Cancer biology B7-4
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
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: discontinuous spread of a malignant neoplasm to distant sites
Growth of Cancer Cells
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
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
The general features associated with benign and malignant tumors are:
Overall Growth Slow Rapid
Tissue Destruction Little Extensive
Vessel Invasion None Frequent
Metastases None Frequent
Overall Effect on the
Very Significant: Often
leading to death
Some general morphologic features can often be used to differentiate
between benign and malignant tumors. These are:
Mitoses Few Many
Differentiation Good Often Poor
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.
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
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
Grade and Stage of Neoplasms
based on degree of differentiation and on estimate of growth rate (mitotic
index)-- thought that less differentiated tumors more aggressive (too
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
Utilizing the T (tumor), N (involvement of LN) and M
(+/- metastasis)-- TNM system
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
Surgical staging-- direct exploration of extent of disease by
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%
TNM System used to determine Tumor Stage and relation to Five-years
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
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;
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.
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
Cancer by H&E staining
Cancer cell by Electron Microscope
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
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
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.
This clearly suggests that these cancer cells have bypassed / disrupted the
senescence regulators within the cell and acquired the capacity for
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
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
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.
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
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
Cell Proliferation protein
Growth factor Receptor
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.
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
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
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
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
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.
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
Level of exposure
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)
Aflatoxin A (mold)- liver cancer
Energy rich radiation
UVB is a skin carcinogen and effects augmented by UVA
Gamma irradiation (x-rays)
DNA and retroviruses (not RNA viruses)
HPV and HIV and EBV and Herpes and Hepatitis B
Rare to cause cancer (link between Helicobacter pylori
infection and stomach cancer may be due to chronic
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
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.
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
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
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
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.
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.
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).
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
Stimulators of Angiogenesis
VEGF= vascular endothelial
•Growth factors: FGF, EGF,
PGF, PDGF, GCSF
•Cytokines: IL 8, TNFα, TGFα
•Small Molecules: adeonosine,
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
angiogenesis and blocks pulmonary metastasis in an experimental animal
lung carcinoma model.388
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.
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.
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.
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
-Generation of antiangiogenic factors as angiostatin from plaminogen
endostatin, tumostatin, arrestin, and canstatin from type XVIII and IV
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.
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.
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.
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
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
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
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–
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;
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
-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
-A zinc ion in the active centre of the protease is required for their
MMPs have roles in many steps of tumor progression
Interaction between tumour cells and the surrounding connective
b) Cell attachment
1-Integrin: cell-matrix adhesion
2- E-cadherin/catenin adhesion complex: cell-cell adhesion
-Heterodimeric transmembrane receptors consists of a and b subunits
-Function to provide interactions between cells and macromolecules in
-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
c) Cell migration
1. Small Rho GTPase family
2. Motility promoting factors
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
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
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.
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.
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 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-
. 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
may develop cancer with less exposure or none at all. Individuals without a predisposing
gene may not develop the cancer as readily.
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