Microthrombi carrying cancer cells and attached platelets may occasionally adhere to the walls of larger vessels. (A) One highly plausible mechanism of attachment depends on the presence of patches of vascular basement membrane that are not covered by a continuous coating of endothelial cells and are therefore directly exposed to the lumen of a blood vessel. In this fluorescence micrograph, the outline of a blood vessel in a mouse is indicated by the configuration of the pericytes and smooth muscle cells around its exterior that have been immunostained with an antibody reactive with α-smooth muscle actin (red). Patches of basement membrane exposed to the lumen of this vessel have been stained through use of a lectin that specifically sticks to the basement membrane (green). (B) The endothelial cells lining the lumen of a rabbit carotid artery have been removed through the procedure of balloon angioplasty, exposing underlying basement membrane. Subsequently, as seen in this scanning electron micrograph, hundreds of platelets, which have a strong affinity for the collagen IV of the basement membrane, adhered to this basement membrane. (The large disks are red blood cells.) This suggests how microthrombi may attach to exposed patches of basement membrane through their halo of platelets. (A, courtesy of J.H.C. Chen and L. Coussens. B, from M.R. Miller et al., Cardiovasc. Res. 57:853–860, 2003.) (B) TBoC2 s14.03 95
  98. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.3 Instruments for detecting circulating tumor cells Devices have been developed that allow the isolation of circulating tumor cells (CTCs) from the blood of cancer patients, and these instruments are currently in a state of rapid further development. Some of these devices rely on the immobilization of monoclonal antibody molecules on the solid (A) © 2014 Garland Science surfaces on the sides of channels through which blood is then forced. These microfluidic devices come in various configurations, two examples of which are provided here (Figure S14.4A and B). Application of such microfluidic devices to the analysis of the blood of normal controls and prostate cancer patients is seen in Figure S14.4C–E. (B) 100 µm (C) log PSA-positive CTC/ml 1000 100 µm (n = 30) 100 10 (n = 8) (n = 19) 200 µm 45 µm (n = 9) 0 healthy controls male female (n = 13) (n = 4) (n = 6) 50 µm prostate cancer patients (n = 55) side view/cross-section 200 µm top view 50 14 12 10 8 40 30 20 10 0 0 6 4 2 0 50 100 150 200 250 300 350 400 day leuprolide probability of survival (%) (E) serum PSA [ng/ml] (D) keratin positive CTC/ml PSA positive CTC/ml 50 µm 1 100 < 5 CTCs/7.5 ml at all times 80 60 40 ≥ 5 CTCs/7.5 ml at all times 20 0 0 4 8 12 16 20 24 28 time from baseline blood draw (months) bicalutamide TBoC2 s14.04 96
  99. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Figure S14.4 Microfluidic devices for capturing circulating tumor cells (CTCs) from the blood of cancer patients Two examples of a variety of microfluidic devices (chips) are shown. By coating the surfaces of these devices with monoclonal antibody molecules that recognize cell-surface antigens displayed by cancer cells, CTCs from a variety of tumors can be trapped. While early versions of such chips did not allow intact cells to be readily isolated, more recent versions do indeed permit the isolation of intact, even viable cells. An alternative means of isolating CTCs, not shown here, involves the coupling of monoclonal antibodies to iron particles, allowing the coupled antibodies to bind to the surfaces of CTCs, and isolating the resulting antibody–CTC complexes with a magnetic field. (A) This chip contained 78,000 cylindrical microposts coated with a monoclonal antibody reactive with EpCam, a cell surface antigen expressed by many carcinoma cells and normal epithelial cell types but not expressed by hematopoietic cells. In the chip visualized here by scanning electron microscopy, human lung cancer cells added to normal human blood were passed through the microfluidic chamber. A captured lung cancer cell, colorized red, is also seen at higher magnification in the inset at the top right. Ninety-nine percent of the blood samples from patients with a variety of metastatic cancers (lung, prostate, pancreatic, breast, and colon carcinomas) yielded CTCs immobilized on this chip. (B) Shown is a microfluidic chip of the herringbone configuration, which allows more efficient capture of CTCs. Moreover, because of the decreased turbulence within the channels, larger aggregates composed of multiple CTCs can be captured. This device, like the one described in panel A, depends on coating the surfaces with a monoclonal antibody directed against EpCam. (C) Here, a microfluidic chip of the type described in panel A was used to screen the blood of patients with metastatic prostate cancer and, as a control, of normal male and female volunteers for the presence of CTCs. In this case, an antibody against PSA (prostate-specific antigen) was used to trap PSA+ prostate cells on the microposts within the chip; hematopoietic cells expressing the CD45 cell surface antigens were excluded from this analysis. The concentration of CTCs per ml is plotted on a logarithmic scale (ordinate). Normal men exhibited between ~0.5 and 10 CTCs per ml (red circles), while the prostate cancer patients, including those with both localized and metastatic disease, had from ~0.5 to ~3000 CTCs per ml of blood. (D) The changing levels of CTCs in response to anti-androgen therapy against metastatic prostate cancer were measured in a number of men, one of whose responses are plotted here. The man was first treated with leuprolide, which interferes with androgen biosynthesis by altering signaling in the pituitary gland and subsequently, toward the end of this course of treatment, with bicalutamide, an androgen receptor antagonist. This patient’s levels of circulating PSA (red curve) dropped dramatically in response to initial treatment, in parallel with a decrease in the levels of PSA-positive CTCs (green curve), while his levels of cytokeratin-positive CTCs dropped more slowly. (E) The concentrations of CTCs from a group of 177 patients with metastatic breast cancer were monitored at intervals after initiation of a new course of therapy. The survival of two subgroups of patients with different concentrations of CTCs/7.5 ml of blood are shown. (A, from S. Nagrath et al., Nature 450:1235–1239, 2007. B, from S.L. Stott et al., Proc. Natl. Acad. Sci. USA 107:18393–18397, 2010. C and D, from S.L. Stott et al., Sci. Transl. Med. 2:25ra23, 2010. E, from D.F. Hayes et al., Clin. Cancer Res. 12:4218–4224, 2006.) 97
  100. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 14.4 Hidden micrometastases are revealed through organ transplantation Organ transplantation first came into widespread use in the early 1960s. The procedure requires that the immune systems of organ recipients be suppressed in order to prevent immune rejection of donor organs. Some of the organ donors at the time had previously been treated, ostensibly successfully, for one or another type of cancer. While gross examination of the donated organs indicated that they were healthy, cancer cells clearly contaminated these tissues, since the transplant recipients occasionally developed tumors of donor origin. These tumors thrived in the recipients and often became life-threatening, because the immunosuppressed state of the recipients prevented their immune systems from rejecting these foreign cancer cells—a theme that we will pursue further in Chapter 15. Today, organ donors are screened far more thoroughly before their organs are transplanted into others. Even so, donors may harbor undetected micrometastases whose existence may be revealed only upon organ transplantation. In some cases, a misdiagnosis of an organ donor’s cause of death results in unsuitable organs being made available for transplantation. For example, a diagnosis of death from a cerebral hemorrhage will obscure the real cause of death—a metastasis to the brain. A worldwide registry of transplantation-associated malignancies, maintained in Cincinnati, Ohio, cataloged cases arising up to 1995 in which undetected cancer cells in the organs of 153 donors generated tumors in the recipients. (About 300,000 transplant procedures had been performed by that time.) In some cases, individual donors provided multiple organs, resulting in the development of identical cancers in several organ recipients. Some of the organ recipients exhibited widespread metastatic disease soon after transplantation; prominent among these were growths that could subsequently be identified as malignant melanoma and carcinomas of the kidney and lung. Because some types of tumors—notably primary brain tumors, squamous cell carcinomas of the skin, and localized (in situ) carcinomas of the cervix—almost never metastasize, organs from donors carrying these particular tumors are currently judged to be safe for transplantation. 98
  101. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.5 Wolves in sheep’s clothing: when carcinoma cells invade the stroma Histopathologists who would like to study the role of the EMT process in, for example, human tumor pathogenesis face a dilemma. In the absence of a specific marker of their epithelial origin, it is generally difficult to distinguish carcinoma cells that have invaded singly into the stroma from the surrounding mesenchymal cells that reside there naturally, notably fibroblasts and myofibroblasts. Certain experimental systems enable the epithelial origin of cells that have undergone an EMT to be traced (Figure S14.5) but demonstrate how difficult it is, using standard histopathological techniques, (A) © 2014 Garland Science to identify these mesenchymal carcinoma cells. In yet other experimental systems, these distinctions can be made with facility. For example, in Figure 14.17B, carcinoma cells that have undergone an EMT can be readily identified, since the tumor being studied is of human origin and is growing as a xenograft in a host mouse. Use of anti-vimentin antibody that recognizes only human vimentin ensured that only cells derived from the engrafted human cancer cells are recognized; conversely, the vimentin made in abundance by the host-derived mesenchymal cells is not immunostained by this antibody because it is of mouse origin. (B) E-cadherin/YFP/DAPI (C) Zeb1/YFP/DAPI Figure S14.5 Carcinoma cells scattered throughout the stroma In a genetically engineered mouse strain that develops pancreatic carcinomas at high frequency, the pancreatic epithelial cells express the yellow fluorescent protein (YFP) transgene, which appears here as green. Importantly, the YFP gene marks both pancreatic epithelial cells that have generated a primary carcinoma as well as their descendants that have undergone an EMT. This “lineage marking” makes it possible to trace the trajectories of these cells as they move throughout the tumor. (A) Here the carcinoma cells that have remained epithelial are seen to express E-cadherin (red-orange, upper right). However, the cells in the remaining regions of this micrograph look morphologically much like stromal cells but lack the clear E-cadherin marking. Their derivation from carcinoma cells is indicated by their display of YFP. Nuclei of cells are stained blue with DAPI. (B) At much higher magnification, the islands of carcinoma cells seem to be surrounded by oceans of stromal cells. However, the fact that the majority of the “stromal” cells seen here express YFP (green) demonstrates their origin in the epithelial compartment of the tumor, more specifically from carcinoma cells that arose in this tissue. True stromal cells of mesenchymal origin are seen here as cells whose nuclei stain blue with DAPI but lack both E-cadherin and YFP expression. (C) The epithelial origins of these EMT-derived cells in the stroma are further supported by staining these cells for ZEB1 (red-orange), a transcription factor that is known to act as a master regulator of the EMT program (see Section 14.8). The ZEB1 protein is often localized to the nuclei of cells that have high levels of YFP in their cytoplasm (arrows). Cells that have neither ZEB1 nor YFP are apparent only by their nuclei, which are stained blue with DAPI. (Courtesy of A. Rhim and B. Stanger.) 99
  102. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 14.6 TGF-β works in conflicting ways during tumor progression The powers of TGF-β to affect tumor development both positively and negatively have been shown dramatically by experiments reported in 1996 using a transgenic mouse model of skin carcinogenesis. In this model, the development of squamous cell carcinoma was favored by the expression of a ras oncogene under the control of a transcriptional promoter that ensured its expression in keratinocytes of the skin. When a transgene favoring TGF-β expression in the skin was also installed in the germ line of these cancer-prone mice, the absolute number of skin tumors that formed was decreased, ostensibly because of the anti-proliferative effects of TGF-β. However, those few tumors that did arise showed the phenotype of aggressive spindle-cell carcinomas (formed from cells that have undergone an EMT) rather than the more benign squamous cell carcinomas that are usually seen in such transgenic, tumor-prone mice. Consequently, once transformed epithelial cells have developed a resistance to TGF-β’s cytostatic effects (for example, through inactivation of the pRb pathway; see Section 8.10), this factor can collaborate with oncogenes residing in the carcinoma cells to trigger an EMT and attendant high-grade malignancy. 100
  103. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.7 Dynamics of EMT induction: the EMT may be controlled in some cancer cells exclusively by their own genomes The descriptions provided here of the EMT and its induction by stromal signals cannot be applied to all carcinomas, if only because key governors of the EMT, such as E-cadherin levels, may occasionally be affected by changes occurring within cancer cells, including promoter methylation and mutational inactivation of the E-cadherin gene. (Experimental shutdown of E-cadherin expression has been shown, on its own, to have a strong EMT-inducing effect, ostensibly because of the liberation of previously sequestered β-catenin and other proteins associated with the C-terminal tail of E-cadherin.) Moreover, following its phosphorylation by a cytoplasmic tyrosine kinase like Src, the E-cadherin protein may be ubiquitylated, endocytosed (internalized), and degraded, and therefore no longer able to form cell–cell adhesions; clearly the activity of such tyrosine kinases can be deregulated by cell-autonomous changes in cancer cells. Under these various circumstances, we can imagine that induction of an EMT is essentially a cell-autonomous process, that is, its induction is not dependent on heterotypic signals arising from elsewhere, notably in the adjacent stroma. In other tumors, carcinoma cells may give the appearance, because they reside stably in the mesenchymal state, of having arrived there through strictly cell-autonomous processes. In many of these cases, however, the mechanism is actually quite different: These cells may have previously acquired the ability to synthesize EMT-inducing signaling proteins, such as TGF-β, EGF, HGF, and TNF-α, in response to certain paracrine signals originating in the stroma; thereafter, the mesenchymal state may be maintained in these carcinoma cells by resulting autocrine © 2014 Garland Science signals (for example, see Figure 14.20). Indeed, in a variety of experimental systems, if epithelial cells (normal or neoplastic) are exposed for a short time (for example, several days) to EMTinducing factors, they will enter into the mesenchymal state but lapse back to the epithelial state once these inducing factors are removed from their culture medium. However, if such carcinoma cells are exposed to these EMT-inducing factors for longer periods of time (for example, several weeks), these cells can maintain their mesenchymal state long after the inducing factors are removed, ostensibly through the actions of self-reinforcing autocrine signaling loops; such dynamics, once again, give the impression of a fully cell-autonomous process, when the reality is that heterotypic signals were required to initially force cells into a mesenchymal state, whereupon these cells (through autocrine signaling) maintained their residence in this state without the need to further communicate with stromal cells. Together, these various observations indicate that there are two classes of cancer cells that appear to reside stably in a mesenchymal state. (1) Many types of cancer cells are likely to be able to initiate and maintain the EMT-induced mesenchymal state in a fully cell-autonomous manner, and the cell-autonomous changes that they have undergone (for example, mutational inactivation of the E-cadherin gene) effectively preclude these cells and their descendants from ever reverting to an epithelial state. (2) Other cancer cells rely on autocrine signaling loops to maintain their residence in the mesenchymal state, doing so in a metastable manner, since such autocrine loops can be interrupted by certain physiologic regulators, causing these cells to revert back to an epithelial state via an MET. 101
  104. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.8 An example of an EMT relatively late in embryonic development A variety of morphogenetic steps occurring long after the earliest processes, such as gastrulation and neural crest migration, continue to depend on EMTs and the transcription factors that orchestrate them. One example (A) (B) Figure S14.6 Palatal morphogenesis and Twist The process of palate formation that generates the roof of the oral cavity in a mouse embryo depends on the fusion of two palatal “shelves” that are initially formed on opposite sides of the future oral cavity, grow toward one another, and then fuse. This fusion process depends on an EMT in the epithelial cells at the point of initial s14.06 TBoC2 contact; as illustrated in this series of micrographs, at the point of fusion the epithelial cells have acquired expression of the Twist EMT-inducing transcription factor, which is detected here by immunofluorescence (green). (Since the growth factor TGF-β is known to induce Twist expression in cultured cells, the fusion of the palatal shelves can be prevented by the application of antiTGF-β antibody.) Cell nuclei were stained with ToPro dye (red). (A) Before fusion, Twist is detected in scattered cells in both the © 2014 Garland Science is the formation of the palate (Figure S14.6), which requires the fusion of embryonic cell layers converging from both sides of the roof of the embryonic oral cavity to form a single tissue—a process that occasionally occurs incompletely and thereby generates the condition of cleft palate. (C) outer epithelium and the underlying mesenchymal tissue. (B) As the two palatal shelves converge, intense Twist expression is induced, largely at the point of future fusion. (C) Once fusion has occurred, Twist expression declines dramatically and the fusion point (still apparent here) will eventually be healed completely, erasing all vestiges of the fusion process (not shown). It remains unclear whether this EMT program enables epithelial cells to migrate bidirectionally out from the point of fusion toward the nasal and oral cavities (above and below the recently formed palate, respectively) or whether it generates large populations of mesenchymal cells that will form the future mesenchyme of the palate. (From W. Yu, H. Kamara and K.H. Svoboda, Dev. Dyn. 237:2716–2725, 2008.) 102
  105. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.9 Relatively rapid metastatic dissemination of advanced primary tumor cells A study published in 2008 attempted to measure the time periods that typically elapsed between distinct stages of colorectal carcinoma progression. This multi-step process was first linked to the genetic evolution of carcinoma cells in 1989 (see Figure 11.10), but the early research did not reveal the time elapsed between the successive steps. This became possible with the advent of large-scale sequencing of cancer cell genomes, which allowed calculations of the rate at which random, unselected point mutations accumulate with time, a rate found to be ~4.6 × 10–10 per base pair per cell generation. This number, taken together with the known rate of doubling of colorectal cells at various stages of tumor progression, allowed a rough calculation of the time required for the major steps of multi-step tumor progression to reach completion. These calculations predict (Figure S14.7) that ~17 years is required for a primary, high-grade carcinoma to develop (via genetic and epigenetic mechanisms) from a relatively large, benign, adenomatous polyp. However, once this high-grade carcinoma forms in the gut, less than 2 years are required for metastases to the liver to arise. The rapidity with which such high-grade primary carcinoma cells disseminate seems to be incompatible with a requirement for additional rare genetic events to occur and become well represented in the genomes of evolving pre-metastatic tumor cell populations. This suggests that the heritable genetic changes that were selected during primary tumor formation and were required for its outgrowth were already sufficient to enable metastatic dissemination to take place. Stated differently, high-grade primary carcinoma cells may already be genetically equipped to disseminate. Accordingly, epigenetic changes acquired by these high-grade primary carcinoma cells—specifically those acquired during passage through an EMT—may suffice to enable these genetically altered cells to disseminate. In light of the information presented in Section 14.5, the process of multi-step tumor progression can be re-depicted: Primary tumor formation depends on the acquisition of a number of critical gene mutations (and hypermethylated transcriptional promoters). In many tumors, once these changes have been acquired, expression of one or several EMT-TFs in these cells may enable them to complete the remaining steps of the invasion–metastasis cascade (up to the stage of colonization) without the need for additional genetic alterations. These considerations have additional implications for the overall process of tumor pathogenesis and metastasis. They suggest three major phases of this process: (1) primary tumor formation through heritable genetic and epigenetic somatic alterations (the latter via promoter methylation); (2) activation of an EMT program in cells and their physical dissemination leading to the formation of micrometastases; and (3) colonization, which demands that disseminated micrometastatic cells undergo a series of adaptations to an unfamiliar tissue microenvironment by still-unknown changes, perhaps largely nongenetic. The subsequent outgrowth of a macroscopic metastasis—the product of successful colonization—may then allow a repetition of steps 2 and 3, in this case involving the newly formed macrometastasis rather than the primary tumor as the source of the participating cancer cells. founding cell of metastasis ? METASTASIS founding cell of high-grade carcinoma ? ADVANCED CARCINOMA SMAD4/TGF-βRII TP53/BAX EARLY CARCINOMA PIK3CA/PTEN founding cell of large adenoma normal colorectal epithelial cells SMALL ADENOMA CDC4/CIN APC/ β-catenin 0 LARGE ADENOMA KRAS/BRAF 34 normal cell MICROADENOMA 40 6 yr small adenoma patient 58 60 63 age (yr) 2 yr 3 yr 17 yr high-grade carcinoma micrometastases macrometastases © 2014 Garland Science Figure S14.7 Timetable of colorectal tumor progression This scheme illustrates the time spans that have been associated with various discrete stages of colorectal tumor progression on the basis of the somatic mutations that have been acquired in the genomes of colorectal epithelial cells at various stages of multi-step tumor progression. The age of the average patient at each stage of colorectal cancer (CRC) progression is indicated below the drawing, as are the time spans required to progress from a fully normal intestinal enterocyte to the formation of a small adenoma (6 yr), from this stage to a highgrade primary carcinoma (17 yr), from this stage to a micrometastasis (2 yr), and from the latter to the formation of a clinically detectable macroscopic metastasis (3 yr), this last step representing the process of colonization. The time of appearance of the founding cells of several of these clonally expanding populations is indicated. Mutant oncogenes and tumor suppressor genes that are found typically in various stages of CRC tumor progression and are responsible hypothetically for driving these clonal expansions are given in red. (From S. Jones et al., Proc. Natl. Acad. Sci. USA 105:4283–4288, 2008.) 103
  106. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.10 Our cells devote an enormous number of genes to regulating protein degradation Surveys of the human genome sequence have yielded a repertoire of protease-encoding genes that is vastly larger than previously anticipated. These enzymes degrade protein substrates both inside and outside of cells. They can be placed into five major functional classes, depending on the identity of the critical functional group in the catalytic sites of these enzymes. Thus, in addition to the metalloproteinases, serine, cysteine, aspartyl, and threonine proteases have been characterized. A 2003 detailed analysis of the human genome identified 553 genes that encode proteases, some which, on the basis of their sequence, appeared to be catalytically inactive but nonetheless serving some still-unknown function. (For comparison, recall that the entire human kinome contains 518 proteins, compared to the 553 proteins that together compose the “degradome” .) 125 of the proteases appeared to membrane bound, although the location of the membrane (i.e., cell surface versus intracellular membrane) was not revealed by this analysis. The majority of these 553 proteins are likely to be secreted proteins or © 2014 Garland Science cell-surface proteins, highlighting the complex regulatory interactions that are mediated by proteases and operate in the extracellular space, about which we know almost nothing. The large super-family of proteases could be grouped into 63 distinct gene families on the basis of their amino acid sequences. This echoes the explosive growth of another group of enzymes known to be involved in protein degradation—the ubiquitylating enzymes. Some surveys have enumerated as many as 527 of these enzymes that are encoded by the human genome (see Supplementary Sidebar 7.5). The actions of the ubiquitylating enzymes are countered by as many as 110 distinct de-ubiquitylating enzymes (DUBs), most of which are cysteine proteases. Altogether, about 5% of the genes in our genome are devoted to one or another aspect of protein degradation. Only a tiny fraction of these genes and their protein products have been studied to date; many are likely to control the concentrations of important cellular growth-regulating proteins. This makes it inevitable that a significant cohort of these regulators will eventually be discovered to play key roles in neoplastic growth control and tumor invasiveness. 104
  107. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.11 Peritoneovenous shunts provide dramatic support for the seed and soil hypothesis The tendency of metastasizing cancer cells from certain types of primary tumors to establish themselves in specific target organs is amply borne out by repeated clinical observations of millions of cancer cases. Clinical observations of 14 cancer patients reported in 1984 revealed just how well adapted these cells must be in order to successfully colonize potential target organs. These cases involved patients with inoperable tumors (11 ovarian plus one each of stomach, pancreatic, and bronchial carcinomas) that were causing abdominal distension and associated discomfort because of the accumulation of large volumes of ascites (see Supplementary Sidebar 13.3). This fluid, which contains large numbers of cancer cells, accumulates in the peritoneal (abdominal) cavity, because (1) the lymphatic vessels draining this cavity become obstructed by cancer cells and (2) the cancer cells often release large amounts of VEGF (also known as vascular permeability factor) into the ascitic fluid, causing microvessels in contact with this fluid to become highly permeable and leak plasmalike fluids into the abdominal cavity. Surgeons have occasionally resorted to treating ascites by implanting a tube (termed a peritoneovenous shunt) that leads from the abdominal cavity into the subclavian vein near the heart; this tube permits the ascites and associated cancer cells to drain into the general circulation. When the circulation reaches the kidneys, the water from the ascitic fluid can be eliminated, while many important solutes are retained and recycled in the body (Figure S14.8). (The alternative—the direct removal of fluid from the abdominal cavity via a needle—can lead to severe depletion of ions and essential metabolites.) The ascitic fluid of ten of these patients was found to contain suspensions of between 105 and 106 viable cancer cells per milliliter. The 2 to 10 liters of ascitic fluid produced by the patients every week drained through these inserted tubes for their remaining lifetimes. The procedure yielded a great reduction in pain and discomfort and, in a number of cases, a clear extension of survival. At the same time, this procedure also resulted in the continuous introduction, over months’ to several years’ time, of vast numbers of highly malignant cancer cells directly into the circulation. After passing through the heart, most of these cancer cells would be trapped in the capillary beds of the lung (see Figure 14.8D); some would pass through the lungs and become trapped in many other tissues throughout the body. However, in spite of billions of malignant cells being seeded in the lungs, these patients developed no macroscopic lung metastases; at most, © 2014 Garland Science venous limb one-way valve perforations peritoneal limb Figure S14.8 The peritoneovenous shunt and the seed and soil hypothesis The peritoneovenous shunt (tube) is inserted into the intraperitoneal space in the abdomen (below), where it collects ascites fluid through perforations in the walls of the tube. The fluid is then drawn through a one-way valve and empties into the venous circulation near the heart (above). While this shunt may transport billions of highly malignants14.08cells into the venous TBoC2 cancer circulation and thus into many parts of the body, few metastases are observed. (From V.T. de Vita, S. Hellman and S.A. Rosenberg, Cancer: Principles and Practice of Oncology, 4th ed. Philadelphia: Lippincott, 2000.) only clinically insignificant, microscopic metastases were found upon autopsy. Hence, the highly malignant cancer cells clearly experienced a tissue microenvironment in the lung that was ill suited to their continued survival and proliferation. These observations provide dramatic testimony of the fact that the acquisition of a highly malignant growth phenotype does not, on its own, enable cancer cells to establish themselves in distant organ sites, and that additional cellular traits are required to allow them to colonize foreign tissue environments. 105
  108. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 14.12 Tooth extractions may occasionally become exceedingly painful A 1993 paper by a group of Israeli oral surgeons summarized 55 cases reported in the scientific literature, in which the extraction of a tooth was followed, within days, weeks, or several months, by the appearance of a metastatic growth at the site of the extraction. (The oral cavity is otherwise a rare site for the appearance of metastases.) In one-third of these cases, these metastases were the first indication of the existence of a cancer in a patient’s body. Almost all of the responsible primary tumors were carcinomas arising in various internal organs. The time between the appearance of these oral lesions and the death of the patient was only four months on average, indicating that the disease was already highly advanced when the oral metastases were discovered. These cases illustrate graphically the fact that in some cancer patients, large numbers of metastatic cells are scattered throughout the body and that the cells’ ability to found new tumor colonies at various sites depends strongly on the localized tissue environment. In a region of wound healing, such as a tooth extraction site, active inflammation and the remodeling of both bone and soft tissue occur. In such an environment, the tissue stroma provides many of the factors that create a hospitable site for wandering cancer cells to settle down and to proliferate rapidly. This echoes our discussion in Section 13.3, in which the activated, desmoplastic tumor stroma was described as a tissue having many of the attributes of a wound site. 106
  109. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.13 Tumor stem cells further complicate our understanding of the metastatic process Earlier in this book (see Section 11.6), we encountered evidence that tumor cell populations arising in the breast and brain (and apparently in many other organs as well) are organized much like normal tissues, in which self-renewing stem cell populations are responsible for spawning the bulk of the cells. In the case of certain well-studied breast carcinomas, the bulk populations (>>95%) of tumor cells behave much like the actively dividing, more differentiated cells found in normal tissues. According to this conceptual model, the cancer stem cells (CSCs), also known as tumor-initiating cells (TICs), are tumorigenic, in that they can seed a new tumor mass when implanted into a host, the nonstem cells in the tumor mass are unable to do so, because they lack self-renewal potential. These observations, if extended and validated for a wide variety of tumor types, have important implications for the process of metastasis: if the non-stem cells in a primary tumor mass are truly non-tumorigenic, then these cells may well succeed in leaving the primary tumor and lodging in distant tissue sites, but they will be unable to colonize these sites because of their limited © 2014 Garland Science self-renewal and thus limited long-term proliferative potential. Accordingly, the ability to create macroscopic metastases may be confined to the relatively small number of CSCs that escape from the primary tumor. These cells are thought to have the ability to replicate to an unlimited extent and therefore are ideally suited to found new metastatic colonies that eventually expand to lifethreatening sizes. Indeed, as noted in Section 14.7, the same EMT program that allows primary carcinoma cells to physically disseminate from a primary tumor also endows many of them with the self-renewal potential of CSCs, which means that it is also likely to influence the fate of disseminated carcinoma cells. For example, those carcinoma cells that maintain their mesenchymal (and associated CSC) phenotype after dissemination are likely to be more qualified to colonize a site of dissemination, while those that rapidly shut down their EMT program, exit from the CSC state, and revert to a more epithelial state may lose colonization ability. These dynamics may well contribute to “metastatic inefficiency”—the failure of the vast majority of disseminated cancer cells to colonize the tissue sites in which they have landed (see Section 14.15). 107
  110. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 14.14 Does Darwinian evolution accommodate metastasis-specific alleles? The conceptual model that was developed in Chapter 11 portrays multi-step tumorigenesis as a process formally akin to Darwinian evolution: a cell within a pre-neoplastic cell population acquires a particular mutant allele that confers additional proliferative advantage, and sooner or later, the descendants of this cell will predominate among the neoplastic cells in the primary tumor mass (see Section 11.5). As a logical extension of this thinking, the final steps of tumor progression would involve the acquisition of mutant alleles that enable the cells within benign tumors to evolve further toward the invasive and metastatic state. Inherent in this model, however, is a conceptual quandary: imagine that mutant alleles exist that confer metastatic ability but not replicative or survival advantage. The descendants of the rare tumor cell (for example, one in 106) that happens to acquire such a purely metastatic allele will, much later, continue to be equally rare among the population of cells in the primary tumor mass (since this allele, by definition, does not confer proliferative or survival advantage). And given the scarcity of these cells, coupled with the inefficiency of the metastatic process, metastases originating from this small clonal population will rarely if ever form. This logic indicates that mutant alleles specialized to confer metastatic potential are unlikely to play a significant role in the last stages of primary tumor progression. The simplest alternative to this model posits that certain mutant alleles arise during tumor progression that are advantageous for evolving, premalignant cancer cells and also incidentally confer increased metastatic ability. Cells carrying such multifunctional alleles may soon dominate the primary tumor mass, simply because of the proliferative advantage conferred by these alleles. This now-large mass of cells may then successfully seed metastases, because its constituent cells all happen to exhibit the additional, unselected trait of metastatic ability. (Implicit in this model is the notion that certain genes and proteins favoring aggressive proliferation within the primary tumor may also impart invasive and even metastatic phenotypes.) A variant of this model involves a delayed phenotypic response to an initially sustained mutation. Imagine that at a certain step early in tumor progression, there are several alternative mutant alleles that can equally satisfy the proliferative needs of an evolving premalignant cell population. For example, relatively early in colon tumor progression, the K-ras oncogene is activated in almost half of all colon cancers; in many of the remaining ones, mutant B-raf genes (~15% of all tumors) or mutant PI3 kinase genes (~30% of all tumors) are often found (see Figure 11.12A). While these three alternative classes of alleles may function similarly early in multi-step tumor progression by promoting cell proliferation © 2014 Garland Science and survival, much later, in the context of subsequent mutational steps in tumor progression (for example, the mutation of a p53 gene), the three genetic subtypes of colon tumors may elicit very different behaviors. Thus, speculatively, in the presence of a subsequently acquired mutant p53 allele, the K-ras oncogene might portend the development of relatively benign tumors, while the mutant B-raf alleles may provoke an invasive outcome. If so, this would indicate that some tumors “start off on the wrong foot” because of the nature of the mutant genes that they happen to acquire early in multi-step tumor progression; years later, when tumor progression generates tumorigenic cells, these initially acquired alleles may strongly influence the invasive and metastatic behaviors of these cancer cells. In all these cases, other types of heritable alterations, notably promoter methylation, may play roles similar to the genetic mutations discussed here. The apparently important role played by the EMT program in invasion and metastasis by carcinoma must also be integrated into this thinking. Thus, if certain mutant alleles (and inherited epigenetic alterations) confer an acquired ability to metastasize (for example, as portrayed in Figure 14.52B), these alleles may not directly drive invasion and metastasis. Instead, they may act less directly by conferring on primary tumor cells a heightened responsiveness to EMTinducing signals, such as those originating in the tumor-associated stroma. 108
  111. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.1 Rearrangements of chromosomal DNA segments generate a vast array of antigen-binding domains in antibodies and T-cell receptors Both soluble antibody molecules (assembled from immunoglobulin subunits) and T-cell receptors (TCRs) are able to develop an extraordinarily large array of antigen-recognizing domains; in the case of the antibodies, as many as 1016 distinct antigen-recognizing domains may be generated by permutations of the coding regions of the genes specifying these proteins. A similar organizational plan is found in both classes of genes (Figure S15.1A). The rearrangements of the antibody and TCR genes are effected by the RAG-1 and RAG-2 proteins. They do so through the deletion of intragenic segments and the attendant fusion of previously nonadjacent DNA segments within each gene. In the case of antibody genes, the variable domains of these genes, which are responsible for recognizing and binding antigens, are composed of amino acid sequences from both the heavy and light chains (see Figure 15.1). A given antibody molecule is assembled from two light and two heavy chains. During the formation of antibody-encoding and TCR genes, a series of deletions in the heavy- and light-chain genes juxtapose small coding segments in a combinatorial fashion to generate a large number of distinct variable region domains with a commensurately large diversity of antigen-recognizing functions. For example, © 2014 Garland Science in the case of the heavy-chain locus, one of ~40 VH segments is fused at random to one of ~25 DH segments and thereafter to one of 6 JH segments; in each case, the particular choice of segments is made semi-randomly. In principle, these fusions can generate 40 × 25 × 6 = 6000 distinct VDJ combinations. Similarly, in the case of the κ light-chain locus, ~40 randomly chosen Vκ segments are fused at random with any one of 5 Jκ segments to generate ~200 combinations of fused VJ segments. Since each antigen-recognizing domain of an antibody molecule is composed of both a heavy- and a light-chain segment, this can generate 6000 × 200 = 1.2 million combinations of heavy- and light-chain variable domains and thus, in principle, an ability of the antibody molecules bearing these various domains to recognize an equal number of antigens. (In practice, many antibody molecules generated in this way may not be able to recognize and strongly bind any antigen at all.) This diversity is further increased by imprecise fusion events between the short coding segments present in the germ-line loci as well as an enzyme, termed AID, that inserts point mutations in the reading frames encoding the variable segments during the maturation of the immune response. A similar series of rearrangements occurs during the formation of TCR-encoding genes (see Figure S15.1C), without the involvement, however, of the AID enzyme. 109
  112. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science (A) λ light-chain locus L1 V λ1 L2 Vλ2 J λ1 L Vλ~30 C λ1 Jλ2 C λ2 Jλ 4 C λ4 κ light-chain locus L1 Vκ1 L2 Vκ 2 L3 Vκ 3 L3 VH3 J κ 1–5 L Vκ ~38 LH VH~40 Cκ heavy-chain locus L1 VH1 L2 VH2 (B) DH1–23 light chain heavy chain V J C L V D L L Cµ J H1–6 V C J DJ germ-line DNA DNA somatic recombination D–J rearranged DNA joined somatic recombination V–J or V–DJ joined rearranged DNA L V J C L V J C L C V DJ L C V DJ transcription RNA primary transcript RNA C AAA AAA splicing mRNA translation L V J C L V DJ C AAA VL AAA CL C H2 CH 3 protein polypeptide chain VH C H1 TBoC2 n15.121/S15.01A,B 110
  113. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (C) Vα n germ-line DNA Vα2 Vα1 Jα © 2014 Garland Science Cα α recombination Vα1 Jα Cα α rearranged DNA transcription splicing translation α protein (T-cell receptor) β translation splicing transcription Vβ1 Dβ1 Jβ Cβ1 β rearranged DNA recombination Vβ n germ-line DNA Vβ1 Dβ1 Jβ C β1 Dβ2 Jβ Cβ2 β Figure S15.1 The origins of antibody and T-cell receptor diversity The immunoglobulin (Ig, antibody) genes and the T-cell receptor genes (TCR) generate diverse antigen-combining regions through similar mechanisms involving the rearrangement of germ-line sequences. (A) Antibody molecules are composed of heterotetramers of two heavy (H) and two light (L) genes (see Figure 15.1). While only a single heavy-chain locus exists, one of two alternative light-chain genes, termed κ and λ, are responsible for specifying the light chains of an antibody molecule. Each of the light-chain loci (above) encodes a series of V segments TBoC2 n15.121/S15.01C (red), ~30 in the case of the Vλ locus and ~40 in the case of the Vκ locus. A small cohort of Jλ and Jκ segments (yellow) and C (constant) region segments (blue) encode the remaining portions of the light chains. The single heavy-chain locus (below) is more complex, carrying ~40 VH (red), ~25 DH (green), and 6 JH (yellow) segments in addition to a number of C (constant) segments in addition to (lying to the right of) the Cμ segment (not shown). The VJ and VDJ segments in the light and heavy gene loci are responsible for the diverse antigen-combining abilities of immunoglobulins. The L segments (open rectangles) encode short N-terminal “leader” segments that enable co-translational insertion of nascent polypeptide chains into the endoplasmic reticulum. (B) During the rearrangements that generate functional antibody genes, V, J, and C segments in the case of the light chains (left), and V, D, J, and C segments in the case of the heavy chains (right), are fused to assemble the coding sequences of these subunits of the mature, functional antibody molecule. (Not illustrated here are fusions to a number of alternative CH segments, which can result in joining of a number of alternative constant regions to a rearranged variable region; these fusions thereby create alternative “classes” of antibody molecules, all of which share in common the same antigen-binding domain.) (C) A similar scheme operates to generate the heavy and light chains of the T-cell receptor (see Figure 15.11). Its two subunits, termed α and β, are encoded on unlinked loci but are organized on the same principle as the immunoglobulins and are rearranged by the same molecular machinery. (From K. Murphy, Janeway’s Immunobiology, 8th ed. New York: Garland Science, 2012.) 111
  114. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.2 Virus-infected cells may not always be recognized by the immune system As described so far, the immune system recognizes virus-infected cells because they display fragments of virus-encoded proteins on their surfaces, using the “hands” of the MHC class I molecules to do so (see Figure 15.10). Subsequent attacks on these cells by the immune system can kill recently infected host cells long before viruses have succeeded in multiplying within them. Responding to this threat, the always-clever viruses, which have co-evolved with vertebrate immune systems for hundreds of millions of years, have developed strategies to anticipate and blunt these immunological counterattacks. Some viruses, notably certain members of the herpesvirus family, encode proteins that block the processing and subsequent presentation of viral antigens by MHC class I proteins at the surfaces of virus-infected cells; hence, the immune system is essentially blinded to the presence of replicating viruses within these cells. An alternative strategy is used by adenoviruses as © 2014 Garland Science well as other members of the herpesvirus family (for example, cytomegalovirus, CMV), which block or substantially reduce the display of all MHC class I molecules on the surfaces of infected cells, thereby obstructing the subset of these MHC molecules that would otherwise carry viral oligopeptides to the cell surface and display them there. This hints at a similar mechanism, described later in this chapter, that is exploited by cancer cells to avoid being attacked by the immune system: many cancer cells succeed in reducing or eliminating expression of their class I MHC molecules, thereby preventing the immune system from recognizing abnormal proteins that these cells may be making. Yet other cancer cells fail to increase their levels of class I MHC molecules in response to interferon-γ (IFN-γ), the factor that is released by immune cells, including NK cells, and normally succeeds in eliciting increased expression of cell surface MHC class I molecules and hence increased antigen presentation by cells throughout the body. 112
  115. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.3 Bizarre tumors reveal how cancer cells can become infectious agents While a number of tumor viruses can be transmitted from one individual to another, cancer cells cannot. Thus, the tumor cells of one person cannot be introduced into the body of a second individual and cause the appearance of a tumor in the latter. This lack of contagiousness reflects the workings of the immune system, which recognizes tumor cells from another person as foreigners, because these cells display allogeneic (foreign) histocompatibility antigens; having identified such cells as foreign, the immune system rapidly and efficiently eliminates them. (One exception to this rule derives from identical twins, who, because they are syngeneic, can in principle exchange cells, including cancer cells, without rapid elimination of the donor cells from the body of a recipient. Another exception derives from those who are immunocompromised for various reasons.) This rule of the non-infectiousness of cancer cells is broken by the canine transmissible venereal sarcoma (CTVS), which was first described by a Russian researcher working in Saint Petersburg in 1876, long before the beginnings of modern cancer research. Cells of this tumor are transmitted during copulation from male to female dogs and in the reverse direction as well. Thereafter, the cancer cells proliferate to form tumors of the male and female reproductive organs. This cancer affects dogs worldwide, and the associated cells appear initially to have lost expression of histocompatibility antigens, preventing the cells from being recognized as foreign (allogeneic) by the immune systems of newfound hosts. However, after 2 to 3 months, the CTVS cells begin to express MHC class I and II molecules (Figure S15.2A) and the tumors eventually regress. The re-expression of these MHC molecules appears to be provoked by tumor-infiltrating lymphocytes (TILs) that accumulate in increasing numbers in the CTVS tumors and release 25 20 15 10 soluble factors that elicit this re-expression in the CTVS cancer cells (Figure S15.2B). Some studies indicate that as the tumors regress, the proportion of TILs that are CD8+ cytotoxic lymphocytes (TC’s) increases progressively. However, the precise source of the MHC-inducing factors is not clear. All the CTVS tumors seen worldwide seem to descend from a common ancestral cell and contain a myc gene that has been activated through insertional mutagenesis by a retrovirus-like transposable element. The tumor has apparently been spreading through canine species for thousands of years. After these tumors regress, a dog is left with a lifelong immunity against subsequent re-infection by CTVS cells. (CTVS is bizarre in yet another respect: it periodically acquires mitochondrial genomes and thus apparently mitochondria from cells of the hosts through which it passes.) In 2006, a similarly behaving transmissible tumor was described in Tasmanian devils, a carnivorous marsupial found only on the Australian island of Tasmania. These animals frequently fight, biting each other in the face. (The resulting Tasmanian devil facial tumors that develop rapidly prevent them from eating, causing death by starvation; it remains unclear whether, if infected individuals were to survive for longer periods, they would eventually develop the ability to reject these tumors.) As is the case with CTVS, tumors isolated from a number (15) of animals all show identical genetic abnormalities, in this case at the karyotypic level, indicating a common origin. The animals of this species comprise a geographically isolated population that exhibits a limited degree of interindividual genetic heterogeneity; accordingly, they may exhibit limited variability in their MHC class I and II alleles. This may help to explain the inability of infected individuals to recognize tumor cells as distinctly foreign, that is, of allogeneic origin. This disease threatens to drive the Tasmanian devils to extinction. (B) tumor regression % of CTVS cells expressing MHC I or II index of MHC I expressed (A) dog 1 dog 2 dog 3 5 © 2014 Garland Science 40 30 MHC I MHC II 20 10 0 0 3 6 9 12 15 18 21 weeks after implantation CTVS cells cultured in + medium from TILs from regressing tumors + medium from TILs from progressing tumors + fresh medium Figure S15.2 Regression of CTVS and re-expression of MHC antigens One exception to the rule of the non-transmissibility of cancer from one organism to another comes from canine transmissible venereal sarcoma; its cells are transferred from one dog to another via sexual intercourse. The transferred cells initially form a vigorously growing tumor, which is, however, rejected after several months. (A) While the canine transmissible venereal sarcoma (CTVS) cells initially express very low levels of MHC class I proteins, after 12 weeks of tumor growth these proteins begin to be re-expressed, as seen here in the tumors borne by three dogs. Once re-expressed, these proteins are the likely cause of the regression of these tumors. (B) This re-expression of MHC antigens results, at least in part, in response to signals (possibly interferon-γ) released by tumor-infiltrating lymphocytes (TILs). Fresh culture medium has little effect on the expression by CTVS cells of MHC class I (green) or class II (red) proteins (left). Moreover, when TILs are extracted from progressing tumors (first 12 weeks) and propagated in culture, factors released into their culture medium by these TILs also have little effect on MHC protein expression (middle). However, factors released by cultured TILs extracted from regressing tumors (12–21 weeks) potently induce the expression of MHC class I and II proteins by CTVS cells propagated in culture (right). (From Y.W. Hsiao et al., J. Immunol. 172:1508–1514, 2004.) 113
  116. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.4 Mice have proven to be far more useful for tumor biologists than chickens The first inbred strain of mice (termed DBA) was produced in 1909. Since that time, numerous other inbred strains have been produced, including frequently used strains such as C57, C3H, AKR, and BALB/c; altogether, 19 major strains and 39 derived substrains are in common use by biologists. Use of an inbred strain makes it possible to transfer cells (including tumor cells) and tissues from one mouse to another without the complications created by immunological rejection of engrafted cells or tissues, since all mice within an inbred strain are syngeneic. Immunocompromised mice, notably the Nude, NOD/SCID, and Rag1/2 mutant mice (see Sidebar 3.2), have proven to be valuable for a variety of critical experiments in cancer biology. For example, the tumorigenicity of cultured cells derived from many types of human tumors has been demonstrated directly by introducing these cells into Nude mouse hosts (after first irradiating these mice to eliminate most of their resident NK cells). Without our ability to establish such xenografts (that is, © 2014 Garland Science grafts in which donor and recipient belong to different species), the tumorigenic powers of cultured human cancer cells would remain only a matter of conjecture because we could not rule out the possibility (1) that these cells might well have lost their tumorigenicity during extended propagation in vitro, or (2) that their tumorigenicity might depend on the collaborative interactions of several cell types in the original tumors from which they were derived, only one of which is present in the cancer cells propagated in vitro. Immunological considerations also help explain why researchers have not made extensive use of chickens in recent decades, even though these birds were prime objects of study during the early years of modern cancer research. Highly inbred strains of chickens have never been developed; hence, transformed chicken cells cannot be engrafted into syngeneic hosts. Moreover, this difficulty cannot be circumvented by the use of genetically immunocompromised chickens, which also do not exist. 114
  117. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.5 An HPV vaccine protects against many cervical carcinomas The ability of the immune system to recognize viral proteins as being foreign has made possible the development of the first vaccine against human cancer that has been proven to be clinically effective and thus approved by the FDA and other authorities for introduction into the clinic. This vaccine, termed Gardasil, is composed of noninfectious HPV virus-like particles that are capable of engendering an antiviral immune response against the four HPV virus types most strongly implicated in the pathogenesis of cervical carcinomas—types 6, 11, 16, and 18 (Figure S15.3). In an initial © 2014 Garland Science clinical trial, 12,000 women were enrolled in a 3-year-long trial; half of these women were exposed to the Gardasil vaccine while the other half were exposed to a placebo. At the end of the trial period, among women who had no indication of previous infection by HPV types 16 and 18, the vaccine was found to have prevented 98% of the HPV types 16 and 18–induced cervical intraepithelial neoplasias (see Figure 2.15) that would otherwise have occurred, as gauged by the frequency of these growths in the control, placebo-treated population. The vaccine had relatively little efficacy against infections that were already well established. (A) viral replication cycle in infected cells L1 pentamers L2 monomers HPV dsDNA genome (B) self-assembly of VLPs in the absence of viral DNA L1 pentamers since virus-like particles (VLPs) that lack viral genomes provoke Figure S15.3 Viral vaccination by virus-like particles a vigorous antiviral immune response. (A) In a normal HPV life formed in vitro Viral vaccination can proceed by either of two cycle, L1 pentamer capsid proteins assemble with L2 monomer strategies. Individuals can be infected with a temperate but capsid proteins, and together they assemble with the circular replication-competent form of a virus that can replicate in the dsDNA of HPV to construct infectious virus particles. (B) The L1 host without causing unacceptable symptoms or pathology while proteins, when produced in large amounts on their own through still provoking an immune response. While often highly effective TBoC2 N15.122/s15.03 recombinant techniques, will self-assemble—as seen in the in exposing various components of the virus to the immune electron micrograph—into VLPs that are highly immunogenic, system, this strategy runs the risk of inadvertently spawning a provoking long-lasting, potent antiviral immunity. A mixture of virulent infectious cycle. The alternative strategy depends on the VLPs generated by the self-assembly of the L1 proteins derived the use of a replication-defective form of the virus that exposes from four different HPV types has proven to be highly effective in the vaccinated person to the virion’s proteins, specifically its generating the quadrivalent vaccine termed Gardasil. (Courtesy capsid proteins. While ensuring that no virulent viral infectious of D. Lowy and from R. Kirnbauer et al., J. Virol. 67:6929–6936, cycle will be launched, this strategy often fails to elicit effective, 1993.) long-lasting antiviral immunity. In the case of HPV vaccination, however, the second strategy has been found to be effective, 115
  118. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.6 An unexpected type of anti-p53 reactivity is often found in cancer patients Possibly the bestdocumented example of a cancer-associated protein provoking an immune response relates to the p53 protein. The great majority of p53 mutations are point mutations that create missense codons in the reading frame of this gene (see Section 9.3). The resulting amino acid–substituted proteins might well be immunogenic. Indeed, one survey has indicated that anti-p53 reactivity is present in the sera of 1600 out of 9489 cancer patients studied. Surprisingly, when the p53 antisera were analyzed, the associated antibodies were found, almost always, to recognize amino acid sequences present in the N- and C-terminal domains of the p53 protein. These locations are generally unaffected by the amino acid substitutions found in mutant p53 proteins, which usually alter the centrally located, DNA-binding domain of p53 (see Figure 9.6). Hence, the novel peptide sequences present in mutant p53 proteins are, unexpectedly, not the cause of the © 2014 Garland Science immunogenicity of these proteins; instead, the amino acid sequences present in wild-type p53 are responsible. These wild-type sequences are present in greatly increased concentrations in most tumors (see Section 9.9) because of the dramatic increases in p53 protein levels in many kinds of cancers. Apparently, the immune system does not become tolerant to a protein that normally is present in vanishingly low concentrations in cells. Moreover, the mutant, tumor-associated p53 proteins may have altered three-dimensional configurations and may therefore display oligopeptide domains that are not normally exposed by the wild-type p53 protein. Such a stereochemical shift may make these domains more susceptible to processing into oligopeptide fragments and display by MHC class I and II molecules. Unanswered by these studies is the question whether the ability to recognize abnormal p53 (as indicated by the anti-p53 serum responses) helped the immune systems of these 1600 patients to control expansion of the tumors that they bore. 116
  119. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.7 Immune recognition of tumors may be delayed until relatively late in tumor progression When does the anti-tumor immune response become activated during the course of multi-step tumor progression? Some cancer immunologists argue that early in tumor progression, nests of tumor cells may be too small to be recognized by the immune system. Accordingly, they propose that immune recognition may occur only much later, after tumors have become large and have acquired the ability to invade adjacent tissues. The evidence favoring a close connection between tumor cell invasiveness and the initiation of immune recognition of these cells is not definitive. In fact, there are indications that a certain degree of immune surveillance may already occur before epithelial cells have become invasive and breached the © 2014 Garland Science basement membrane. For example, in some epithelial tissues, such as the gut, there is evidence of intraepithelial lymphocytes (IELs); these immunocytes are responsible for monitoring epithelial cell populations for the presence of invading pathogens. Such IELs might also serve as early-warning sentinels, allowing the immune system to detect neoplastic cells long before they have become invasive. In addition, as seen in Figure 15.20C, macrophages can ingest cancer cells on the epithelial side of a basement membrane (in this case in the lumen of a duct formed by an adenocarcinoma). It remains unclear whether such macrophages, having ingested cancer cells, migrate to lymph nodes and present cancer cell–associated antigens to the helper T cells in these nodes, thereby initiating adaptive humoral and cellular immune responses. 117
  120. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.8 Some paraneoplastic syndromes reveal defective tolerance and overly successful immune responses to tumors Paraneoplastic syndromes are those in which a tumor acts to perturb host tissues that are located at some distance from primary tumors and derived metastases. For example, some localized tumors are able to secrete into the circulation, in a deregulated fashion, endocrine hormones such as parathyroid hormone (PTH) or adrenocorticotropic hormone (ACTH). These hormones may then proceed to profoundly perturb specific normal organs located at some distance from the tumors, creating serious physiologic imbalances. Another form of paraneoplastic disease involves the class of responses labeled paraneoplastic neurological degeneration (PND). In these syndromes, previously normal patients may initially appear in the clinic with various types of neurological disorders, including loss of vision, sensation, coordination, memory, and various types of cognition. Detailed diagnostic workup reveals that these individuals carry previously unrecognized tumors that are located somewhere outside their central nervous system. In other cases, patients with an already-diagnosed tumor develop these relatively rare neurological symptoms (seen overall in about 1 per 10,000 cancer patients). For instance, 1 in 1000 patients suffering from small-cell lung cancer (SCLC) exhibit encephalitis and sensory neuropathy; neuroblastoma patients may also exhibit such symptoms. Breast and ovarian carcinoma patients may occasionally exhibit cerebellar degeneration and associated loss of motor coordination. In all these cases, an active autoimmune attack on the nervous system can be detected that has been provoked, in some way, by a tumor (Figure S15.4). These paraneoplastic syndromes appear to represent the tip of a far larger immunological iceberg. Thus, as many as © 2014 Garland Science 15–20% of small-cell lung cancer patients develop antibody to the Hu antigen. This antigen is expressed by their tumor cells and represents the antigen that, in a small fraction of these patients, incites autoimmune attack on their central nervous system (CNS), in which this Hu antigen is normally expressed by several distinct cell types. The CNS is shielded from intensive immune surveillance by several mechanisms, resulting in the absence of immune tolerance of many of its proteins; this absence of tolerance likely explains the vigor of the immune attacks on CNS function seen in many of these cancer patients diagnosed with a PND. In some cases, the immune systems of patients suffering from these paraneoplastic neurological syndromes have also been found to mount a cell-mediated attack on their tumor cells. We see here situations in which the immune system has been successful, indeed overly successful, in mounting an attack on cells displaying tumor-associated antigens. Patients suffering from these paraneoplastic syndromes often carry tumors that are confined to a small size by their highly effective immune systems; such patients often succumb to the effects of the paraneoplastic neurological degeneration, which can be far more threatening for their survival than the tumors they carry. 62 kD normal serum (A) cerebellum ovarian carcinoma patient serum + Purkinje cell lysate (B) patient serum + ovarian carcinoma lysate her cerebellar Purkinje cells and tumor cells and was subsequently Figure S15.4 Paraneoplastic autoimmune disease Certain identified as the Yo antigen. Serum from a normal, healthy tumors, often small-cell lung carcinomas (SCLCs), are able to patient did not recognize this protein. (The Yo antigen is an provoke an autoimmune disease in the form of paraneoplastic intracellular protein localized to the cytoplasm, and it appears that neurological degeneration (PND). In this case, a patient presented much of the damage suffered by cerebellar Purkinje cells derives with widespread cerebellar dysfunction as indicated by her ataxia from presentation of Yo fragments by MHC class I molecules (loss of muscle coordination). (A) Analysis of the patient’s serum and resulting attack on these cells by cytotoxic T lymphocytes. revealed antibodies that recognized and bound an antigen that Accordingly, the specific contribution to cerebellar degeneration was displayed by the Purkinje cells of her cerebellum (red-brown TBoC2 supp18/s15.04 of the humoral immune response and its anti-Yo antibodies is cells, left); subsequent clinical evaluation revealed that she bore unclear.) (From R.B. Darnell and J.B. Posner, N. Engl. J. Med. an ovarian carcinoma to which her serum also reacted (right). 349:1543–1554, 2003.) (B) Use of her serum in an immunoblot analysis showed that it reacted with a protein of about 62 kD that was present in both 118
  121. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.9 TSTAs can arise as by-products of chemical and physical carcinogenesis The potent carcinogen 3-methylcholanthrene (3-MC) has been used frequently over the past half century to induce sarcomas in BALB/c mice. One of these tumors, termed CMS5 (see Figure 15.23), was found to induce a particularly strong cytotoxic T-cell response, which led eventually to the identification of the responsible antigen. The antigen was found to reside in one of the tumor cells’ MAP kinase proteins (see Section 6.5), specifically, Erk2, which had sustained a single lysine-to-glutamine substitution that created an immunogenic oligopeptide. Ectopic expression of this mutant Erk2 in normal cells gave no evidence of any transforming activity, suggesting that its encoding gene was originally mutated as an incidental by-product of the mutagenesis that led to the creation of the initially formed sarcoma cell. Cloning of the gene encoding another 3-MC–induced mouse sarcoma antigen from the Meth A tumor cells described in Figure 15.23 revealed that it specifies one of the many proteins that form the large subunit of the ribosome; this mutant protein, which differs from its wild-type counterpart in a single amino acid residue, also seems to confer no benefit on the cancer cells that express it. © 2014 Garland Science Similarly, the antigenicity of an ultraviolet light–induced mouse skin tumor was traced to a mutant RNA helicase protein; this enzyme normally operates in the cell nucleolus, and the tumor-associated mutant version carries an amino acid substitution because of a point mutation induced by repeated UV exposure. Once again, as an incidental by-product of its mutagenic powers, the actions of a carcinogen led to the synthesis of a mutant, antigenic protein that does not appear to have contributed to tumor pathogenesis. Accordingly, the mutant alleles that encoded these various antigenic proteins can be portrayed as the products of “passenger” mutations—adventitious byproducts of tumor formation rather than the “driver” mutations that impel tumor progression forward (see Sidebar 11.3). (In the case of the Meth A sarcoma, the identification of the mutant ribosomal protein as a TSTA could be validated by immunizing mice either with entire ribosomes from Meth A sarcoma cells or with a chemically synthesized oligopeptide that replicated the mutant sequence within the ribosomal protein. In both cases, such immunized mice rejected subsequently implanted Meth A sarcoma cells. Wild-type ribosomes and wild-type oligopeptide failed to induce this response.) 119
  122. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.10 Are melanomas more antigenic than other tumors? The tumor antigens associated with melanomas have been analyzed in great detail over the past two decades. This has created the impression that these tumors are more likely to be immunogenic and therefore more likely to be recognized and rejected through the activation of an anti-tumor immune response. In truth, this focus on melanomas may be a consequence of a variety of factors that have no connection with their intrinsic immunogenicity. Most melanomas are readily visible because they are pigmented and are located in the skin. As a result, their progression and occasional spontaneous regression have been more apparent than the growth and disappearance of other types of solid tumors. Moreover, melanoma cells, in contrast to most other types of human cancer cells, are readily adapted to tissue culture. Consequently, large numbers of these cells can be produced through culture in vitro. This makes it possible to prepare melanoma cell proteins for biochemical analysis and to gauge the responsiveness of these cells in vitro to growth inhibition by cellular and humoral immune responses. © 2014 Garland Science These factors have conspired to make melanoma antigens far more intensively studied than those of other solid tumors. As these other tumor types are analyzed more closely, the immunological distinctions between melanomas and other solid tumors are receding, and it becomes increasingly plausible that other tumors, such as carcinomas, are intrinsically as immunogenic as melanomas. Still, one factor has emerged that has persuaded some tumor immunologists that melanomas are indeed intrinsically more immunogenic than most other tumor types: the density of point mutations in the genomes of melanoma cells is at much as 10-fold higher than in many other types of cancer cells (see Figure 12.16), ostensibly as a consequence of long-term exposure of the normal melanocyte precursors to decades of UV radiation. These point mutations create many genes with altered reading frames and, accordingly, a variety of novel tumor-specific antigens that may attract, in turn, the attentions of the adaptive immune system. 120
  123. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.11 Strategies for cloning genes encoding melanoma TATAs The isolation of genes encoding various melanoma-associated tumor antigens has proven to be challenging. The strategy shown in Figure S15.5 ultimately yielded a cDNA clone that, following sequencing, revealed the identity of an encoded TATA protein. Subsequent analyses were then required to identify the 8 to 11 amino acid–long oligopeptide cleavage fragment of this protein that functions as the actual TATA. Thus, oligopeptides are eluted from the MHC class © 2014 Garland Science I proteins of the melanoma cells and sequenced to identify the sequence of the TATA oligopeptide that has provoked the CTL response. Provocatively, the cloning of TATA genes has demonstrated that as many as one-fourth of the tumor antigens identified through this strategy derive from the use by tumor cells of alternative reading frames, from the use of cryptic transcriptional promoters located within introns, or from the translation of intronic sequences. MEL A antigenic protein mRNAs prepare mRNAs reverse transcription to make cDNAs SV40 replication origin insert into expression vector eukaryotic promoter class I MHC peptide antigen TCR cytotoxic T lymphocyte CTL 159/5 add 159/5 CTLs release of TNF-α isolation of MEL Aencoding plasmid identify cultures that produce tumor necrosis factor (TNF-α) when 159/5 CTLs recognize peptide antigen E. coli HLA-B44 expression vector split into subpopulations monkey cells co-transfection into monkey kidney cells cDNA together with expression HLA-B44 expression vector library pools of 100 plasmids co-transfected into the monkey cells with a vector that expresses Figure S15.5 Cloning of genes encoding melanoma the specific variant MHC class I molecule (in this case HLA-B44) antigens One strategy for cloning a melanoma tumor-associated that was previously found to be expressed by the melanoma cells transplantation antigen (TATA) begins (top left) with the and to be responsible for presentation of the antigen that originally identification of a patient whose cytotoxic T lymphocytes (in this provoked (top left) the CTL attack. 159/5 CTLs are then added case the 159/5 CTL cell clone) are capable of lysing the melanoma TBoC2 supp19/s15.05 to subpopulations of transfected monkey kidney cells in order to cells cultured from this patient, which are termed MEL A cells. identify the pool(s) of cells that express and present the TATA that mRNA is then prepared from the cultured melanoma cells and provoked the CTL attack; this identification is made by measuring reverse-transcribed into cDNA, and the collection of resulting the tumor necrosis factor (TNF-α) that is released by CTLs into the cDNAs is introduced into the genome of a mammalian expression vector that can be amplified as a plasmid in E. coli. Pools of 100 of supernatant medium whenever they recognize an MHC class I– the vectors that have been cloned and amplified in E. coli (lower presented antigen. This measurement of TNF release identifies the right) are then introduced via transfection into COS-7 monkey particular pool of monkey cells that carries a cDNA expressing the cells that carry the SV40 large T oncoprotein, which functions in TATA, and this pool of monkey cells is then separated into subpools this case to drive rapid replication and thus amplification of the and the entire procedure is repeated in order to narrow down the introduced, cloned vector molecules (which possess SV40 originidentification to a specific cDNA, which can then be amplified and of-replication sequences; step not shown). These cloned vectors, sequenced. (Courtesy of T. Boon.) which together constitute a cDNA library, are actually 121
  124. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.12 Anti-CD47 therapies hold promise in treating lymphomas and other hematopoietic malignancies In order to prevent attack by macrophages, nonHodgkin’s lymphomas (NHLs) generally express higher levels of cell surface CD47 relative to the normal B cells in the circulating blood and in the germinal centers of the lymph nodes. The Raji Burkitt’s lymphoma cells studied here express high levels of CD47 and, because they are B-cell tumors, also express CD20, a B-cell marker. This suggests a form of immunotherapy in which both an anti-CD47 antibody is used to neutralize CD47 function, rendering the Raji cells vulnerable to attack by macrophages, and an anti-CD20 MoAb (rituximab) is used, resulting in attacks on B cells because of complement-dependent cytotoxicity (CDC; see Figures 15.9 and 15.35) as well as antibody-dependent cellular cytotoxicity (ADCC; see Sidebar 15.4). Strategies like these do not depend on antigens that are particular to one or another patient’s tumor and thus may be used to treat all tumors of a given type (Figure S15.6). 400 200 0 600000 400000 200000 47 i-CD ant 45 i-CD ant pe oty 1 is lym Raji cells 3.0 2.5 2.0 1.5 1.0 0.5 0 0 4 7 11 14 18 21 25 control IgG anti-CD47 anti-CD20 anti-CD47 + anti-CD20 -Ho dgk in’s min 4.0 3.5 non ger IgG ma pho ent al c l bl normal B cells s ers 0 era iph per (C) 800000 tumor volume (cm3) tumor cell fluorescence (B) 1600 1400 1200 1000 800 600 ood CD47 expression (A) © 2014 Garland Science an anti-CD45 antibody, which recognizes the major leukocyte Figure S15.6 Anti-CD47 therapy Levels of the CD47 antigen antigen and serves here as a second control antibody. However, if expressed on the surface of cells can determine whether or not they were coated with an anti-CD47 antibody prior to injection, they are attacked by macrophages. (A) The expression levels they failed to engraft, ostensibly because they were unable to of CD47 are markedly higher on the surface of non-Hodgkin’s TBoC2 n15.123/s15.06 display the CD47 “don’t eat me” signal and were therefore lymphoma (NHL) cells relative to the normal B cells found in consumed soon after injection by host macrophages. (C) Raji cells both the peripheral blood and the germinal centers in the lymph were injected into subcutaneous sites in these mice and allowed nodes. (B) Raji cells are a line of Burkitt’s lymphoma cells that to form tumors of ~0.1 cm3 volume before the onset of treatment in this case were engineered to express a fluorescence-emitting protein. They were treated with an immunoglobulin gamma by systemic injection of a control IgG antibody (brown); rituximab, 1 (IgG1) control antibody, an anti-CD45 monoclonal antibody an anti-CD20 MoAb (red; see Sidebar 15.4 and Section 15.19); (MoAb), or an anti-CD47 MoAb prior to injection into severe an anti-CD47 MoAb (green); or a combination of both (purple). combined immunodeficient (SCID) mouse hosts, and the growth The combination of rituximab and the anti-CD47 MoAb entirely of tumors was monitored in living mice two weeks later using blocked the subsequent growth of the implanted tumors. (From bioluminescent imaging. Raji cells formed vigorously growing M.P. Chao et al., Cell 142:699–713, 2010.) tumors if treated with the control IgG1 antibody or coated with 122
  125. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.13 Cancer cells may thwart extravasation by circulating T cells Squamous cell carcinomas (SCCs) of the skin are induced by the mutagenic powers of UV radiation and become particularly abundant in patients whose T-cell functions have been suppressed by various pharmacologic treatments. Normally, T cells will attach to the luminal surfaces of endothelial cells via one of several selectins, of which E-selectin may be the most important. Thus, E-selectin displayed by endothelial cells will recognize and bind sialic acid–containing moieties displayed on the surfaces of T cells (as well as monocytes and certain granulocytes); such binding and resulting close apposition of these T cells to the vessel walls is a prelude to their extravasation through the process of diapedesis (see Sidebar 14.1). In many human cutaneous SCCs, the carcinoma cells are apparently able to elude attack by T cells CD31 (endothelial cells) © 2014 Garland Science simply by causing the endothelial cells in the tumor-associated microvessels to down-regulate expression of E-selectin (Figure S15.7). This is likely to reduce or eliminate T-lymphocyte attachment to the microvessel walls and thereby greatly reduce the rate of extravasation by these cells. As a consequence, the tumor cells are never threatened by the cytotoxic effects of these lymphocytes. This down-regulation of luminal E-selectin expression appears to be provoked by a subtype of myeloid cells termed myeloid-derived suppressor cells (MDSCs), which are still incompletely characterized. The MDSCs infiltrate these tumors and release nitric oxide (NO), which appears to impinge on the tumor-associated endothelial cells, causing them to suppress their expression of E-selectin. This therefore seems to represent yet another strategy used by tumors for evading attacks by various types of immunocytes. E-selectin peritumoral tumor Figure S15.7 Squamous cell carcinomas evade immune attack by reconfiguring nearby endothelial cells In the peritumoral regions surrounding a human cutaneous squamous cell carcinoma (SCC; top row), the endothelial cells forming microvessels are detected with an antibody reactive with CD31 (red-brown stain, left). As is the case with the microvessels in most normal tissues throughout the body, these capillaries express E-selectin (red-brown stain, right). However, in microvessels deep within the SCC (bottom TBoC2 n15.124/s15.07 row), the microvessels are once again apparent using an anti-CD31 stain (left) but E-selectin is at very low or undetectable levels (right). By depriving passing immune cells, including T lymphocytes, of a means of tethering themselves via E-selectin to microvessel walls, these tumors apparently succeed in reducing or eliminating certain types of immune attack. (At present, it is unclear precisely how the tumor cells surrounding microvessels are able to modulate the expression by nearby endothelial cells of E-selectin.) (From R.A. Clark et al., J. Exp. Med. 205:2221–2234, 2008.) 123
  126. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.14 Herceptin can be modified to potentiate cancer cell killing Newer versions of the Herceptin treatment strategy have been developed to enhance the antitumor efficacy of this monoclonal antibody. Because antibody molecules are bivalent (carrying two identical antigen-binding pockets), techniques can be used to construct antibodies that are bi-specific, that is, react with two distinct antigens simultaneously. Thus, a bi-specific antibody has been constructed in which one antigen-binding pocket recognizes and binds to the HER2 antigen on the surfaces of breast cancer cells while the other pocket binds to CD3 (see Figures 15.19 and 15.21), a © 2014 Garland Science protein that is a subunit of the large complex of proteins forming the T-cell receptor (TCR); this receptor complex is displayed on the surfaces of a variety of T cells, including conventional T cells and a subset of NK cells called NKT cells. Moreover, CD3 binding by the monoclonal antibody leads to functional activation of the NKT cells. Consequently, the bi-specific antibody molecule can cross-link NKT cells (that it has activated) to HER2-overexpressing breast cancer cells. This close proximity can potentiate killing of the breast cancer cells by the cytotoxic NKT cells. 124
  127. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.15 Bone marrow transplantation and the treatment of hematopoietic malignancies Bone marrow transplantation (BMT) offers the possibility of curative treatments of a subset of hematopoietic malignancies. This treatment depends on the ability to ablate a patient’s entire hematopoietic system under conditions that would, on their own, be lethal for the patient. This collapse of the entire hematopoietic © 2014 Garland Science system is avoided by the transplantation of hematopoietic stem cells from another individual. Accordingly, the patient becomes the recipient of donor bone marrow cells. This rescue depends, in turn, on the ability of the hematopoietic stem cells (HSCs) of donor origin to regenerate the entire complex hematopoietic system in the recipient (Figure S15.8). NK cell THYMUS T cell common lymphoid progenitor B cell dendritic cell dendritic cell multipotent hematopoietic stem cell multipotent hematopoietic progenitor macrophage monocyte osteoclast neutrophil eosinophil common myeloid progenitor basophil platelets megakaryocyte erythrocyte STEM CELL COMMITTED PROGENITORS DIFFERENTIATED CELLS that this ability to regenerate an entire hematopoietic system can Figure S15.8 Hematopoietic differentiation Our current be traced to the properties of a single cell type—the multipotent understanding of hematopoietic cell differentiation teaches a HSC. (2) It shows that a single stem cell type can spawn multiple number of lessons. (1) It indicates that a single cell type—the types of “committed” stem progenitor cells, in this case, the two multipotent hematopoietic stem cell (HSC; left)—is capable stem cell types that are committed to generating the lymphoid and of generating all of the cell types in the blood and in the myeloid cell branches of the hematopoietic system. (3) It shows immune system. This is demonstrated most dramatically during that self-renewal ability (curved arrows) is not confined to a single the procedure of bone marrow transplantation, when the stem cell type in a tissue; instead, in certain tissues such as this one, entire hematopoietic system is ablated, often with a potent “committed progenitors” (i.e., the lymphoid and myeloid stem cells chemotherapeutic drug, such as cyclophosphamide, together shown here) as well as some of their descendants have self-renewal with whole-body radiation. The resulting lethal collapse of the TBoC2 capability hematopoietic system can be prevented by transplantation of b12.04/s15.08 (see Figure 11.18). (From B. Alberts et al., Molecular Biology of the Cell, 5th ed. New York: Garland Science, 2008.) donor bone marrow. Parallel experiments in mice demonstrate 125
  128. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 15.16 Whole genome sequencing allows a new attack on tumor cells The rapid advances in the sequencing of entire human genomes, including tumor cell genomes, has made it possible to attack cancer cells from an entirely new angle: Entire human genomes can be sequenced in less than a week. The tumor exome (that is, the collection of genomic sequences encoding exons) should reveal the identities of dozens, even hundreds of mutant protein-encoding sequences that have been generated as a consequence of the widespread genetic instability of many tumor genomes. Thus, a significant subset of adult tumors carry high numbers of somatic mutations scattered throughout their genomes (see Figure 12.16). Indeed, in some human melanomas, as many as a thousand mutations altering amino acid sequence can be found through exome sequencing. Among these mutant protein-encoding genes are the subset that are being actively expressed by a patient’s tumor cells, which can be determined by RNA-seq (which reveals mRNA expression by sequencing of the entire repertoire of cDNAs reverse transcribed from the patient’s tumor RNA). Among the countless “bystander” mutations that are present within these expressed proteins, some are likely to yield MHC class I-binding peptides that are strongly immunogenic. A bioinformatics algorithm can be used to predict how the expressed mutant proteins will be cleaved (i.e., the precise sites of cleavage) by the (immuno) proteasomes, and which of the resulting oligopeptides can, in principle, be bound and presented by human MHC Class I and Class II proteins. © 2014 Garland Science The genomic sequences of the patient’s tumor DNA will also reveal the identities of the MHC Class I and Class II alleles expressed by the patient’s cells. Another algorithm can then be used to determine which of the mutant oligopeptides identified above are actually likely to be avidly bound by the particular MHC Class I and II allelic variants that the patient’s cells express, yielding in turn a prediction of which oligopeptides are likely to be strongly immunogenic. These identified oligopeptides may then be synthesized chemically and directly administered to the patient to stimulate uptake by the patient’s dendritic cells. Alternatively, and likely more effective, the DNA sequences encoding the potentially immunogenic, mutant oligopeptides can be fused, using recombinant DNA, with sequences encoding proteins that facilitate uptake by dendritic cells; the resulting chimeric proteins expressed by these fused DNA sequences can then be harvested and administered to the patient. By either route, resulting antigen-loaded dendritic cells can then process the internalized protein, presenting the derived mutant oligopeptides to the patient’s TH and TC cells via their MHC Class I and Class II proteins (see Figure 15.11) and triggering, in turn, a concerted attack on the patient’s tumor cells. Such an attack can be further potentiated by using either the anti-CTLA-4 (see Figure 15.43) or the similarly acting, even more potent anti-PD-1 antibody. This and similar strategies are being actively pursued in a number of laboratories. Its utility in efficiently eliminating tumor cell burden in patients remains to be demonstrated. 126
  129. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.1 Modern cancer therapies have had only a minor effect on the overall death rate from the disease It is instructive to examine the causes of declining rates of cancer-associated mortality and their connection with improvements in detection and therapy, the latter including surgical resection of primary tumors as well as various forms of chemo- and radiotherapy. Figure 16.1A reveals that stomach cancer—once a major killer—has declined by a factor of more than 6 in the United States over the past three-quarters of a century, an apparent reflection of better food storage and preservation practices and associated declines in fungal contamination; declines in Helicobacter pylori infection rates may also have contributed to this decrease. Deaths from carcinomas of the uterus and lower intestinal tract have declined because of increased screening using the Pap smear and colonoscopy, respectively; these have allowed early-stage tumors to be removed before they became malignant. Lung cancer mortality in males has begun to decrease (Figure 16.1B) because of declining rates of tobacco use. (In the United States, in the years 2004–2008, 20% of the overall decline in cancer-associated mortality came from decreases in lung cancer–associated deaths, which were due almost entirely to reductions in smoking.) None of these important declines in death rate seems to be attributable to improved therapeutic treatment of already-established aggressive cancers. For example, 226,000 new cases of lung and bronchus cancer were predicted to occur in the United States in 2012, while mortality associated with this disease was © 2014 Garland Science expected to be 160,000. Forty-four thousand new cases of pancreatic cancer were anticipated, with 37,000 associated deaths. Diagnosed cases of liver cancer and associated mortality were predicted to be almost 29,000 and 20,500, respectively. Hence, diagnosis of these killer diseases continues to carry a very poor prognosis. Figure 16.1B also reveals that most of the major sources of cancer-associated mortality have remained steady or increased somewhat. Notable exceptions are breast and prostate cancers, where age-adjusted mortality rates have declined 25–30% over the past two decades—clear tributes to the effectiveness of modern therapeutic attacks on these tumors; the major causes of this improvement appear to be traceable to surgery, adjuvant chemotherapy (that is, treatment with cytotoxic drugs following surgery), and adjuvant radiotherapy, as well as tamoxifen and anti-androgen treatments, and, in the case of breast cancer, Herceptin, cessation of hormone-replacement therapy and, quite possibly, increased screening by mammography. Still, as outlined above, the decline in overall cancer mortality cannot be attributed to therapeutic attack on advanced tumors. Instead, these statistics indicate that prevention strategies (including lifestyle changes) as well as screening for certain types of cancer are likely to yield the greatest benefits in reducing deaths from cancer, at least for the foreseeable future. They also hint at the considerable challenges still facing the researchers who are developing therapeutics directed against alreadyestablished aggressive primary tumors and derived metastases. 127
  130. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.2 Prostate cancers usually do not require aggressive intervention—a tale of two countries By some estimates, the percentage of elderly American men harboring prostate carcinomas, as judged upon autopsy, is roughly equal to their age in years. Thus, even though almost 80% of American men develop prostate carcinomas by the age of 80 (as judged upon autopsy), only about 3% of them die of the disease. In many American states, the number of cases of prostate cancer diagnosed each year (in men of all ages) exceeds by a factor of about 6 the number of deaths caused by this disease. In very large part, these large numbers of registered cases (that is, disease incidence) are artifacts of diagnostic bias— reflections of our improved ability to detect neoplasms whose existence would previously have gone unnoticed because they would not have progressed to life-threatening malignancies (Figure S16.1). For example, in countries such as Denmark, where screening for prostate cancer using the prostate-specific antigen (PSA) blood test was uncommon, the estimated ageadjusted annual incidence rate of this tumor was 31 cases per 100,000 in the year 2000, while in the United States, where use of this test was widespread, the registered incidence was 104 per 100,000 that year. (In one large study cohort in the United States, the proportion of patients bearing tumors that suggested low risk of invasion and metastasis rose from 29.8% in 1989–1992 to 45.3% in 1999–2001.) Because clinicians have been unable © 2014 Garland Science to accurately discriminate between tumors that require therapy and those growths that can be left alone, aggressive treatment of diagnosed tumors became the norm in the United States. By one estimate, fewer than 10% of patients with prostate cancer in the United States are subjected to “watchful waiting,” that is, no immediate clinical treatment with periodic surveillance every several years. In contrast, watchful waiting has been the norm in Denmark with relatively little value attached to aggressive treatment of the great majority of growths detected in the prostate. Still, the aggressive overtreatment practiced in the United States compared with the watchful waiting practiced until recently in Denmark has had significant differential effects on the age-adjusted prostate cancer–associated mortality rates: it declined by >25% in the United States during the period 1975–2005 while having increased by ~50% during this period in Denmark. Averaged over the 2000–2006 period, the prostate cancer–associated mortality rate was ~75% higher in Denmark than in the United States. Taken together, these numbers suggest that even though the great majority of prostate cancer patients in the United States are treated with one or another form of unnecessary therapy, this aggressive scattershot approach to treating almost all men diagnosed with prostate cancer—treating many who do not really require aggressive treatment along with the few that do—has yielded substantial benefits in terms of reducing age-adjusted mortality associated with this disease. age-adjusted rate per 100,000 250 widespread adoption of PSA test incidence 200 prostate carcinoma 150 breast carcinoma 100 widespread cessation of HRT 50 0 1973 1987 1999 2008 Figure S16.1 Incidence of prostate vs. breast carcinomas in the United States The incidence of prostate cancer briefly and abruptly increased in the United States in 1991– 1992, when a clinical report demonstrating the utility of testing for serum levels of prostatespecific antigen (PSA) was published, leading to the test’s widespread clinical adoption. Shortly thereafter, the disease rate returned to its historic, gradually increasing trend, indicating that the spike in prostate cancer incidence was an observational (i.e., diagnostic) artifact rather than a reflection of changes in the real frequency of life-threatening disease. By 2007, an estimated 20 million PSA tests were being performed annually in North America. Five years later, the lifetime TBoC2 an American man being diagnosed with risk of Supp20/S16.01 prostate cancer was ~16%, while the risk of dying from this disease was 2.9%. Similar dynamics have governed breast cancer incidence, which peaked at 142 (age-adjusted) cases per 100,000 population in 1999. (Here there was also a dramatic readjustment of incidence, but one traceable to a real biological process: the impressive decrease of ~7% in breast cancer incidence observed between 2002 and 2003 was attributed to widespread reduction in the use of hormone replacement therapy, or HRT.) By 2012, it was concluded that PSA testing had measurable benefit only for younger men with a longer life expectancy and that for all others, the disadvantages of this test (resulting from the morbidity caused by aggressive clinical treatment following positive PSA test results) outweighed the potential benefits. (Before 1999, from A. Jemal et al., CA Cancer J. Clin. 55:10–30, 2005.) 128
  131. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.3 Clinical practice and our understanding of disease pathogenesis have often been poorly aligned, leading to sub-optimal, often tragic outcomes The treatment of breast and prostate cancers over the past century reveals the strong disconnect between how cancers develop and how they have been treated. To begin, we recognized the overriding fact that virtually all breast and prostate cancer–associated mortality derives from metastases, not from the outgrowth of primary tumors. Hence, much attention has been focused on preventing the dissemination of cancer cells and the outgrowth of metastases. Radiotherapy was used sporadically throughout much of the first half of the twentieth century to treat various types of cancer. In the case of breast cancer, however, surgery was the treatment of choice until the 1980s. In 1894, two American surgeons—Willy Meyer in New York and William Halsted in Baltimore—simultaneously reported the procedure that came to be called the radical mastectomy. It involves excision not only of the entire breast in which a tumor has been detected but also of underlying chest wall muscles and nearby lymph nodes, including those in the armpit. Until the 1970s, as many as 90% of women treated surgically for breast cancer in the United States underwent this procedure. In addition to disfigurement, a significant percentage of treated women experienced lymphedema due to the inability of lymphatic fluid to properly drain from the arm on the side where the axillary (armpit) lymph nodes were excised. In many patients, it was and is a lifelong affliction. The logic behind radical mastectomy derived from, among others, Rudolf Virchow, the father of modern pathology and the popularizer in 1852 of omnis cellula e cellula—all cells arise from other cells, a fundamental concept underlying all modern biology. Virchow argued that the lymph nodes draining a tissue (see Figure 14.42) served as filters to trap disseminating cancer cells. Thereafter, others concluded that metastases growing in these nodes represented staging areas from which a second wave of dissemination would occur to more distant sites in the body, eventually spawning life-threatening metastases. Hence, removal of draining lymph nodes, including those at some distance from the primary tumor, were thought to be a critical means of preventing distant metastatic spread. This thinking excluded an alternative model of metastasis: that breast cancer cells metastasize simultaneously to draining lymph nodes and to distant tissues. Accordingly, the presence of significant numbers of lymph nodes carrying shed breast cancer cells might be only an indication of a parallel spread of primary tumor cells directly to distant anatomical sites. In effect, according to this parallel dissemination model, the lymph nodes served only as “canaries in the mine”—providing signs that distant dissemination had already occurred, rather than being temporary staging areas for more distant travel. If correct, then attempts to prevent metastasis by treating the primary tumor and draining nodes would be doomed to failure. Beginning in the 1970s, there was increased awareness that the Halsted radical mastectomy might not confer substantial benefit on many breast cancer patients. Although staunchly resisted by American surgeons, who argued that to do less would put breast cancer patients at grave and unnecessary risk, a more conservative procedure termed a lumpectomy was developed; it involved removing the tumor and immediately surrounding breast tissue but leaving the remainder of the breast and adjacent tissue intact. Likewise, there was resistance to the notion © 2014 Garland Science of conducting a controlled clinical trial, in which the benefits of radical mastectomy versus lumpectomy would be compared in two similarly sized clinical populations. When such controlled trials were nonetheless performed, it became clear that breast cancer patients fared equally well following a lumpectomy as after radical mastectomy, in terms of their disease-free survival and long-term survival. In aggregate, between 2 and 3 million Halsted procedures were performed in the United States over the course of the 20th century, and it is plausible that few if any women carrying aggressive primary tumors ever received significant, long-term benefit from this procedure, or at least more benefit than they would have received from the far less disfiguring lumpectomy. A similar dynamic describes the trajectory of prostate cancer surgery. In this case, a different preconception drove surgical practice: that a high percentage of primary prostate carcinomas have the potential to metastasize. In many clinical practices, however, radical prostatectomy has been pursued as the most prudent means of preventing metastatic relapse for all patients diagnosed with various forms of this disease, including still-benign, localized tumors. In the case of this surgical procedure, the side effects are once again significant: radical prostatectomy often leads to lifelong incontinence and impotence. A 2012 study analyzed the clinical outcomes of prostate cancer patients with localized, still-benign tumors who were treated with radical prostatectomy or, alternatively, underwent “watchful waiting,” that is, periodic monitoring of the prostate gland in the years after initial diagnosis. This study demonstrated that the prostate cancer–associated mortality in the decade following surgery was essentially equivalent for both those who did and those who did not undergo prostatectomy. Its conclusions need to be qualified by the fact that patients who have lowgrade tumors (and represent the great majority of prostate cancer patients) are not likely to benefit from prostatectomy, while a minority—those with high-grade, malignant tumors—may receive substantial benefit. The breast and prostate cancer treatments cited above are clearly influenced by the concept that most primary tumors that are detected will sooner or later become aggressive. For example, many medical and surgical oncologists express the clear opinion that invasive breast cancers, if left untreated, will inevitably develop into large, life-threatening malignancies that can no longer be controlled by available therapies. Hence, in the absence of any clinical intervention, rather than the 39,500 breast cancer–related deaths that were predicted to occur in 2012, there might be 227,000 deaths—the number of invasive breast cancers that were predicted to be diagnosed in the United States that year. Breast oncologists will also argue that small tumors, if treated early, are curable, while large tumors are far less curable and more prone to generate metastatic relapses. This thinking is once again strongly influenced by the mechanistic model in which all small tumors expand sooner or later into large, clinically significant tumors. The alternative to this thinking is radically different: most small tumors will never develop into large, life-threatening growths within the expected lifetimes of patients. With diagnostic imaging technology that detects tumors of smaller and smaller size, the majority of the currently detected breast cancers may well fall into this second class. Moreover, the “curability” of small tumors implies that if they are caught early, they can be successfully treated. The 129
  132. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg alternative explanation is that it is unimportant when these small tumors are detected during the course of their development, since their small size testifies to their intrinsic indolent nature and “curing” them represents the elimination of growths that would never become life-threatening, whether or not they had been detected in the first place. The current inability to predict accurately whether a given tumor will or will not © 2014 Garland Science eventually become life-threatening means that in the United States virtually all breast cancers that are detected are treated aggressively with chemotherapy (including, when appropriate, tamoxifen and Herceptin) and radiotherapy as well as surgery. Importantly, the above conjectures on the nature of breast and prostate cancer progression remain topics of heated debate. 130
  133. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.4 The ability to assign tumors to specific disease subtypes is critical to the success of drug development Tumors that have traditionally been placed together in a single group, such as leukemias, constitute a very heterogeneous collection of diseases, each having its own route of pathogenesis, its own set of acquired genetic and epigenetic alterations, and its own distinct responses to a particular therapy. For example, the actions of Gleevec (also known as Glivec and imatinib), a drug discussed extensively in this chapter, are focused on antagonizing, among several molecular targets, the Bcr-Abl oncoprotein, which is found in virtually all (>95%) cases of chronic myelogenous leukemia (CML) but is uncommon in most other kinds of leukemias (see Figure 4.16). During its initial clinical development, Gleevec might well have been tested in patients suffering from all types of leukemia, in which case 5% or less of the treated population (that is, the 5% suffering from CML) would have been found to benefit from this therapy—a success rate that is usually far too low to justify further investment in the development and clinical introduction of a drug. Instead, in early clinical trials, Gleevec treatment was limited to only those patients whose leukemias had been certified to carry the specific chromosomal translocation that is the hallmark of CML and causes production of the Bcr-Abl oncoprotein. © 2014 Garland Science This strategy yielded extremely favorable clinical responses in the great majority of treated CML patients, which encouraged its further, accelerated development and its rapid introduction into oncology clinics worldwide. (As an aside, Gleevec’s early and resounding clinical successes in the late 1990s were viewed as a harbinger of things to come—a strong argument for the powers of rationally designed drugs. As it turned out, CML is a rather unique condition, in that the chronic phase of this disease seems to be driven entirely by the Bcr-Abl oncoprotein, making the leukemia cells highly vulnerable to the shutdown of this protein. This contrasts with most adult neoplasias, in which complex genetic alterations are present in the cancer cells’ genomes and multiple, distinct oncogenic events collaborate to drive disease progression, as discussed in Chapter 11. The multiple genetic alterations and the plasticity of the genomes of the cells forming the more common tumors appear to underlie their far lower response rates—and the lower reductions in disease-related mortality—following administration of a variety of newer rationally designed drugs. Hence, most unfortunately, the brilliant success of Gleevec did not foretell similar successes of other targeted drugs in the years that followed.) 131
  134. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.5 p53 germ-line polymorphisms and somatic mutations can complicate the induction of apoptosis by drugs The induction of tumor cell apoptosis by anti-cancer drugs is often influenced by the configuration of the p53 gene in a patient’s tumor genome. Thus, in addition to the somatic mutations that have altered this gene during the course of tumor progression, the inherited germ-line version of p53 may affect responsiveness to treatment. Indeed, it is possible that, in general, the configuration of the p53 alleles in a patient’s tumor represents the single most important determinant of responsiveness to various forms of therapy. To begin, we note that the “wild-type” human gene comes in two polymorphic variants that affect the identity of the amino acid in residue position 72 of the p53 protein; one encodes arginine (72R), while the other specifies proline (72P). Cultured, otherwise isogenic cells that express either the 72P, the 72R, or no p53 protein exhibit dramatic differences in their apoptotic responses following exposure to two commonly used chemotherapeutic drugs (Figure S16.2A). And patients whose tumors retained the two copies of the wild-type 72R allele showed substantially greater survival than those whose tumors carried a wild-type 72P allele (see Figure S16.2B). Observations like these © 2014 Garland Science point to challenges in the development of apoptosis-inducing therapies that reflect a patient’s inherited germ-line alleles. The somatic mutations that affect p53 also are determinants of a tumor’s responsiveness to chemotherapeutic drugs and thus the clinical course of the patients carrying these cancers. As is indicated in Figure S16.2C, the retention of a wild-type p53 allele in patients’ head-and-neck squamous cell carcinomas dramatically improved overall survival. Actually, p53 can have conflicting effects on responsiveness to chemotherapeutics, depending on the status of other genes in the signaling network with which it interacts. In particular, the ATM protein, which is responsible for activating wild-type p53 protein in response to DNA damage (see Figure 9.13), is an additional important factor that operates together with p53 to determine responsiveness to cytotoxic drugs that target the DNA of cancer cells (see Figure S16.2D and E). Taken together, these diverse observations lead to the conclusion that p53 (and ATM) are critical regulators of cancer cells’ responses to chemotherapy, and that these responses are shaped by both the germ-line configuration of the genes that encode these proteins and the somatic mutations that alter these key regulators. Figure S16.2 Effects of p53 polymorphisms on the induction of apoptosis (A) Human H1299 lung cancer cells, which have lost expression of their endogenous p53 gene, were forced to express either the 72P or the 72R polymorphic variant of wild-type human p53 protein. They were then exposed to various concentrations of cisplatin (left) or doxorubicin (right), two commonly used anti-tumor chemotherapeutic drugs (see Tables 16.1 and 16.2), and their survival following drug-induced apoptosis was gauged. Note that the ordinate is a logarithmic scale. As is apparent, those cells that expressed the 72R variant of p53 were far more sensitive to killing by both agents than were those expressing the 72P variant protein. Moreover, as anticipated, those expressing no p53 protein were far more resistant to killing by both agents. (B) Seventy patients suffering from inoperable head-and-neck squamous cell carcinomas (HNSCCs) were treated with cisplatin. Forty-eight of these patients carried tumors with wild-type p53 alleles (either 72P or 72R heterozygotes or homozygotes). Of these, 31 had tumors that were of the 72R/72R genotype, 12 had tumors of the 72P/72P genotype, and 5 had tumors of the 72P/72R genotype. The survival of these three groups is shown here. Those patients who were R/R homozygotes (whose p53 proteins favored drug-induced apoptosis—see Panel A)—enjoyed far better survival than those with the the other two genotypes. (C) For the 70 patients mentioned in panel B, the survival of the p53-positive and -negative groups in the years following initial diagnosis and treatment is shown in this Kaplan–Meier plot. Hence, independent of the germ-line configuration of their p53 alleles, retention in their tumors of a wild-type gene copy (either the P or R allelic variant) was associated with far better long-term survival than was a state in their tumors in which both wild-type gene copies were lost. (Subsequent research on a larger group of patients has extended these results and solidified the conclusions, having made them statistically more robust.) (D, E) The responses to chemotherapy and thus survival of breast cancer patients are shown here in these Kaplan–Meier graphs, in which the patients are segregated according to the p53 and ATM status of their tumor cells (see Figure 9.13). Their clinical courses can be explained by the responses of their cancer cells to genotoxic chemotherapeutic agents, such as doxorubicin. As is apparent, patients whose tumors were p53 wild-type but lacked ATM function fared far worse than did those whose ATM function was intact. Thus, cancer cells that possessed intact p53 and ATM function were able to respond to chemotherapy by activating a cytostatic or apoptotic response, eliminating these cells and conferring clinical benefit; in contrast, those with defective ATM function could not activate their p53 apoptosis-inducing protein, thereby permitting these cells to survive, and leading to a poor clinical course. Conversely, those patients whose tumors lacked p53 function and also lacked ATM function fared far better than those whose tumors lacked p53 but expressed wild-type ATM. In this case, it seems that in the absence of these two DNA damage–response proteins, cancer cells that were exposed to genotoxic chemotherapeutic agents lacked critical G1/S and G2/M cell cycle checkpoint controls (see Figure 8.4). Following chemotherapy-induced DNA damage, their cancer cells, rather than halting and repairing this damage, proceeded to advance into M phase, thereby stumbling into mitotic catastrophe (see Figure 16.8); the resulting death of these cancer cells explained the better clinical course of these patients. In contrast, in the presence of functional ATM, p53-mutant breast cancer cells still retained a critical checkpoint control protein, enabling these cells to halt cell cycle advance in response to DNA damage and repair their DNA before they re-entered into the cell cycle; this allowed the cancer cells to survive and proliferate, resulting in the poor clinical course of these patients. (A–C, from A. Sullivan et al., Oncogene 23:3328–3337, 2004. D and E, from H. Jiang et al., Genes Dev. 23:1895–1909, 2009.) 132
  135. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg (A) MDM2 p300/CBP p53 structure proline rich R OR ubiquitylation P 0 no p53 72P 72R –2 no p53 72P 72R –1 –2 log10 survival –1 log10 survival acetylation sequence-specific DNA binding 0 –3 –3 –4 –4 –5 –5 –6 –6 cisplatin (µg/ml) doxorubicin (µg/ml) (C) 100 100 72R/72R 75 72R/72P 50 72P/72P 25 1 2 (D) 3 years 4 % of patients surviving % of patients surviving (B) tumors retain a wild-type allele 75 50 tumors carry no wild-type allele 25 5 1 2 years 3 4 (E) 1.0 0.8 1.0 ATM wild-type 0.6 0.4 ATM low 0.2 p53 wild-type 0.0 cumulative survival cumulative survival © 2014 Garland Science ATM low 0.8 0.6 ATM wild-type 0.4 0.2 p53 mutant 0.0 0 20 40 60 80 100 120140 follow-up time (months) 0 20 40 60 80 100 120 follow-up time (months) TBoC2 Supp21/S16.02 133
  136. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.6 Ras function can be inhibited by interfering with the enzymes responsible for the maturation of the Ras protein The involvement of Ras oncoproteins in more than 25% of human tumors has made these proteins highly attractive targets for drug development. Since the GTPase activities of these proteins cannot be targeted, drugs have been developed that intervene instead at a critical point in the posttranslational maturation of newly synthesized Ras proteins. These proteins all seem to be tethered to intracellular membranes, largely but not exclusively to the plasma membrane, via one or more C-terminal lipid tails; without this membrane anchoring, they are unable to interact with their signaling partners (see Chapter 6). Much effort has been focused on farnesyltransferase inhibitors (FTIs), which block the enzyme that links the 15-carbon farnesyl lipid group to recently synthesized Hand K-Ras protein molecules (Figure S16.3). In general, these FTIs have not fared well in pre-clinical development and clinical trials. The difficulties illustrate issues surrounding drug design that we encounter repeatedly throughout this chapter. First, of the four Ras proteins (H-, N-, and two K-Ras proteins), only H-Ras depends absolutely on its farnesyl group for function, while the others have additional or alternative lipid groups (for example, geranylgeranyl) attached to their C-termini that suffice to support reasonably normal function. Second, the H-Ras oncoprotein—the only one of these proteins that depends absolutely on farnesylation for function—is activated in only a small minority (<5%) of tumors carrying ras oncogenes. This limits the utility of any FTI that might be successfully developed. (When farnesylation of the N- and K-Ras proteins is blocked by an FTI, these two proteins acquire, (A) © 2014 Garland Science instead, geranylgeranyl groups—see Figure S16.3A—which seem to afford them full function.) Third, an FTI might also prove toxic to normal cells that rely on the normal H-Ras protein for their metabolism. Fourth, this lack of selectivity is compounded by the fact that at least 20 other, unrelated cellular proteins have been found to depend on farnesyltransferase enzymes for their proper post-translational maturation; examples of this are provided by other small G proteins, notably the Rheb proteins, which, when farnesylated, become critical positive regulators of the centrally important mTOR kinase (see Figure 16.41), as well as certain Rho proteins. Hence, FTIs often have nonspecific toxicities toward the cells in normal tissues because these cells depend on other farnesylated proteins for their viability. Together, these factors help explain why the development of FTIs has proven to be so unrewarding to date, and why, with rare exception, these compounds have not fared well when used as monotherapies (that is, as singly administered agents) in clinical trials. In spite of the complications listed here, FTIs do have clear, albeit poorly understood, anti-tumor effects. For example, when used on its own, an FTI called variously tipifarnib or Zarnestra has shown clear therapeutic activity in chronic myelogenous leukemia (CML) and acute myelogenous leukemia (AML) patients, acting through biochemical mechanisms that remain obscure. Another FTI, termed BMS-214662, shows potent synergistic activity together with a tyrosine kinase inhibitor of Bcr-Abl in pre-clinical experiments involving treatment of chronic myelogenous leukemia cells; once again, the precise mechanism of the cytotoxic action(s) of this FTI is not well understood. (B) S S-farnesyl CH2 protein CH NH O CH2 CH3 S CH2 protein CH N H S-geranylgeranyl O CH2 CH3 farnesyl protein transferase Figure S16.3 Farnesyltransferase inhibitors The Ras proteins are modified by lipid chains at the C-termini, which allow them to become tethered directly or indirectly to membranes. (A) The farnesyl and geranylgeranyl groups are the main lipid moieties involved. (B) The enzyme farnesyl protein transferase (FPT) is responsible for the farnesylation of the H-Ras protein and as many as 20 other cellular proteins and is the target of farnesyltransferase inhibitors. The structure of the two subunits (red, light blue) of the rat FPT is shown in this ribbon diagram. Also illustrated (within circle) are an activated farnesyl substrate (yellow), a peptide substrate to which farnesyl becomes linked (white), and a zinc atom in the catalytic site of FPT (red sphere). (B, from C.L Strickland et al., Biochemistry 37:16601–16611, 1998.) TBoC2 Supp22/S16.03 134
  137. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.7 Chemical synthesis, compound libraries, and high-throughput screening Currently available synthetic organic chemistry techniques make it possible to generate some 10200 distinct molecular species by linking various combinations of chemical groups to one another in various orders and configurations—the technology of “combinatorial synthesis.” Of these organic molecules, only a tiny fraction— about 1062—have a molecular weight below 500, and only 1% of these are thought to possess the chemical properties, such as solubility, lipophilicity, chemical stability, and molecular weight, that would make them plausible drug candidates (Table S16.1). Instead of exploring the entire, still-huge “chemical space” of 1060 organic molecules synthesized by such combinatorial methods (Figure S16.4A), it is far more productive in practice to search through collections (“libraries”) of compounds that have been synthesized for previous drug development programs and contain, typically, 105 or more distinct chemical species. (About a quarter million distinct molecular species are estimated to be present in aggregate in the chemical libraries currently screened by the pharmaceutical industry.) These compound libraries are stored in industrial facilities (see Figure S16.4B) from which individual compounds can be retrieved robotically. The technology of high-throughput screening (HTS) makes it possible to search through libraries of existing diverse organic molecules in order to find the small number having the functional properties of the desired drug, for example, a chemical species that inhibits a certain tyrosine kinase while leaving other tyrosine kinases unaffected. Thus, in vitro enzymological assays or cellbased assays can be devised in which 105 or even 106 distinct compounds can be individually assayed, using robotic machinery (see Figure S16.4C), to measure a functional property, such as the ability to inhibit the kinase of interest; these screens can often be accomplished in several days. High-throughput screening of such libraries often yields a small number of lead compounds that have some of the desired functional and chemical properties. Researchers can then explore the chemical space in the immediate vicinity of a lead compound by incrementally modifying it, doing so by adding certain chemical groups to the initially identified molecule and © 2014 Garland Science removing others. This process of derivatizing a lead compound may result in the discovery of variant chemical structures with even better properties, such as improved solubility, ability to inhibit a targeted enzyme at lower concentrations, improved permeation into cells, or the ability to be taken orally (see Figure S16.4D). [For example, “compound 1” in Figure 16.11C was found to bind avidly to Bcl-XL but was unfortunately absorbed by the abundant human serum albumin (HSA) in the plasma, precluding its use as a drug. Consequently, compound 1 was further modified to reduce its binding to HSA and to enhance its binding to Bcl-XL, which finally yielded the drug ABT-737.] However, even with such modifications of existing compounds, only tiny corners of the total chemical space of 1060 possible drug compounds have been explored by the pharmaceutical industry. Table S16.1 Predictors of poor drug absorption Properties of a molecule that predict its poor absorption or permeation into tissues and cellsa 1. No more than 5 hydrogen bond donors 2. Molecular weight over 500 3. Octanol/water solvent partitioning > 5 (lipophilicity) 4. More than 10 hydrogen bond acceptors (N’s and O’s) These rules stipulate that in order for a drug to be orally active, its properties cannot violate more than one of the four listed conditions. Conformance to these rules is said to determine a drug’s pharmacokinetics, which derive in turn from (1) its absorption via passive diffusion through mucous membranes and plasma membranes into the general circulation; (2) its distribution via the circulation to various tissues throughout the body; (3) its metabolism, often via cytochromes operating in the liver; and (4) its excretion via the kidneys, gastrointestinal tract, or lungs. In fact, only about half of the drugs that have been approved by the U.S. Food and Drug Administration and are in clinical use conform to these properties, which are often referred to as “Lipinski’s Rule of 5” or simply “the Lipinski rules.” a From C.A. Lipinski et al., Adv. Drug Deliv. Rev. 46:3–26, 2001. 135
  138. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg descriptor 2 (A) © 2014 Garland Science (B) 125 100 75 50 25 0 -25 -50 -75 -100 125 - 100 descriptor 3 - -75-50 -25 0 25 50 75 100 7 10 5 0 5 125 12 2 50 5 -12 - -10 5 -50 75 0 0 25 descriptor 1 (C) III F H N F F Gli–luc: N O H N F (D) O I O IV N NH O II O H N H N Gli–luc: 1130 nM O 271 nM O N O N N H N N O Gli–luc: H N H N O N H N 256 nM N N H N 246 nM O N N O 4385 nM Gli–luc: 409 nM N 3914 nM 334 nM 71 nM O CN OCF3 V VI Z H N O Gli–luc: Z = Me 7 nM = Cl 14 nM H N O N O NVP-LDE225 IC50 = 7 nM N N O TBoC2 Supp23,new/S16.04 136
  139. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Figure S16.4 Generation of compound libraries and screening of large numbers of compounds (A) The diversity of chemical species including natural and synthetic organic products can be portrayed graphically by depicting a three-dimensional “chemical space” in which each of the three dimensions indicates a specific chemical attribute (a “descriptor”) of the molecules being cataloged. In reality, because the number of descriptors (each referring, for example, to the presence of a specific side chain in the molecules being plotted) is vastly larger than three, the actual chemical space is multidimensional, amounting possibly to several hundred dimensions. (B) The drug library of a typical large pharmaceutical company is stored, as seen here, in a facility from which individual compounds can be retrieved robotically. (C) Computer-driven robots, such as the one pictured, can be used to process thousands of samples daily, often using the 96-well microtiter plates shown in this photograph. By introducing enzyme- or cell-based assays into these plates, the biochemical and biological activities of large numbers of compounds can be measured over a short period of time—the technology of highthroughput screening (HTS). (D) The optimization of a drug designed to antagonize Smoothened, as described here, reflects the general strategy of lead-compound optimization. In this case, the drug development began with a lead compound (I, above, left) that had measurable activity against a targeted molecule but only at an unacceptably high concentration (IC50), in this case 1130 nM. (In this case, a cell-based assay was used, in which the ability of a test compound to reduce the expression level of the luciferase protein, expression of which was being controlled by the Gli transcription factor.) Its derivatization began with a major modification of the northernmost ring (yellow field) and the addition of a methyl group (II, pink field) on its western side. Subsequent derivatizations of area in the green field of molecule II yielded four compounds (III) that exhibited either substantial increases or minimal decreases in the IC50 (concentration needed to achieve 50% inhibition). However, further derivatizations of one of these (IC50=of 271 nM) led to three more compounds (IV, far right), one of which showed an IC50 of only 71 nM following the addition of two methyl groups in the southeastern part of this molecule. This improvement was incorporated into the next versions of the drug (V, left, below), which carried two alternative additions. These involved initially removing the previously added methyl group on the western side of the molecule (see II) and derivatizing instead the same ring (side group Z of V) with two alternative side groups (below). One of these, involving the addition of a methyl group (Me), yielded a molecule with an IC50 of 7 nM. Further derivatizations of the northernmost ring and an alteration of the heterocyclic ring (red arrows, VI), undertaken to improve pharmacokinetics and oral bioavailability, yielded the final drug molecule, termed NVP-LDE225. This drug was then introduced into pre-clinical studies focused on the treatment of small-cell lung cancer (SCLC) and medulloblastoma. (A, courtesy of S.L. Schreiber. B, courtesy of U. Schopfer, Novartis Pharma AG. C, courtesy of Beckman–Coulter Co. D, courtesy of X. Wu and from S. Pan et al., ACS Med. Chem. Lett. 1:130–134, 2010.) 137
  140. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science Supplementary Sidebar 16.8 Evolution can generate huge collections of structurally similar proteins Evolution generally proceeds via the incremental modification of existing morphological structures or existing molecules. In the case of the latter, the invention of totally novel protein structures is a very rare event, even when measured over the course of hundreds of millions of years. Accordingly, the structures of almost all modern metazoan proteins are clearly discernible in the proteins encoded by the genomes that served as the common ancestors of modern metazoa. Therefore, once a protein structure is invented and achieves functional activity, the overall molecular scaffold of its evolutionarily derived relatives usually remains relatively unaffected, while small domains or individual amino acids may be altered in these derived proteins to generate novel functions or activities. This specialization and acquisition of novel function usually depends, over the course of evolutionary time periods, on repeated cycles of gene duplication followed by sequence divergence of the duplicated genes. Thus, each of the resulting diverged genes will specify a protein that has its own distinctive features but nonetheless continues to exhibit the basic, commonly shared structure. In the case of the functionally diverse set of kinase molecules, almost all members of the vast repertoire of modern mammalian kinases can trace their ancestry to a common founder enzyme that must have been invented long before the Cambrian era and thus before the metazoan radiation (Figure S16.5). Figure S16.5 The human kinome As illustrated here, the great majority of the protein kinases of mammalian cells, including serine/threonine and tyrosine kinases, share substantial structural similarity, indicating that they all descend from a primitive protein kinase that existed long before the radiation of metazoan life forms. The serine/threonine and tyrosine kinases diverged from one another relatively early and have been further diversified over the past approximately 109 years. Altogether, the human genome sequence reveals 518 distinct genes encoding protein kinases, which, as a group, have been called the “human kinome.” Of these, 90 phosphorylate tyrosine residues, while the remainder phosphorylate serine or threonine residues of substrate proteins. All of the tyrosine kinases (TKs) and 318 of the serine/threonine kinases show clear structural relatedness to one another and can be arrayed on an evolutionary tree that depicts how they appear to have evolved from one another through repeated gene duplications followed by diversification. (A small number of “atypical” protein kinases, not shown, appear to represent independent evolutionary inventions.) The shared origins of the kinases located on this tree dictate that many of these enzymes are structurally similar to one another, which complicates the creation of drugs that interfere selectively with only a few members of this large family of enzymes. The TKs (top left) represent relatively recent evolutionary inventions, as they are absent in prokaryotes and are present in only very small numbers (e.g., 3) in the genomes of single-cell eukaryotic protozoa sequenced to date. Their great diversification and specialization appear to have been critical to the evolution of anatomically complex metazoa. The remaining groupings on this tree are TKL, tyrosine kinase-like; STE, homologs of yeast sterile 7, 11, and 20 kinases; CK1, casein kinase-1; AGC, members of protein kinase A, kinase G, and kinase C families; CAMK, calcium/ calmodulin-dependent protein kinases; and CMGC, containing CDK, MAPK, GSK-3, and CLK families. (Courtesy of Cell Signaling Technology, Inc.) 138
  141. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg © 2014 Garland Science 139
  142. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.9 Large-scale screen of the inhibitory effects of a drug on various kinases Attempts at identifying all of the kinases that might be affected by a drug of interest have, until recently, involved assays of the responses of only a small proportion of the large cohort of protein kinases known to be present in human cells. Consequently, over the past decade, certain off-target effects are likely to have eluded drug developers. This has begun to change with the advent of more systematic screening of a far larger portion of the kinases that might be affected by these inhibitors. One biotechnology company has developed an assay (Figure S16.6A–D) for measuring the binding affinities of a test drug for 442 distinct kinases that are located on various branches of the kinome tree (see Figure S16.5). As gauged by (A) © 2014 Garland Science this assay, two EGF receptor inhibitors—Iressa and Tarceva— are indeed found to bind preferentially the EGF-R tyrosine kinase, while staurosporine, which is thought to inhibit a wide spectrum of protein kinases of all types, is confirmed to exhibit a wide-ranging kinase-binding ability. (The binding affinity of a test drug for a kinase, as measured in this assay, has been found to predict the ability of this drug to inhibit the activity of the kinase.) This assay can be scaled up to enable the systematic screening of large numbers of kinase inhibitors, which greatly accelerates the rate at which candidate drugs of this class can be evaluated for their potency and their specificity in targeting a kinase of interest while having minimal effects on other closely related kinases (see Figure S16.6E). construction of fusion gene phage coat kinase gene protein gene test compound measure displacement of phage from bead by test compound using either a biological assay of phage particles (plaque assay) or PCR measurement of phage DNA kinase calculate Kd microbead phage coat amplifiable protein containing kinase fused to phage coat protein (B) (C) RTK (D) EGF-R RTK TK EGF-R RTK TK TK TKL CLK TKL TKL CLK CK MAPK CLK CK MAPK PKA GAK PKA GAK CAMK Iressa 10 µM CAMK Tarceva 1 µM 100 nM 10 nM generate kinase specificity profile CDK PKA CAMK Kd: CK MAPK CDK CDK immobilized inhibitor ATP-site dependent competition binding assays staurosporine 1 nM 140
  143. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Figure S16.6 Effects of kinase inhibitors on a wide spectrum of protein kinases The individual responses to various kinase inhibitors of an array of 156 distinct tyrosine and serine/threonine kinases have been tested by measuring the binding affinities of each inhibitor for each of these kinases. This test depends on the fact that almost all kinase inhibitors bind to the ATP-binding sites of targeted enzymes. (A) A cDNA encoding the kinase domain of a protein is cloned into a bacteriophage vector so that some of the phage capsid (coat) proteins (green) are synthesized as fusion proteins with the kinase (purple). An ATP analog that is known to bind the ATP-binding sites of many kinases (red pentagons) is then immobilized by being linked to microbeads (light blue). This allows phage particles to become linked via the fusion proteins in their capsids to the ATP analog on the beads. The assay then measures the ability of a test compound (e.g., a candidate tyrosine kinase inhibitor, yellow) to compete with the immobilized ATP analog (red), thereby blocking association of the phage to the bead. The number of phage particles released from the beads (determined by using a phage plaque assay or a polymerase chain reaction for the phage DNA) indicates the reduction in binding of the phage to the beads and therefore the binding affinity of the test compound for the kinase present in the fusion protein. The binding affinity is represented by the displacement constant Kd for the fusion protein, i.e., the concentration at which 50% of the phage is displaced from the beads. © 2014 Garland Science The remaining panels show the evolutionary relationships of the kinases in the form of kinome trees (see Figure S16.5) on which the binding affinities of tested inhibitors for the kinases are noted. Each kinase that showed a Kd of less than 10 μM is indicated by a red circle whose diameter varies inversely with the Kd (binding affinity constant). (B, C) These panels show the effects of Iressa and Tarceva, respectively, two inhibitors of the EGF receptor (EGF-R) that have already been licensed for clinical use. Both, reassuringly, show greater specificity for the EGF-R than for the 155 other kinases tested. However, Iressa also binds GAK (cyclin G–associated kinase; see Sidebar 5.1) at about a 10fold higher concentration, while Tarceva affects GAK at an even lower concentration, barely higher than that required to inhibit the EGF-R itself. (D) In contrast, staurosporine, a widely used experimental reagent that is thought to inhibit many kinases, is seen here to indeed bind a large number of these enzymes, some while it is present at subnanomolar concentrations. (E) The technology described here has been extended to systematically survey the effects of 38 kinase inhibitors, one of which (staurosporine) was analyzed in panel D. (A, from J.D. Griffin, Nat. Biotechnol. 23:308–309, 2005. B–D, courtesy of P.R. Zarrinkar and D.J. Lockhart, Ambit Biosciences; see also M.A. Fabian et al., Nat. Biotechnol. 23:329–336, 2005. E, from M.W. Karaman et al., Nat. Biotechnol. 26:127–132, 2008.) (E) 141
  144. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.10 Epidermal growth factor receptor expression levels predict little about a tumor’s susceptibility to receptor antagonists Elevated expression of the EGF-R is seen in a diverse array of tumors, including glioblastomas, head-and-neck squamous cell carcinomas, and bladder, kidney, esophageal, and lung cancers, often without associated amplification of the encoding gene. In many of these cancers, autocrine signaling loops driven by ligands such as TGF-α and amphiregulin are also activated. Tumors displaying elevated EGF-R expression and/or activity would seem to be obvious targets for treatment with receptor antagonists, specifically antireceptor monoclonal antibodies and tyrosine kinase inhibitors. However, the clinical results of such attempts to date have been disappointing. In colorectal cancers (CRCs), for example, the responsiveness of a tumor to monoclonal antibody therapy is not predicted by the extent of receptor overexpression. And while colorectal cancers as a class have been relatively unresponsive to treatment with low–molecular-weight tyrosine kinase inhibitors, such as Iressa and Tarceva, non-small-cell lung carcinomas have occasionally responded in a striking fashion (see Section 16.13). In the case of metastatic CRCs, the presence of a mutant K-ras allele in the genome of the corresponding primary tumors is a predictor of nonresponsiveness to anti-EGF-R MoAb therapy, © 2014 Garland Science and the direct demonstration of the absence of such an allele in a patient’s tumor is now a prerequisite in Europe for MoAb treatment of the tumor. Similarly, the presence in the primary CRC genome of mutant, activated B-raf and PI3K alleles (the latter involving exon-20 mutations) or inactivated PTEN alleles has also been associated with poor response to MoAb therapy, as measured by time-to-progression (interval between inception of therapy and evidence of worsening of symptoms and tumor growth) and overall survival (percentage of patients surviving at a given time, independent of other clinical responses). Conversely, elevated expression of amphiregulin and epiregulin—two EGF-R ligands—and the implied activation of autocrine signaling loops have been associated with increased time-toprogression and overall survival. Significantly, in the presence of a mutant, activated K-ras oncogene, the expression of these ligands was not predictive of a positive response to MoAb therapy, indicating that the effects of the K-ras oncogene dominate and overrule any other factors that might affect responsiveness to antibody therapy. In the case of anti-EGF-R monoclonal antibody therapies, the most useful indicator of a positive clinical response is not some biochemical or genetic marker but rather the skin rash developed by some of the patients under treatment (Figure S16.7). Taken together, this large body of clinical experience has not been reassuring for the advocates of rational drug therapy. Figure S16.7 Skin rash—a surrogate marker for treatment efficacy The responses of most solid tumors to anti-EGF receptor monoclonal antibody therapy have been modest. However, the minority of patients who do show significant clinical responses often develop an acne-like skin rash of the sort seen here. This rash may in some way reflect the importance of the EGF-R in maintaining normal skin (see Figure 16.19A), but the precise mechanistic reasons for its correlation with positive clinical responses and its utility as a surrogate marker of tumor-killing activity are obscure. (From K.J. Busam et al., Br. J. Dermatol. 144:1169–1176, 2001.) TBoC2 Supp24/S16.07 142
  145. Supplement to The Biology of Cancer, Second Edition by Robert A. Weinberg Supplementary Sidebar 16.11 Akt/PKB function is controlled by multiple upstream signals Recall that Akt/PKB activity is regulated by the actions of three enzymes (see Section 6.6). (1) PI3 kinase creates the PIP3 tethering site on the inner surface of the plasma membrane to which inactived Akt/PKB can attach. (2) PDK1, also tethered to PIP3, can then add a single phosphate group to Akt/PKB. (3) PDK2, another kinase, adds a second phosphate group to Akt/PKB, completing the activation of Akt/PKB and allowing Akt/PKB to phosphorylate a number of key regulatory proteins in the cell (see Table 6.3). “PDK2” was simply a functional designation, since the precise identity of this kinase molecule was long a mystery, indeed a bone of much contention. The elusive PDK2 was finally identified as the TORC2 complex, which is composed of mTOR, Rictor, GβL, and a number of other regulatory subunits that are not illustrated in Figure 16.41. This means that mTOR activity is critical for the activation of Akt/PKB and that suppression of © 2014 Garland Science TORC2 firing will cause cells to slow their growth and proliferation and even become susceptible to apoptosis. Inhibition of mTOR activity is also known to shut down ribosome biosynthesis and, under certain conditions, to induce autophagy (see Figure 9.37). In many types of cancer cells, we know that Akt/PKB activation depends on the hyperactivity of PI3 kinase or the inactivity of the PTEN phosphatase (both of which yield elevated levels of PIP3). The resulting Akt/PKB activation must benefit the cancer cells, since the activities of PI3K and PTEN are altered in a wide variety of human cancers (see Table 6.4). Still, changes in PI3K or PTEN function do not on their own suffice to activate Akt/ PKB, which depends also on mTOR function (see above). This logic dictates that mTOR must be highly active in many cancer cell types, enabling them to realize the benefits of PI3K activation or PTEN inactivation. 143

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