Cancer biology b7 4 lecures


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Cancer biology b7 4 lecures

  1. 1. 1 Cancer biology B7-4 Definitions Cancer is an abnormal growth of cells caused by multiple changes in gene expression leading to dysregulated balance of cell proliferation and cell death and ultimately evolving into a population of cells that can invade tissues and metastasize to distant sites, causing significant morbidity and, if untreated, death of the host. Tumor: any swelling or mass; classically tumor was one of the four signs of inflammation. In contemporary usage, the term is used as a synonym for neoplasm. Metaplasia: is an adaptive substitution of one type of adult tissue to another type of adult tissue under stress a more vulnerable type of tissue will be replaced by another more capable of withstanding stress. Dysplasia: An abnormality in cell size, appearance, with or without a disorganized growth pattern Neoplasia : Neoplasia is an abnormal type of tissue growth. Cells proliferate in an uncontrolled and mostly autonomous fashion (without external growth factor stimulation). Malignant: a descriptive term applied to neoplasms having an aggressive natural history, generally characterized by rapid and invasive growth and metastasis. Metastasis: discontinuous spread of a malignant neoplasm to distant sites
  2. 2. 2 Growth of Cancer Cells Cancer classification  There are >100 different types of cancers.  Arise from abnormalities in cell growth and division.  Originate from different types of normal cells.  Vary in rates of growth and ability to spread
  3. 3. 3 Classification of the cancer according to the morphology One of the most important aspects of the diagnosis of neoplasia is differentiation between benign and malignant tumors. The following are features that differentiate a malignant tumor from a benign tumor: 1. Malignant tumors invade and destroy adjacent normal tissue; benign tumors grow by expansion, are usually encapsulated, and do not invade surrounding tissue. Benign tumors may, however, push aside normal tissue and may become life threatening if they press on nerves or blood vessels or if they secrete biologically active substances, such as hormones, that alter normal homeostatic mechanisms. 2. Malignant tumors metastasize through lymphatic channels or blood vessels to lymph nodes and other tissues in the body. Benign tumors remain localized and do not metastasize. 3. Malignant tumor cells tend to be ‘‘anaplastic,’’ or less well differentiated than normal cells of the tissue in which they arise. Benign tumors usually resemble normal tissue more closely than malignant tumors do. The general features associated with benign and malignant tumors are: Benign Malignant Overall Growth Slow Rapid Local Growth By Expansion By Invasion Tissue Destruction Little Extensive Vessel Invasion None Frequent Metastases None Frequent Overall Effect on the Patient Usually Minor Very Significant: Often leading to death
  4. 4. 4 Some general morphologic features can often be used to differentiate between benign and malignant tumors. These are: Benign Malignant Mitoses Few Many Differentiation Good Often Poor Capsule Usually Present Absent Nuclear Chromatin Character Similar to Normal Increased Staining Intensity Classification of cancers based on cell/tissue origin Carcinomas: cancers of epithelial cells.  Epithelia: cell that cover surfaces, for example skin, lining of digestive tract. Glands also are epithelia.  Make up 90% of human cancers. Sarcomas: tumors of connective tissues.  Connective tissues lie below epithelia and hold things together or support the body. Some examples are muscle and bone.  Cancers are rare, because these cells do not reproduce very often. Leukemias and Lymphomas: Cancers of blood cells.  Leukemias are cancers of circulating blood cells or stem cells of the bone marrow.  Lymphomas are usually solid tumors in lymphatic organs (system that cleanses the blood).  Comprise 8% of all human cancers.
  5. 5. 5 Further classification of cancers (1) Site of origin: lung, breast carcinomas, Liver Hepatocarcinoma (2) Cell type: squamous cell carcinoma: cancer of flat epithelial cell  Skin cancer: 3 types o Basal cell carcinoma o Squamous cell carcinoma Account for 90% of skin cancers; cure rates 99%; rarely metastasize. o Melanoma--Cancer of pigment forming cells. Can metastasize rapidly. Fatal to 20% of patients. Cancers of glands: prefix adeno- Example adenocarcinoma Cancers of embryonic tissues: suffix -blastoma Neuroblastoma: Childhood cancer of neurons. Retinoblastoma: Childhood eye cancer. Classification of cancers according to its cell arising Can be monoclonal or polyclonal tumors
  6. 6. 6 Incidence of Cancer The 5 most common types of cancer account for about 80% of all cancers. They are Skin, Prostrate, Breast, Lung and Colon Cancer. From L. J. Kleinsmith, Principles of Cancer Biology. Copyright (c) 2006 Pearson Benjamin Cummings. Geographic Variation it differs from developed and developing countries
  7. 7. 7 Grade and Stage of Neoplasms Grading based on degree of differentiation and on estimate of growth rate (mitotic index)-- thought that less differentiated tumors more aggressive (too simple). Grade I-- 75% to 100% differentiation Grade II-- 50%-75% differentiation Grade III-- 25%-50% differentiation Grade IV-- 0%-25% differentiation Also based on amount of infiltration and amount of stromal tissue in and around tumor Staging Utilizing the T (tumor), N (involvement of LN) and M (+/- metastasis)-- TNM system Tumor Staging Four methods involved in staging; Clinical-- estimation of disease based on physical exam, clinical lab tests, x-ray films and endoscopic examination Radiographic staging-- evaluation of progression based on radiography Surgical staging-- direct exploration of extent of disease by surgery Pathologic staging-- use of biopsy to determine degree of spread, depth of invasion, and involvement of LN Stage I-- (T1N0M0) Primary tumor limited to the organ of origin. No evidence of nodal or vascular spread. Tumor usually can be removed by surgery. LTS (long term survival) is from 70-90% Stage II--(T2N1M0) primary tumor spread into surrounding tissue and LN immediately draining area. Tumor may be operable but not completely resectable. LTS 45-55% Stage III--(T3N2M0) primary tumor is large with invasion into deeper tissues. Not resectable. LTS 15--25% Stage IV-- (T4N3M+) Large primary tumor (>10 cm), invading adjacent tissues. Extensive LN involvement and distant metastases. LTS < 5%
  8. 8. 8 TNM System used to determine Tumor Stage and relation to Five-years survival rate. — Phenotypic characteristic of cancer cells There are several features that can be used to differentiate normal cells from malignant cells.  Invasion: Malignant cells do not respect tissue boundaries, and can be seen infiltrating or invading into surrounding structures.  Increased mitotic rate: Mitoses are rarely seen in normal tissues. Malignant cells will often have increased numbers of mitoses. Mitoses are typically counted 'per high power field'. More aggressive tumours typically have a higher mitotic rate; however these tumours are typically more sensitive to radiation.  Differentiation and Anaplasia: Normal cells are usually structured in a particular way that corresponds with their function. This is known as differentiation. Malignant cells may become less differentiated as part of their path to malignancy. This is known as anaplasia. o Well differentiated maligant cells show features similar to the parent tissue. For example, well differentiated adenocarcinoma cells will tend to form gland-like structures;
  9. 9. 9 well differentiated squamous cell carcinomas may show intercellular bridging or keratin formation. o Poorly differentiated cells have lost most of their resemblance to the parent tissue, which may be difficult to identify without special staining techniques. o Anaplastic cells have no resemblence to their parent tissue, and usually indicate a very aggresive malignancy. Anaplastic Features  Loss of normal tissue architecture: Normal cells are usually arranged in an orderly fashion. Epithelial cells often have polarity, with their nuclei at a specific location. Malignant cells lose this architecture and are arranged haphazardly.  Pleomorphism: Malignant cells may show a range of shapes and sizes, in contrast to regularly sized normal cells. The nuclei of malignant cells are often very large (often larger than the entirety of a normal cell) and may contain prominent nucleioli.  Hyperchromatic nuclei: The nuclei of malignant cells typically stain a much darker colour than their normal counterparts.  High nuclear-cytoplasmic ratio: The nuclei of malignant cells often take up a large part of the cell compared with normal cell nuclei  Giant cells: Some malignant cells may coalesce into so-called giant cells, which might contain the genetic material of several smaller cells. Cancer by H&E staining Cancer cell by Electron Microscope
  10. 10. 10 Hallmarks of Cancer There are six classical hallmarks of malignancy:  Immortality- Unlimited replicative capacity - normal cells may only multiply a set number of times before they become senescent (unable to divide further). Malignant cells circumvent this limit through activation of telomerase.  Sustained growth signals- Self sufficiency in growth signals - malignant cells are able to grow without an external stimulus to do so,  Bypass anti-growth signals- Lack of response to growth inhibition - this is often due to loss of tumour suppressor genes, which would normally put the growth of the cell on hold  Avoidance of apoptosis - normal cells trigger apoptotic pathways in response to uncontrolled growth signalling. Apoptosis is often suppressed by malignant cells to avoid this fate  Angiogenesis - malignant tumours must form new blood vessels in order to expand locally. Angiogenesis is also important for allowing malignant cells to metastasise  Invasion and Metastasis - malignant tumours invade surrounding normal tissues and may also spread throughout the body. Immortality: Continuous cell division, All organisms have a defined size and shape, both at the tissue and cellular level. Cancer cells are typically defined by their capacity to divide uncontrollably. In contrast to normal cells, cultured cancer cells have the capacity to dramatically exceed normal doubling times to almost indefinite levels.
  11. 11. 11 This clearly suggests that these cancer cells have bypassed / disrupted the senescence regulators within the cell and acquired the capacity for unlimited division. Telomeres are the aglets of chromosomes. Telomeres are repeat DNA sequences that protect the linear end of chromosomes. After every round of cell division, telomere lengths get progressively shorter, until it provokes the cell to stop dividing and enter senescence. Cancer cells prevent telomere shortening by producing the enzyme, telomerase, which keeps extending telomeres,thus preventing senescence. Cancer cells on the other hand, maintain their telomere lengths without any loss of DNA base pairs. The main strategy used by cancer cells to maintain telomere lengths is by activating an enzyme called telomerase. Almost 85-90% of all cancers have an active telomerase. Telomerases add non-coding, hexanucleotide repeats onto the ends of telomeric DNA, thus maintaining the required lengths above the critical threshold, preventing erosion and allowing unlimited replicative capacity. Unlike cancer cells, actively dividing normal cells have levels of telomerase that are extremely low or undetectable. Tumours circumvent senescence pathways by activating telomerases and therefore therapeutic strategies aimed at inhibiting telomerases will preferentially kill tumour cells and have no toxicity on normal cells. Telomeric DNA Telomeric DNAChromosomal DNA Cell division senescene
  12. 12. 12 However, there is some debate that senescence is an artifact of cell culture conditions and not a true representation the phenotype in the body (in vivo). Resolution of this debate will be useful in understanding how replicative potential and tumour progression are linked. Sustained growth signals No cell can survive in isolation. Every cell is part of a community, which forms a tissue or organ. Cell behaviour is almost always dependent on growth signals from the surrounding (mitogenic), which trigger cell division. These external growth factors (or ligands) bind to membrane- bound glycoprotein receptors that transmit the message via a series of intracellular signals that promote or inhibit the expression of specific genes. Examples of growth signals include diffusible growth factors, extracellular matrix proteins and cell-cell adhesion / interaction molecules. If these growth signals are absent, any typical normal cell will change to a quiescent state instead of active division. This dependence on exogenous growth factors is a critical homeostatic mechanism to control cell behaviour within a tissue. Cancer cells, on the other hand, generate mutant proteins (oncogenic proteins) which mimic these normal growth signals (proto-oncogenic proteins). Transformation of proto-oncogenes into oncogenes is brought about by several factors such as mutations, chromosomal rearrangements, viral insertion, gene amplifications etc. The consequence of oncogenic transformation is that tumour cells become independent of these external growth signaling factors in any normal tissue microenvironment. This acquired feature by tumour cells can be demonstrated empirically in vitro.
  13. 13. 13 There are three main cellular strategies used by cancer cells in achieving growth factor autonomy, based on the growth factor signaling pathway as shown in figure; a) Changes in extracellular growth signals b) Changes in transcelluar mediators of those signals (receptors) c) Changes in intracellular signaling messengers that stimulate proliferation. Bypass anti-growth signals The balance between cell proliferation and quiescence is brought about by a complex interplay between these two signaling pathways. Typically, anti-growth signals work in two distinct ways; a) Forcing actively dividing cells into the quiescent (G0) phase of the cell Extracellular Signalling transducers Transcriptional Factor Cell Proliferation protein Growth factor Receptor Growth Factor Intracellular
  14. 14. 14 cycle, which can be atemporary measure until there is a change in proliferative capacity (either a change in microenvironment conditions or there is a GF signal). b) Cells may be induced into a permanent post-mitotic (non-dividing) state as a result of development. For example the specific terminal differentiation of neurons or the denucleation state of mature erythrocytes Cancer cells, on the other hand, bypass or evade these anti-growth signals to enable their own growth and proliferation. For example, mutations in genes that normally inhibit cell proliferation would result in increased cell division. These tumour suppressor genes (TSGs) constitute a large group of genes that encode proteins whose normal role is to restrain cell division. Mutations in these genes lead to a loss-of- function and typically, both copies (alleles) of the gene need to be altered to enable tumour formation (unlike oncogenes, which are gain-of-function mutation). Avoidance of apoptosis Apoptotic cell death is part of normal growth and development. Tissue homeostasis is a balance between cell division and cell death, wherein the number of cells in that tissue is relatively constant. If this equilibrium is disturbed, the cells will either a) divide faster than they can die, resulting in cancer development. Cancer cells can bypass apoptosis in many ways. The most common method involves mutations of the p53 tumor suppressor gene resulting in the loss of proapoptotic regulators. More than 50% of all human cancers (and 80% of squamous cell carcinomas) show inactivation of the p53 protein. P53 is also known as the ‘guardian of the cell’ because of its pivotal role in cell response to stress.
  15. 15. 15 Extrinsic and intrinsic apoptotic pathways. 1) The extrinsic signals are triggered by binding of the ligand (e.g. CD95L) to its receptor (CD95). This activation of the receptor leads to the activation of FADD, which in turn activates DED. DED activation initiates apoptosis via initiator caspase 8, which leads to irreversible apoptosis either directly or through effector caspases (caspase-3). Active caspase-8 cleaves BID to tBID, which translocates to the mitochondrion to release of SMAC/DIABLO. SMAC/DIABLO sequesters IAPs resulting in apoptotic induction through caspase 3. 2) The intrinsic apoptotic pathway is initiated at the mitochondrion by diverse stimuli. a ) Irreparable DNA damage signaling through the p53
  16. 16. 16 proteins removes suppression of apoptosis by BCL2, leading to membrane permeabilization and the release of cytochrome c (Cyto c), SMAC/DIABLO, AIF (apoptosis- inducing factor) b ) Cytochrome c interacts with APAF1 to recruit and activate caspase 9, forming the apoptosome, which activates the downstream executioner caspases 3 and 7. AIF causes DNA degradation. Abbriviations; FADD - Fas-associated death domain; DED – Death Effector Domain; tBID – truncated BID;BID - [BH3 (BCL2 homology domain 3)-interacting agonist domain]; IAP - inhibitor of apoptosis; BCL2 - B- cell leukaemia/lymphoma-2; SMAC/DIABLO - second- mitochondrial-derived activator of caspases; APAF-1 - apoptosis protease activating factor-1.
  17. 17. 17 Angiogenesis Cell and tissues need oxygen and nutrients to survive and grow and therefore most cells lie within 100 μm of a capillary blood vessel. Under most conditions, cells that line the capillaries – the endothelial cells- do not grow and divide. However, certain conditions such as wound healing, trigger endothelial cell division and growth of new capillaries and this process is termed angiogenesis or neovascularisation. Tumours can also ‘turn on’ angiogenesis. In fact, it is a key transition step to convert a small, harmless cluster of mutant cells (an in situ tumour) into a large malignant growth 1-Tumour cells release pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which diffuse into nearby tissues and binds to receptors on the endothelial cells of pre-existing blood vessels, leading to their activation. 2- Such interactions between endothelial cells and tumour cells lead to the secretion and activation of proteolytic enzymes such as matrix metalloproteinases (MMPs) which degrade the basement membrane and extracellular matrix. 3- Degradation by basement membrane allows activated endothelial cells — which are stimulated to proliferate by growth factors-to migrate towards the tumors. 4- Integrin molecules cells such as vβ 3-integrin, help to pull sprouting the new blood vessels forwards 5- The endothelial cells deposit a new basement membrane and secrete growth factors such as, platelet-derived growth factor (PDGF), which attract supporting cells to stabilize the new vessel.
  18. 18. 18 Metastasis
  19. 19. 19 Solid tumours are usually part of normal tissues, and under optimal conditions, can invade adjacent tissues or pass out through the circulatory system to colonise distant sites in the body. These secondary tumours – metastases – are responsible for almost 90% of cancer-related deaths. This capacity of tumour cells to invade and metastasize is the final of the six hallmarks of cancer. Metastasis enables tumours to survive and grow in new environments where there are no restrictions of space or nutrients. The newly formed secondary tumours can contain cancer cells and also some normal support cells recruited from host tissue. Causes of Cancer (Carcinogenesis) Tumor Initiators vs Tumor Promotors vsWhole Carcinogen Initiators- cause minimum of two genetic mutations Promotors- are not mutagenic themselves and do not cause cancer, but stabilize mutations by inducing cell replication Whole carcinogen- has both properties (can induce and promote) Exogenous chemical, physical and biological carcinogens Humans vary in ability to cope with each different inducer Genetics Stress Level of exposure Chemical carcinogens Inorganic compounds; encountered in workplace environments Nickel, cadmium, arsenic Organic compounds ; Nitrosamines (smoked & pickled foods) tri- chloroethylene (cleaning), aromatic compounds (benzopyrenes & arylamines) generated from burning (cigarettes, coal and fuel)
  20. 20. 20 Natural compounds Aflatoxin A (mold)- liver cancer Hormones Medical drugs Physical Carcinogens Energy rich radiation UVB is a skin carcinogen and effects augmented by UVA Gamma irradiation (x-rays) Radioactive elements Radon Uranium/plutonium Iodine Biological Carcinogens Viruses DNA and retroviruses (not RNA viruses) HPV and HIV and EBV and Herpes and Hepatitis B Bacteria Rare to cause cancer (link between Helicobacter pylori infection and stomach cancer may be due to chronic inflammation) Endogenous Carcinogens Involved in cancer development through modulation of the response to exogenous carcinogens Also through strictly endogenous pathways: Normal metabolism- generation of nitrosamines, aromatic amines, reactive aldehydes and reactive O2 species Level of these dependent upon diet, exercise DNA repair mechanisms- damage all the time- repair
  21. 21. 21 effected by age or cells removed by apoptosis- if the mechanisms affected then cancer may arise Recognition by immune response (immune surveillance) Chronic infection (replication of cells [liver]) Predisposition to cancer -involves interaction of genetic and environmental factors -xeroderma pigmentosum (extreme sensitivity to light and incidence of skin cancer of 100%) -autosomal recessive trait (homozygous recessive) -defect in DNA repair -defects in ability to metabolize foreign chemicals (xenobiotics) especially those that are carcinogenic �Avoidability of cancer *life style accounts for 80% a cancers and therefore can be avoided *ideal life style • do not smoke or drink • should eat a diet low in fat, rich in fiber and yellow vegetables • should protect from hazardous chemicals in work and home and avoid unneeded x-rays, avoid excessive exposure to sunlight • woman should do above and have at least one child early in reproductive life and avoid multiple sex partners *cannot avoid pollution or infections.
  22. 22. 22 Angiogenesis Development of a functional vasculature is a key event in normal embryonic development as well as in the adult for such things as wound healing, corpus luteum angiogenesis during the female reproductive cycle, and development of the placenta. The process of new blood vessel formation from mesodermal stem cells during embryonic development is called vasculogenesis. Angiogenesis, by contrast, is the term used to describe development of new blood vessels from pre-existing ones. This is the process that takes place during wound healing, the reproductive cycle, and in tumors. In growing tumors, endothelial cells that will form the rudiments of new blood vessels may proliferate 20 to 2000 times faster than normal tissue endothelium in the adult. Initiation of the angiogenesis response is triggered by several factors. Among these are VEGF family members, basic FGF (bFGF or FGF-2), PDGF, angiopoietins, and factors that facilitate blood vessel formation by modulating extracellular matrix (ECM) production or differentiation of cell types involved in blood vessel formation. These latter factors include TGF-b, avb3 and avb5 integrins, ephrins, and plasminogen activators. the growing tumors elicited the proliferation of new capillaries in the host tissue, indicating the release of a diffusible substance by the tumor that stimulates capillary growth. This factor was called tumor angiogenesis factor (TAF). Folkman and colleagues showed that tumor cells transplanted into the cornea of rabbits initially grew slowly, but after about a week, small capillaries began to grow outward from the iris toward the tumor and when the capillaries reached the tumor, it began to grow rapidly. A wide variety of tumors have been examined for TAF activity, and many tumors have been found to contain it. The ability to
  23. 23. 23 induce angiogenesis, however, is not restricted to neoplastic cells. Angiogenesis can also be induced by spleen lymphocytes, thymocytes, peritoneal macrophages, and testicular grafts from newborn mice and by leukocyte invasion of the cornea. It is now known that the induction of capillary growth by tumors is, in fact, the result of a combination of factors. As noted above, angiogenesis is also a normal process by which new blood vessels are formed, for example, in development of the placenta, in vascularization of developing organs, and in wound healing. Under these conditions, however, angiogenesis is highly regulated, being turned on for specific periods of time and then shut off. It is an unregulated form of angiogenesis that occurs in tumors and in certain other diseases, such as arthritis, age-related macular degeneration (AMD), diabetic retinopathy (DR), and hemangiomas. A number of steps are required for angiogenesis to occur: (1) local dissolution of the subendothelial basal lamina of the existing vessels; (2) proliferation of endothelial cells; (3) migration of endothelial cells toward the angiogeni stimulus; and (4) laying down of a basal lamina around the nascent capillary. Different angiogenesis factors modulate different parts of this cascade. For example, FGFs and VEGF are directly mitogenic for endothelial cells; TGF-b stimulates ECM deposition to help form a basal lamina; and angiogenin may help create new ‘‘tracks’’ for vessel formation by ribonucleolytic action. The first purification of an angiogenesis factor was based on affinity of such factors for heparin and this led to the identification of basic and acidic FGFs as angiogenesis factors. Since then many others have been isolated and characterized, a number of such factors having been shown to be produced and secreted by human tissues. For example, VEGF is
  24. 24. 24 produced by human gliomas and epidermoid carcinoma cells. In some cases, angiogenesis factors are found in the urine or effusion fluids of cancer patients and their presence relates to conversion of hyperplasia to neoplasia and to tumor progression. Both tumor cells themselves and the surrounding stroma can produce angiogenic factors. Indeed, there is much evidence to suggest that neovascularization or conversion to the ‘‘angiogenic phenotype’’ is involved in tumor progression. Most cancers in humans are of epithelial origin and may grow slowly and remain localized (in situ) for many years before they become invasive and metastatic. Evidence suggests that part of this change from in situ carcinoma to invasive malignant cancer involves neovascularization of the tumor. There are data indicating that tumors of 1 to 2mm in diamter can persist in tissue without a tumor-derived vasculature. Epithelial cancers do not develop normal vascular beds like normal tissues and depend to a large extent on diffusion of oxygen and substrates for growth. When tumor cells are too far away from the capillary blood supply for diffusion to provide the needed nutrients the cells may die. This explains why the core of large solid tumors is often necrotic. As long as the tumors remain small, they can obtain sufficient nutrients by diffusion; as they grow and progress to a more malignant cell type, however, this process becomes limiting. At that point, tumors may be stimulated to release angiogenic factors that induce capillary outgrowth from the host’s surrounding normal tissues into the tumor. As noted above, tumor vascular beds are structurally and functionally abnormal. The vascular system in tumors is disorganized, tortuous, and dilated, leading to chaotic blood flow and variable regions of hypoxia. Thus, although full vascularization of the tumor does not occur, it does provide nutrients for their growth. Since this process of angiogenesis is believed
  25. 25. 25 to be part of the process involved in converting in situ carcinomas to aggressive malignant tumors, blocking the process could inhibit or significantly slow this conversion. This concept led to a search for antiangiogenic agents, some of which are described below. Vascular Endothelial Growth Factor Vascular endothelial growth factor (VEGF) appears to play a critical rate- limiting role in physiological angiogenesis. It is also important in pathological angiogenesis, including that associated with tumor growth and invasion. There are a number of members of the VEGF family, including VEGFs A, B, C, and D, and placental growth factor (PLGF). VEGFA is a key regulator of blood vessel growth and development, whereas VEGFC and D regulate lymphatic angiogenesis (see below). VEGFA is mitogenic for ECs derived from arteries and veins and acts as a survival factor for them in vitro and in vivo. It does so by activating the PI3K-Akt signal transduction pathway and by inducing the expression of the anti-apoptotic proteins Bcl-2 and A1. VEGF also acts as a vascular permeability factor and its unopposed action causes vessel leakiness, which is part of the pathophysiology of AMD and DR. The VEGFA gene has eight exons, and alternative splicing produces four different isoforms: VEGF-121, -165, -189, and -206 (containing those numbers of amino acids). VEGF-165 is a heparin-binding form and plays a key role in EC mitogenesis, which is significantly decreased when the heparin-binding domains are deleted. VEGF-121 is a freely diffusible form and VEGF- 189 and VEGF-206 are sequestered in the ECM. VEGF-165 is secreted by cells, but a significant amount remains bound to cell surfaces and the ECM. Hypoxia plays a critical role, via HIF-1a induction, in enhancing VEGF gene expression.
  26. 26. 26 Several growth factors and oncogene proteins up-regulate VEGF gene expression. Stimulating growth factors include EGF, TGF-a, TGF-b, keratinocyte growth factor, IGF-1, FGF, and PDGF. Inflammatory cytokines including IL- 1a and IL-6 also induce expression of VEGF in synovial fibroblasts and some other cell types. Moreover, the Ras and Myc oncogenic pathways up-regulate VEGF gene expression. In this latter case of oncogene-mediated angiogenesis, the repression of the critical anti-angiogenic factor thrombospondin-1 (Tsp-1) is key. Ras induces the sequential activation of PI3K, Rho, ROCK, and Myc. Myc in turn represses Tsp-1 gene expression. In addition, Ras can activate VEGF expression through activation of the Raf-Mek- Erk-AP1 pathway. These data support the concept that angiogenesis is under tight regulatory control in normal tissues through a baseline expression of angiogenesis inhibitors such as Tsp-1. Loss of this regulatory control is what occurs in cancers. The data also suggest that development of agents that mimic Tsp-1 could provide a new approach to anti-angiogenic therapy for cancer. VEGFA signals through two related receptor tyrosine kinases, VEGFR-1 and VEGFR-2. A third receptor, VEGFR-3 (Flt-4) binds VEGFC and VEGFD. VEGFR-1 (FLT-1) is up-regulated by H1F-1a and binds VEGFA, VEGFB, and PLGF. VEGFR-1 activation induces expression of matrix metalloproteinase-9 (MMP-9) in lung ECs and facilitates lung cancer metastasis. VEGFR-2 (KDR or Flk-1) is the major mediator of the mitogenic and permeability effects of VEGF. VEGFA, by its binding to VEGFR-2, induces EC proliferation via the Raf-Mek-Erk pathway and increases EC survival via the PI3K-Akt pathway.
  27. 27. 27 VEGF mRNA expression is up-regulated in a wide array of human cancers, including, perhaps somewhat surprisingly, hematopoietic malignancies. Antibodies to VEGF and small-molecule VEGFR inhibitors block human tumor xenograft growth in nude mice. As noted above, cancer cells are the major source of VEGF production in tumors, but the tumor stroma also produces VEGF, thus there are at least two targets for anti-angiogenic therapy. A number of clinical trials are under way with anti-VEGF agents (discussed below).
  28. 28. 28 Platelet-Derived Growth Factor The platelet-derived growth factor (PDGF) family has angiogeneic effects in vitro and in vivo. The four PDGF polypeptides PDGF-A, -B, -C and - D can form homodimers and heterodimers upon ligand binding. Of these, PDGF-BB is one that plays a key role in angiogenesis and is expressed in a number of cell types including ECs and many tumors. PDGF-BB acts via the PDGF-receptor b to enhance pericyte proliferation and migration. PDGF-BB also up-regulates VEGF expression in vascular smooth muscle cells, promoting EC proliferation and survival. Thus, anti-PDGF approaches to therapy may provide a way to do two things: (1) inhibit EC proliferation and survival, and (2) decrease formation and stabilization of an EC-friendly environment provided by pericytes and vascular smooth muscle cells. Stimulators of Angiogenesis VEGF= vascular endothelial growth factor •Growth factors: FGF, EGF, PGF, PDGF, GCSF •Cytokines: IL 8, TNFα, TGFα •Small Molecules: adeonosine, nicotinamide, prostoglandins
  29. 29. 29 The angiopoietins The angiopoietins (Ang-1 and Ang-2) were discovered as ligands for the Tie family of receptor tyrosine kinases that are selectively expressed in the vascular endothelium. Ang-3 and Ang-4 have also been discovered but are less well characterized than Ang-1 and Ang-2. Studies in gene knockout mice have defined many of the functions of the angiopoietins and their receptors. Mouse embryos lacking Ang-1 or Tie 2 develop a fairly normal vasculature; however, ECs in such embryos fail to associate properly with the underlying stroma, leading to defects in heart vasculature. Thus, Ang-1, acting with Tie 2 receptors, is thought to facilitate EC– stromal interactions. Overexpression of Ang-1 by transgene expression results in hypervascularization in skin, mostly due to increased vessel size. This is in contrast to VEGF overexpression, which leads to increased vessel number. Combining the two in transgene overexpression experiments leads to profound hypervascularity. Another contrast between Ang-1 and VEGF is that VEGF expression by itself produces leaky vessels, but Ang-1 plus VEGF produces more mature, non-leaky vasculature. Thus, both VEGF and Ang-1 appear to be required in normal angiogenesis. Ang-2 was found on the basis of its homology to Ang-1 in cloning experiments. But Ang-2 has turned out to be a Tie 2 antagonist and is involved in vasculature remodeling. This concept is supported by experimental data from the remodeling vasculature in the ovary and in Ang-2 gene knockout experiments in mice. It is also supported by Ang-2-mediated vessel remodeling in tumors, where Ang-2 expression correlates with host vessel destabilization that allows tapping into the host’s blood supply and facilitating VEGF-mediated endothelial proliferation. Ang-1 and Ang-2 are expressed in tumor cells and play a role in tumor angiogenesis. Ang- 3, by contrast, inhibits tumor
  30. 30. 30 angiogenesis and blocks pulmonary metastasis in an experimental animal lung carcinoma model.388 Angiogenesis Inhibitors A large number of potential therapeutic targets that could inhibit tumor angiogenesis have been identified. They can be divided into a number of subcategories: (1) inhibitors of proangiogenic factors (VEGF, Ang-1, bFGF, PDGF) or their receptors; (2) protease inhibitors (MMPs) that block vascular remodeling; (3) inhibitors of ECM production or cell– ECM adhesion needed for vessel stabilization (TGF-b, aVb3 and avb5 integrins); (4) natural inhibitors (thrombospondin, angiostatin, endostatin); and (5) agents that block HIF-1a production.
  31. 31. 31 Inhibitors of Proangiogenic Factors The most common proangiogenic factor implicated in cancer growth is VEGF. It is mitogenic for endothelial cells and facilitates their survival. It is also a permeability factor, causing vessels to leak, and it is expressed in a high percentage of human tumors. Anti-VEGF agents inhibit in vivo tumor growth in a number of animal and xenograft tumor models. Inhibitors of VEGF action include antibodies to VEGF or its receptors, RNA aptamers, VEGF-Trap (a decoy receptor based on VEGFR-1 and VEGFR-2 fused to an Fc segment of IgG1), and small-molecule inhibitors of VEGF receptor-mediated signal transduction. Some tumors are more sensitive than others to anti-VEGF agents. For example, the Wilms’ renal tumor is very sensitive to anti- VEGF antibody, whereas human neuroblastoma xenografts are only moderately sensitive and metastases are still formed. The reason for this relative resistance is that neuroblastomas more tenaciously hang onto blood vasculature co-opted from surrounding tissues than do Wilms’ tumors. Co-option of pre- existing host blood vessels occurs early in tumor development in a number of cancers. Later on, as tumors grow and become hypoxic, tumors express VEGF and other proangiogenic factors and neoangiogenesis is induced. Co-opted vessels then regress. While persistent existence of co-opted vasculature appears to be the resistance mechanism in experimental neuroblastomas, high doses of VEGF-Trap lead to tumor regression, suggesting that this agent also blocks tumor utilization of coopted vessels. Inhibitors of other proangiogenic factors such as PDGF, FGF, and EGF are also under development and some of these are in clinical trial.
  32. 32. 32 The angiopoietins Ang-1 and Ang-2 have also been shown to regulate tumor angiogenesis. As noted above, Ang-1 activates the receptor tyrosine kinase Tie-2, resulting in activation of the PI3K-Akt pathway and promoting endothelial cell survival. Ang-2 is the naturally occurring antagonist of this Ang-1 effect. An effect of Ang-2 is to cause vessel destabilization, thus the ratio of Ang-2 levels to Ang-1 may initiate tumor angiogenesis. However, there is evidence that Ang-1 inhibits angiogenesis in human colon cancer xenografts in nude mice. These effectors may have different effects in different cancers. Metalloproteinases Remodeling of the ECM by tissue proteases is an initiating event in vascular invasion and angiogenesis. The family of matrix metalloproteinases (MMPs) is key to this remodeling, as evidenced by the fact that mice deficient in MMP2 and MMP9 have reduced angiogenesis and decreased tumor progression in vivo. There are also endogenous tissue inhibitors of metalloproteinases (TIMPs) that regulate the action of MMPs and have an anti-angiogenic mechanism. For example, TIMP3 has been shown to inhibit MMP action and to block the binding of VEGF to VEGFR-2, thus blocking VEGF’s downstream signaling and angiogenesis in mouse tumor in vivo. -Cleavage of ligand-binding domains of FGFR1 and uPAR (inhibits FGFR signaling and uPA localization) -Inhibition of MMP-2 binding to αVβ3 integrin by release of MMP-2 PEX domain -Generation of antiangiogenic factors as angiostatin from plaminogen endostatin, tumostatin, arrestin, and canstatin from type XVIII and IV collagen
  33. 33. 33 Integrins Endothelial cell (EC) adhesion molecules are key to EC–extracellular matrix interactions required for capillary tube formation. The integrins aVb3 and aVb5 are adhesion factors involved in this. As such, they are attractive targets for angiogensis inhibitors. Neoangiogenic blood vessels in many species, including humans, express aVb3, but normal quiescent vasculature does not express significant amounts. Expression of both aVb3 and aVb5 is up-regulated in cancer cells. Antagonists to aVb3 are potent angiogenesis inhibitors, and they include monoclonal antibodies, synthetic peptides, small organic molecules, and antisense RNA to shut off aVb3 expression. Endogenous Inhibitors Thrombospondin is an endogenous factor, which when added in soluble form to a culture of ECs inhibits their proliferation. This effect may result from thrombospondin’s ability to bind TGFb and to modulate protease activity. Low thrombospondin levels in patients with invasive urinary bladder cancer have been associated with increased recurrence rates, high microvessel density, and decreased overall survival. Two other members of the endogenously produced anti-angiogenic proteins are angiostatin and endostatin. Angiostatin is an internal polypeptide fragment of plasminogen, and endostatin is a proteolytic fragment of collagen XVIII. These two anti-angiogenic fragments were discovered in Judah Folkman’s lab and have shown anti-angiogenic activity in a number of prelinical models. They have also been tested for activity in clinical trials with mixed results. Their mechanism of action isn’t totally clear, but endostatin appears to act by binding to aV- and a5- integrins on the surface of ECs.
  34. 34. 34 HIF-1a Activation of HIF-1a by hypoxia or other stimulatory factors leads to enhanced expression of a number of genes, including VEGF. Ironically, at least for cancers at an early progressing stage, anti-angiogenic therapy for cancer may actually increase HIF-1a expression, leading to increased expression of a number of HIF-1a-activated genes that foster increased tumor cell proliferation, survival, invasion, and metastasis. Increased metastatic dissemination of human melanoma xenografts has been observed after subcurative radiation treatment, most likely through a radiation-induced increase in hypoxic cells and hypoxia-induced up- regulation of urokinase-type plasminogen activator receptors. This compensatory tumor response to lower blood flow and increased hypoxia may also facilitate the development of drug-resistant cancer cells. Thus, a combination of antiendothelial agents plus anti-HIF-1a drugs is an attractive therapeutic approach. Anti-HIF-1a agents could prevent a compensatory turn on of genes favoring tumor progression and also prevent hypoxia-driven selection of resistant cells.
  35. 35. 35 Cancer invasion and metastasis Metastasis is the process by which a tumor cell leaves the primary tumor, travels to a distant site via the circulatory system, and establishes a secondary tumor. Tumor cells can spread by direct extension into a body cavity, such as the pleural or peritoneal space, or the cerebrospinal fluid. In these cases, tumor cells released into the body space can seed out onto tissue surfaces and develop new growths where they become embedded. Examples of cancers that spread in this way are lung cancers that enter the pleural cavity, ovarian cancers that shed cells into the peritoneal cavity, and brain tumors that shed cells into the cerebrospinal fluid. Tumor cells metastasize by invading blood vessels or lymphatic channels. Although it has frequently been said that carcinomas metastasize primarily through the lymphatic system and sarcomas through the blood vessels, this distinction is somewhat arbitrary, since the blood and lymph systems communicate freely, and it has been shown that cancer cells that invade lymphatic channels enter the bloodstream and vice versa. Capillaries, venules, and lymph vessels offer little resistance to penetration by tumor cells because of their thin walls and relatively ‘‘loose’’ intercellular junctions. Arteries and arterioles, by contrast, are surrounded by dense connective tissue sheaths made up of collagen and elastic fibers, and hence are rarely invaded by tumor cells. The mechanisms for invasion of tumor cells through tissue barriers and into blood and lymphatic vessels are not well understood, but they appear to involve both mechanical and enzymatic processes. As a tumor grows, the pressure exerted on surrounding tissue tends to force tumor cells between intercellular spaces. It is unlikely that this process, in itself, could explain the penetration of cancer cells through tissue barriers such
  36. 36. 36 as basement membranes. For this to occur, the release of certain degradative enzymes appears to be necessary. Indeed, tumors are known to contain and secrete a variety of proteolytic enzymes that may be involved in this step. The enzyme activities released by growing tumors destroy surrounding cells and degrade tissue barriers, allowing tumor cells to penetrate. After tumor cells invade the lymphatic or vascular vessels, they may form a local embolus by interaction with other tumor cells and blood cells and by stimulating fibrin deposition. Individual cells or clumps of cells are then shed from these sites and spread to distant organs by the lymph or blood vessels. Tumor cells that enter the lymphatic system travel to regional lymph nodes in which some tumor cells may be trapped and produce a metastatic growth. However, all the tumor cells are not necessarily trapped or ‘‘filtered out’’ in the first few lymph nodes draining an area of tissue containing a cancer cells The presence of tumor cells in blood does not invariably mean that distant metastases will form. The vast majority of circulating tumor cells shed from solid tumors do not survive in the blood, and only about 0.1% live long enough to form a distant metastasis. During circulation in the vascular system, tumor cells can undergo a variety of interactions, including aggregation with platelets, lymphocytes, and neutrophils, which lead to the formation of emboli that can become lodged in the capillary bed of a distant organ. These clumps of cells adhere to the capillary endothelium and elicit the formation of a fibrin matrix that appears to favor the survival of the cancer cells. A number of years ago, adherence of cancer cells to capillary endothelium and subsequent thrombus formation are involved in metastasis. The adhesion of tumor cells to capillary endothelium in susceptible organs appears to damage the vessel walls and to lead to the accumulation of neutrophils that may penetrate the spaces between endothelial cells and
  37. 37. 37 open up a channel through which tumor cells can also penetrate. Moreover, platelets that aggregate at the site of the thrombus release mediators, such as histamine, which promote capillary permeability, allowing the migration of tumor cells through the endothelium. The role of platelets in this process is implied from several lines of evidence. Many murine tumors aggregate platelets in vitro and in vivo. Addition of fibroblasts to a tumor cell inoculum enhances platelet aggregation and the number of metastases, whereas induction of thrombocytopenia in the host animal or treatment with aspirin, at doses that decrease platelet aggregation, decrease tumor metastases. Aggregation of platelets and release of their contents can be induced by a number of factors, including collagen, thrombin, and arachidonic acid. Platelets accumulate in areas of endothelial cell regeneration following trauma, and platelet-released factors have a mitogenic effect on a number of different cell types, including endothelial cells. Elastase and collagenase are released from platelets, thus altering the connective tissue of the vessel wall. Platelet aggregation also produces an increase in serum thrombin, which in turn increases the amount of fibrin deposited on the endothelial wall. This deposition of fibrin stimulates the release of plasminogen activator from neutrophils, macrophages, and other cells to induce fibrinolysis through plasmin, thus generating more proteolytic activity in the area of the tumor thrombus. Once tumor cells migrate through the vascular wall, they quickly establish themselves in the new environment and begin to proliferate. This is fostered by the release of angiogenesis factors from tumor cells or host lymphocytes and macrophages that promotes vascularization of the nidus of tumor cells. In the presence of platelets or platelet-released factors, the mitogenic activity of angiogenesis factors for endothelial cells growing on a collagen substratum is greatly enhanced. Thus, the local aggregation of platelets in the area of a tumor cell–
  38. 38. 38 containing thrombus activates a whole cascade of events that can promote the extravasation and new growth of tumor cells at a metastatic site. Somewhat paradoxically, the presence of immune lymphocytes that recognize tumor cells may enhance the colonization of metastatic sites. Tumor Invasion as the first step of metastasis cascade;
  39. 39. 39 1-Translocation of cells across extracellular matrix barriers 2- Lysis of matrix protein by specific proteinases 3- Cell migration Components of invasion a) Matrix degrading enzymes -Required for a controlled degradation of components of the extracellular matrix (ECM) -The proteases involved in this process are classified into serine-, cysteine-, aspartyl-, and metalloproteinase (MMPs). Matrix metalloproteinases (MMP) -16 members, subdivided into 4 groups, based on their structural characteristics and substrate specificities -Soluble and secreted groups; collagenase, gelatinase and stromelysins -Membrane type (MT-MMP) group are anchored in the plasma membrane -A zinc ion in the active centre of the protease is required for their catalytic activities.
  40. 40. 40 MMPs have roles in many steps of tumor progression
  41. 41. 41 Interaction between tumour cells and the surrounding connective tissue b) Cell attachment 1-Integrin: cell-matrix adhesion 2- E-cadherin/catenin adhesion complex: cell-cell adhesion
  42. 42. 42 1) Integrin -Heterodimeric transmembrane receptors consists of a and b subunits -Function to provide interactions between cells and macromolecules in the ECM -Integrin can affect the transcription of MMP genes Integrin signalling pathways to mediate the invasion 2) E-cadherin and catenin complex -Most important cell-cell adhesion molecules -Reduce expression of E-cadherin and catenin increase the invasiveness of tumor cells
  43. 43. 43 c) Cell migration 1. Small Rho GTPase family 2. Motility promoting factors
  44. 44. 44 Cancer genetics Definition A continuous process in which multiple alterations occur in genes that control cell division and differentiation that leads to cancer. These genetic alterations are referred to as mutations, which are changes in the normal DNA sequence of a particular gene. Mutations may include deletions, chromosomal translocations, inversions, amplifications, or point mutations. Description Nearly all cancers originate from a single cell and are the result of genetic alterations, although most of them are not inherited. Individuals who are genetically predisposed to a particular cancer will not necessarily develop the disease in the absence of somatic mutations. Somatic mutations occur in non-sex determining cells, meaning they will not be passed on to offspring. These mutations can be influenced by environment and other causes, such as an individual's habits (i.e. smoking). A single genetic error or mutation in a cell does not typically induce malignancy; instead it develops after a series of mutations over a period of time. Oncogenes and tumor suppressor genes The continuous cell proliferation in cancer may either be due to over-activation of a specific gene that promotes cell division or due to the improper functioning of a gene that will otherwise restrain growth. Genes that promote cell division are proto-oncogenes—positive regulators of cell division. Overexpression of proto-oncogenes results in uncontrolled cell growth. Genes that suppress or restrain growth are tumor suppressor genes and loss of their function results in unregulated cell division. An alteration in the function of genes in each of these classes is due to a change, or mutation, in the DNA within the cell. The different types of mutations include point mutations, amplifications, and chromosomal alterations. Point mutations A point mutation is a single nucleotide change in a DNA strand. This may alter the genetic code, thus altering the function of the protein. In the above example, a point mutation in the thymine base of the second triplet would look like: CAG-AAA-CCA-GCG. Changing the code from TAA to AAA could alter the function of a protein and thus could cause a predisposition to disease such as cancer. One example of a point mutation that has been identified is the ras family of oncogenes (such as H ras, K-ras, N-ras ), present in 15% of all human cancers. DNA amplification
  45. 45. 45 Another mechanism of oncogene activation—DNA amplification—results in an increase in the amount of DNA in the cell. A large number of genes are amplified in human cancers. Chromosomal alteration Chromosomal alteration may involve translocations and is often seen in lymphoid tumors. Translocation is the transfer of one part of a chromosome to another chromosome during cell division and may involve transcription factors (i.e., nuclear factors), signal transduction proteins, and cellular regulatory molecules. DNA repair genes In addition to oncogenes and tumor suppressor genes, DNA repair genes may lead to cancer. DNA repair genes are capable of correcting the errors that occur during cell division. Malfunction of these repair genes, either through inherited mutation or acquired mutation, may affect cell division resulting in malignancies. RNA and DNA viruses Malignancies are known to be associated with RNA or DNA viruses. DNA viruses are implicated in human malignancies more often than RNA viruses. Human papilloma virus is related to human cervical cancer , and hepatitis B and C are related to hepatocellular carcinoma (liver cancer). Mendelian cancer syndromes Some forms of cancer are classified as hereditary cancers, or familial cancers, because they follow the Mendelian pattern of inheritance, the more familiar form of inheritance in which genetic material is passed from the mother or father to the offspring during reproduction. Cancer-related genes may be inherited as autosomal dominant, autosomal recessive, or x- linked traits. . Some of the known tumor suppressor genes responsible for familial cancer syndromes are BRCA1, which is associated with breast, ovarian, colon, or prostate cancers. Complex inherited cancer syndromes Several types of cancer do not follow a simple Mendelian pattern of inheritance. In many instances, environmental factors can affect the outcome of disease expression in conjunction with genetic alterations. One such example is lung cancer. Cigarette smoke is an environmental factor that may result in lung cancer for individuals frequently exposed to the toxins in the smoke. However, individuals who possess a gene that predisposed them to lung cancer are genetically more susceptible than the rest of the population to these toxins, and
  46. 46. 46 may develop cancer with less exposure or none at all. Individuals without a predisposing gene may not develop the cancer as readily. Genetic testing Genetic testing examines the genetic information contained inside an individual's DNA, to determine if that person has a certain disease, is at risk to develop a certain disease, or could pass a genetic alteration to his or her offspring. Individuals who seek genetic testing are usually family members believed to have a predisposition or susceptibility to cancer as known from the personal family medical history. The identification of genes associated with certain types of cancers such as BRCA1, BRCA2, HNPCC (colon cancer), and RB improves the accuracy of DNA testing to predict cancer risk. Often a positive test result indicates that the individual does carry the abnormal gene and is more likely to get the disease for which the test was performed than the rest of the population.