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Molecular Biology of Cancer

INTRODUCTION
SLIDE 1, 2 In the developed countries cardiovascular diseases are the leading causes of death
(34%), followed by cancer (23%). Cancer is a group of diseases characterized by unregulated
cell growth and the invasion and spread of cells from the site of origin, or primary site, to other
sites in the body. Cancer is a genetic disease meaning, that alteration of DNS function results in
tumor formation. Only 10% of cancer is inherited, 70% is sporadic, that is, the mutations are
not inherited but occur during individual life. Twenty percent of cancer is resulted by infection
by pathogens, mainly viruses. Tumor is not synonymous with cancer. A tumor can be benign,
pre-malignant, or malignant, whereas cancer is by definition malignant. Earlier many scientists
were confident that science found the final answer on cause of tumorigenesis: cancer is the
result of cumulative mutations that change proteins encoded by cancer-related genes. Over 100
types of cancer have been classified. Approximately 85% of cancers occur in epithelial cells
and are classified as carcinomas. Cancers derived from mesoderm cells (e.g. bone, muscle) are
called sarcomas, and cancers of glandular tissue (e.g. breast) are called adenocarcinomas.The
mutations affect two kinds of cancer genes. The first are called tumor suppressors, which
normally restraints cells ability to divide, and mutations permanently disable the genes. The
second type of cancer genes, known as oncogenes, stimulates cell division. Mutations lock
oncogenes into an active state. Many researchers still take it as axiomatic that such growth-
promoting changes to a small number of cancer genes are the initial event and root cause every
human cancer. For the past few years, however, prominent oncologists have increasingly
challenged that theory. No one questions that cancer is ultimately a disease of DNA, but many
investigators think differently about the causes.

TUMOR FORMATION
Cancer can be perceived as a disease of communication between and within cells. Two major
classes of genes are thought to be involved in the formation of tumors, which are the proto-
oncogenes and tumor suppressor genes.

SLIDE 3 Intelligent tumors Biologists estimate that more than 100 trillion (1014) cells must
cooperate to keep a human being healthy over the course of an 80-year life span. If any one of
those myriad cells could give rise to a tumor, why is it that fewer than half the population will
ever contract a cancer serious enough to catch a doctors attention? One explanation is that a
cell must acquire several extraordinary skills to be malignant. At least seven different
regulatory systems must be perturbed in order for a normal cell to grow as a cancer. (1) Push
the gas pedal Cancer cells continue dividing in situations in which normal cells would quietly
wait for a special chemical signalsay, from an injured neighbor. Somehow they counterfeit
these progrowth messages. (2) No break Conversely, tumor cells must ignore stop dividing
commands that are sent out by the adjacent tissues they squeeze and by their own internal aging
mechanisms. (3) No suicide All cancerous cells have serious problems of some sort with their
DNA, and as they double again and again, many cells in the resulting colony end up far from
the blood vessels that supply oxygen and nutrients. Such stresses trigger autodestruct
mechanisms in healthy cells. Tumor cells find some way to avoid this kind of suicide. (4)

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Attraction of blood vessels Then they have to persuade nearby blood vessels to build the
infrastructure they need to thrive. (5) Immortality A fifth derangement that almost all cancers
acquire is immortality. A culture of normal human cells stops dividing after 50 to 70
generations. That is more than enough doublings to sustain a person through even a century of
healthy life. But the great majority of cells in tumors quickly die of their genetic defects
reproduce indefinitely if the tumor is to grow. The survivors do so in part by manipulating their
telomeres, gene-free complexes of DNA and protein that protect the ends of each chromosome.
(6) Invasion Tumors that develop these five faculties are trouble, but they are probably not
deadly. It is a sixth property, the ability to invade nearby tissue and then metastasize to distant
parts of the body that gives cancer its lethal character. Local invasions can usually be removed
surgically. But nine of every 10 deaths from the disease are the result of metastases. (7)
Evasion of immune system detection The anti-tumor activity of immune system rapidly
eliminates cells with uncontrolled growth. Successful tumor cells therefore have to block these
mechanisms.
Proto-oncogenes and tumor suppressors
(1) Oncogenes SLIDE 4 Most cancers have mutations in proto-oncogenes, the normal genes
involved in the regulation of controlled cell growth. These genes encode proteins that function
as growth factors, growth factor receptors, signal-relaying molecules, and nuclear transcription
factors (proteins that bind to genes to start transcription). When the proto-oncogene is mutated
or overregulated, it is called an oncogene and results in unregulated cell growth and
transformation. At the cellular level, only one mutation in a single allele is enough to trigger an
oncogenic role in cancer development. The chance that such a mutation will occur increases as
a person ages.
(2) Tumor suppressor genes SLIDE 5 Most cancer susceptibility genes are tumor suppressor
genes. Tumor suppressor genes are just one type of the many genes malfunctioning in cancer.
These genes, under normal circumstances, suppress cell growth. Some do so by encoding
transcription factors for other genes needed to slow growth. For example, the protein product of
the suppressor gene TP53 is called p53 protein. It binds directly to DNA and leads to the
expression of genes that inhibit cell growth or trigger cell death. Other tumor suppressor genes
code for proteins that help control the cell cycle. Both copies of a tumor suppressor gene must
be lost or mutated for cancer to occur. A person who carries a germline mutation in a tumor
suppressor gene has only one functional copy of the gene in all cells. For this person, loss or
mutation of the second copy of the gene in any of these cells can lead to cancer.
The two-hit hypothesis SLIDE 6 In 1971, Alfred Knudson proposed the two-hit hypothesis to
explain the early onset at multiple sites in the body of an inherited form of cancer called
hereditary retinoblastoma. Inheriting one germline copy of a damaged gene present in every
cell in the body was not sufficient to enable this cancer to develop. A second hit (or loss) to the
good copy in the gene pair could occur somatically, though, producing cancer. This hypothesis
predicted that the chances for a germline mutation carrier to get a second somatic mutation at
any of multiple sites in his/her body cells was much greater than the chances for a noncarrier to
get two hits in the same cell. So, accordint to the two-hit hypothesis, tumor suppressors act
recessive at the phenotypic level (both alleles must be mutated/lost for cancer to develop).
However, it turned out that this hypothesis is not true for the inherited form of retinoblastoma
itself, because it is actually inherited in an autosomal dominant fashion. However, other tumor
supressor genes can be inherited in a recessive manner.
Loss of heterozygosity SLIDE 7 In hereditary cancer syndromes, individuals are called
heterozygous (having one or more dissimilar gene pairs) because they start life with a mutation

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(inherited from one of the parents) in one of the alleles linked to cancer susceptibility, but it is
balanced by a normal counterpart. These individuals are predisposed to cancer because all their
cells have already sustained the first hit to cancer-linked genes. If the critically needed normal
suppressor gene that balances this germline mutation is lost at some time during an individuals
life, a condition called loss of heterozygosity occurs.
The multiple-hit hypothesis SLIDE 8 The classical example of the principle that several
consecutive mutations are needed for the development of most tumors were demonstrated by
the development of colon carcinoma. In familial adenomatous polyposis (FAP), inheritance of
a single mutant tumor suppressor FAP allele followed by the mutation of the originally normal
allele results in multiple benign polyps (adenomas) of the colon lining. Progression toward an
adenocarcinoma begins with the activation of the K-ras (Kristen rat sarcoma) oncogene and
the deletion of p53 gene. Deletion of the DDC (deleted in colorectal carcinoma) gene initiates
cell surfice changes and further mutations promotes metastasis. The specific mutations
described are characteristic, but can occur in any order.

The role of inflammation in the cancer formation SLIDE 9 Some viral and bacterial infections
induce a chronic inflammatory response that contributes to the process of carcinogenesis. Inflammation is a host
defense mechanism against infectious agents and injury, as it is a consequence of wound healing. Under normal
conditions it is highly regulated and short lived: such acute inflammation typically resolves itself with the help of
anti-inflammatory factors. By contrast, recent evidence suggests that it is lingering, chronic inflammation that
plays an important role in causing cancer. In addition, there is evidence that chronic inflammation, in the absence
of an infectious agent, leads to an increased risk of cancer. A key mediator of the inflammatory response is the
transcription factor NF- B. It is induced by several cell types, such as macrophages, target cells of inflammation,
and cancer cells. In addition to inflammation, NF- B has other downstream effects that contribute to
tumorigenesis, such as the inhibition of apoptosis and the promotion of metastasis and angiogenesis. Thus, NF- B
provides a molecular link between inflammation and cancer. Oncogenic activation of the NF- B gene has also
been identified in human tumors, including multiple myeloma, acute lymphocyte leukemia, prostate, and breast
cancers. Helicobacter pylori is a bacterium capable of inducing chronic inflammation in the stomach and initiating
carcinogenesis. However, only less than 1% of those infected will develop gastric cancer. It is a multi-step process
where inflammation is considered the initial and required step in the process. Barry Marshall and Robin Warren
were awarded a Nobel Prize at 2005 for their discovery of the bacterium Helicobacter pylori and its role in
gastritis and peptic ulcer disease (NOT in cancer formation!).

ONCOGENES, PROTO-ONCOGENES
SLIDE 10 Oncogenes (tumor-causing genes) are cellular genes that can trigger uncontrolled cell proliferation if
their sequence is altered or their expression is incorrectly regulated. Oncogenes were originally identified in RNA
tumor viruses (retroviruses) as genes (v-onc) that could transform cells into an altered state of control of cell
proliferation, often resulting in a tumor, mainly in chicken, mice, and rats. More than 20 different viral oncogenes
are known to have a counterpart in normal cells (c-onc), called proto-oncogenes or cellular oncogenes. These
cellular genes are highly conserved in evolution because they have important functions in all eukaryotic cells.
They encode proteins that are required at defined sites throughout the cell where they regulate the ordered
progression through the cell cycle, cell division, and differentiation. In virtually every case where their functions
are known, oncogenes lie along the signaling pathways by which cells receive and execute growth instructions.
The mutations that activate these genes are either (1) structural mutations that lead to the constitutive activity of a
protein without an incoming signal (e.g., the protein kinases or Ras) or (2) regulatory mutations that lead to the
expression of the gene at an elevated level or at the wrong place and time (e.g., growth factors or transcription
factors).

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Viral oncogenes
SLIDE 11 (1) Rous sarcoma virus (retrovirus of chicken) Oncogenes were first discovered as viral (v-)
oncogenes in retroviruses that cause tumors. Evidence that viruses could cause cancer first came from a series of
studies by Peyton Rous beginning in 1911. He excised fibrosarcomas (connective tissue tumors) from chickens,
ground them up, and removed cells and debris by centrifugation. After passing the supernatant through filters with
very small pores, which retained even the smallest bacteria, Rous injected the filtrate into chicks. Most of the
injected chicks developed sarcomas. The transforming agent in the filtrate eventually was shown to be a virus,
called Rous sarcoma virus (RSV). Some 50 years later, in 1966, Rous was awarded the Nobel Prize for his
pioneering work. Later generations of molecular biologists showed that RSV is a retrovirus whose
RNA genome is reverse transcribed into DNA, which is incorporated into the host-cell genome.
Nontransforming retroviruses contain the genes gag, pol, and env, which encode the virus structural proteins and
the reverse transcriptase. In addition to these normal retroviral genes, oncogenic transforming viruses like RSV
contain the v-src gene. Subsequent studies with mutant forms of RSV demonstrated that only the v-src gene, not
the gag, pol, or env genes, was required for cancer induction. Thus, the v-src gene thus was identified as
an oncogene. The next breakthrough came in 1977 when Michael Bishop and Harold Varmus showed that normal
cells from chickens and other species contain a gene that is closely related to the RSV v-src gene. This normal
cellular gene, a proto-oncogene, commonly is distinguished from the viral gene by the prefix c (c-src). The
landmark discovery of the close relationship between a viral oncogene and cellular proto-oncogene fundamentally
reoriented thinking in cancer research because it showed that cancer may be induced by the action of normal, or
nearly normal, genes. RSV and other oncogenic viruses are thought to have arisen by incorporating a normal
cellular proto-oncogene into their genome. Subsequent mutation in the transduced gene then converted it into
an oncogene.
The text for the slide: A typical retrovirus contains an RNA genome that codes for three genes or groups of
genes: gag (group-specific antigen), pol (polymerase), and env (coat protein, envelope). As with all genes of
higher organisms, a cellular oncogene (c-onc) consists of exons and introns with defined structure and sequence,
as in the gene src (the name is derived from sarcoma, a tumor that is induced by a mutation in this gene). The
virus may contain parts of the cellular oncogenes (c-src). This is designated viral oncogene (v-src) (Rous sarcoma
virus). In chickens, it induces a malignant tumor (a sarcoma) by stimulating uncontrolled mitosis of the host cells
thereby providing abundant cells for fresh infection. Since many cellular oncogenes are also known in an altered,
viral form, it is assumed that the viruses have integrated parts of the respective cellular oncogenes into their own
genomes. Because its genome carries the v-src oncogene, Rous sarcoma virus induces tumors within days. Most
oncogenic retroviruses, however, induce cancer only after a period of months or years. The genomes of the slow-
acting retroviruses differ from those of transducing viruses such as RSV in one crucial respect: they lack
an oncogene. These viruses integrate to close vicinity of a cellular proto-oncogene thereby altering its gene
expression properties

SLIDE 12 (2) Human papilloma virus (HPV) The human papilloma virus is a double-
stranded DNA virus that infects the epithelial cells of skin and mucosa. The epithelial surfaces
include all areas covered by skin and/or mucosa such as the mouth, throat, tongue, tonsils,
vagina, penis, and anus. Transmission of the virus occurs when these areas come into contact
with a virus, allowing it to transfer between epithelial cells. In 1976 Harald zur
Hausen published the hypothesis that HPV plays an important role in the cause of cervical
cancer. Indeed, in 1984 zur Hausen and his collaborators identified two types of them (HPV16
and HPV18) in cervical cancer. The viral oncogenes, E6 and E7 are thought to modify the cell cycle so as to
retain the differentiating host keratinocyte in a state that is favorable to the amplification of viral genome
replication and consequent late gene expression. Viral E6 protein in association with host E6 associated protein,
which has ubiquitin ligase activity, act to ubiquitinate p53, leading to its proteasomal degradation. E7 protein acts
as the primary transforming protein. E7 competes for retinoblastoma protein (pRb) binding, freeing the
transcription factor E2F to transactivate its targets (genes encoding enzymes involved in DNA replication), thus
pushing the cell cycle forwards.

(3) Epstein-Barr virus (EBV) is a gamma-herpesvirus, and encodes several viral proteins that affect host gene
expression. One of these, oncoprotein LMP1, is able to transform cells in culture. Its multi-functional effects
include activation of genes important for promoting cell proliferation (e.g. EGFR epidermal growth factor
receptor) and inhibiting apoptosis (e.g. Bcl-2). It also activates a nuclear transcription factor called NF- B. EBV
has been shown to act as a causative factor in B-cell lymphoproliferative diseases and probably

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nasopharyngeal carcinoma. However, it is thought to be only a contributing factor for a type of cancer called
Burkitt's lymphoma (it acts along with malaria to generate this cancer).
(4) Hepatitis B virus (HBV) There is a strong association between hepatitis B virus infection and liver cancer
(hepatocellular carcinoma). Host-viral interactions evoke an immune response that results in liver necrosis,
inflammation, and regeneration. These effects are sustained in the 10% of adults who develop chronic infections.
It has been proposed that increased proliferation and/or oxidative stress from inflammation may lead to oncogenic
mutations. Inflammation caused by Hepatitis B virus is important for the development of hepatocellular carcinoma.
The multifunctional viral protein, HBV X, is thought to be important for HBV-induced carcinogenesis and it was
shown to induce liver cancer in transgenic mice. It is thought to function by activating proto-oncogenes via
various signaling cascades, including many kinase cascades (e.g. the RAS-RAF-MAPK pathway); interacting with
NF-KB; and binding to and inactivating p53.

(5) Kaposi's sarcoma-associated herpesvirus (KSHV; or human herpesvirus 8; HHV8) is a co-factor of

Kaposis sarcoma, a cancer of skin and connective tissues. Kaposi's sarcoma is associated frequently with AIDS
patients. KSHV is thought to contribute to tumorigenesis through several mechanisms. Some viral products induce
the production of cytokines and growth factors that can influence uninfected neighboring cells. It has been
suggested that KSHV tumorigenesis involves a paracrine process whereby uninfected cells are transformed by
these induced cytokines and growth factors secreted by the infected cells. In addition, KSHV produces several
viral anti-apoptotic proteins (e.g. vBcl-2) and a viral protein called LANA that interferes with the function of RB
and p53.

The role of inflammation in cancer formation

SLIDE 13 Cellular proto-oncogenes and oncogenes When expressed normally, cellular
oncogenes are referred to as proto-oncogenes. Proto-oncogenes have been extraordinarily
conserved evolution, suggested, that in their normal context, these genes must have important
functions. Most proto-oncogenes encode proteins that participate in signal transduction
pathways through which signal to divide (or not divide) are relayed from outside the cell to the
regulatory machinery within. The aberrant expression of oncogenes results in entry of the cell
into the cell cycle with abnormal cell growth, suggesting a gain of function. There are two
mechanisms by which proto-oncogenes can be converted to cellular oncogenes: (1)
Quantitative (regulatory): Tumor formation is induced by an increase in the absolute number
of proto-oncogene products (resulted in enhanced gene expression) or by its production in
inappropriate cell types (e.g. by chromosomal translocation thereby bringing a growth
regulatory gene to the control of a different promoter). (2) Qualitative (structural):
Conversion from proto-oncogene to transforming gene (c-onc) with changes in the nucleotide
sequence (by e.g. point mutation) which are responsible for the acquisition of the new
properties leading to an increase of the stimulatory effect of oncoproteins on cell growth.
SLIDE 14 Hunting for oncogenes (1) Discovery of c-src proto-oncogene. The src gene (v-src) was isolated
from the genome of Rous sarcoma virus (RSV) followed by radiolabeling and hybridization with the chicken
genome. Hybridization signals in Southern blot analysis indicate the presence of chicken genes (v-src)
homologous to that of virus oncogene. SLIDE 15 (2) Cloning of a human oncogene. Transfer of DNA from
human bladder tumor cells to cultured mouse cells yielded transformed cells. In the process the transformed cells
acquired an activated human oncogene, and approximately a million base pairs of irrelevant human DNA from the
tumor cells. Human DNA can be detected in mouse cells that by using a radiolabelled probe for the Alu family of
repetitive sequences, which is present every few thousand base pairs in the human genome (but not in the mouse
DNA). Therefore, an Alu probe hybridizes to many restriction fragments in a Southern blot of DNA from a
primary transfectant (After a second round of transfection into mouse cells (using the primary transformants as the
source of DNA), however, the human DNA is distributed in many recipient cells. Only a few nearby Alu
sequences remain with the oncogene (lane 2). Upon further investigation the sarc gene could be found in many
organisms including e.g. fruitflies. Following further examination, a fundamental principle of cancer biology was
revealed: almost all known oncogenes are altered forms of normal genes or proto-oncogenes.

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DIA 16 Proto-oncogenes An extracellular growth factor stimulates cell growth by transmitting

a signal into the cell, and ultimately to the nucleus, to regulate gene expression in order to
produce proteins that are essential for cell division. There are six types of proteins involved in
the transduction of a growth factor signal: (1) growth factors, (2) growth factor receptors, (3)
intracellular signal transducers, (4) nuclear transcription factors which elicit the mitogenic
effect through the regulation of gene expression, (5) cell cycle regulators, and (6) many types
of microRNAs. Many growth factor receptors are tyrosine kinases. Tyrosine kinases
phosphorylate tyrosine residues in target proteins. Serine/threonine kinases phosphorylate
serine and threonine residues. The addition of the phosphate group may serve as a recognition
site for new protein-protein interactions and/or may cause a conformational change resulting in
the activation or inactivation of an enzymatic activity.
Epidermal growth factor (EGF) signaling serves as an important model for how a signal
from an extracellular growth factor can be transduced through a cell, regulate gene expression,
and trigger cell proliferation. The EGF receptor (EGFR) is a tyrosine kinase receptor and was
the first to be discovered. EGFR receptor contains an extracellular ligand-binding domain, a
single transmembrane domain, and a cytoplasmic protein tyrosine kinase domain. Getting the
signal from a growth factor outside the cell to inside the nucleus where gene expression is
regulated requires several steps: binding of the growth factor to the receptor, receptor
dimerization, autophosphorylation, activation of intracellular transducers (including RAS) and
a cascade of serine/threonine kinases, and regulation of transcription factors for gene
expression.
(1) Growth factors (GFs) Overexpression of these polypeptide growth hormones can elicit (in
cells normally in the G0 phase) an aberrant transition from G0 to G1, with subsequent
uncontrolled growth. (a) Fibroblast growth factors (FGFs) make up a large family of
polypeptide growth hormones normally expressed during proliferation of cells required for
normal wound healing, but overexpression can lead to tumor formation. (b) Platelet-derived
growth factor (PDGF) is a polypeptide that is normally important for extracellular matrix
production, but overexpression may result in proliferation as a result of autocrine stimulation.
Moreover, PDGF is a component of a wound response and its normal role is to stimulate epi-
thelial cells around the wound edge to proliferate and repair the damage. An early evidence for
the role of proto-oncogenes came from analysis of the viral oncogene v-sis. Its protein product
was cytoplasmic and was found to be a truncated version of PDGF. The significance of the
oncogenic form may be its aberrant location (cytoplasmic rather than secreted) and the
subsequent activation of the PDGF signal pathway at inappropriate times (e.g. other than in
response to a wound), leading to unregulated growth.
(2) Growth factor receptors (GFRs) The normal binding of GFs to their receptors results in
signal transduction in response to receptor dimerization. Several GFRs have been identified
that are capable of activation, even in the absence of specific ligand. Many of these receptors
have intracellular domains that function as tyrosine kinases. (a) Epidermal growth factor
receptors (EGFRs): There are at least three members of the family of tyrosine kinase receptors,
erbB-1, erbB-2, and erbB-3, which when mutated, lead to errant signaling and growth in the
absence of cognate ligand. Overexpression of erg b-1 (also known as HER2; human epidermal
growth factor receptor 2) is associated with breast cancers. These patients can be treated with a
monoclonal antibody to HER2 (Perception), which blocks signaling through this oncogenic
pathway. The viral erbB gene isolated from chicken retrovirus encodes a form of the EGF
receptor that is shortened at both ends. This truncated receptor acts as if it is constantly bound
to its ligand and therefore, constitutively sends growth-promoting signals through continual
tyrosine kinases activity. Increasing the amount of normal c-erbB product by gene

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amplification is another mechanism that contributes to carcinogenesis, particularly breast

cancer. Gene amplification involves multiple duplications of a DNA sequence due to errors at
DNA replication forks. (b) The proto-oncogene ret, another growth factor tyrosine kinase
receptor, heterodimerizes with cell-surface receptors GFR- l-4 in order to transduce the signal
for glial-derived neurotrophic factor (GDNF). It plays an important role in kidney development
and neuronal differentiation. Papillary thyroid carcinoma cells often carry somatic
chromosomal re-arrangements involving the amino-terminal parts of numerous genes and the
sequences of ret that code for the tyrosine kinase domain. The fusion protein products display
kinase activity that is independent of GDNF signaling.
(3) Signal transduction proteins The next level at which defects in cell growth and
development can occur is at the level of downstream signal transduction proteins.
(a) The oncogenic activation of ras is observed in about 30% of human tumors. The majority
of mutations are located in codons 12, 13, and 61. The consequence of each of these mutations
is a loss of GTPase activity of the RAS protein, normally required to return active RAS-GTP to
inactive RAS-GDP. The effect is constitutive activation of RAS protein, even in the absence of
mitogens. Some specific mutations in the ras gene are characteristic for specific cancers. A
point mutation within codon 12 that results in the substitution of valine (GTC) for glycine
(GGC) is characteristic of bladder carcinoma, while substitution of serine (AGC) is common in
lung cancer. Ras is the most commonly mutated oncogene in cancer, with 10-20% of tumors
harboring mutations in Ras. Ras mutations are found in large number of tumors of the colon,
pancreas, and thyroid. In the inactive state, Ras binds GDP. In stimulation of the cell by growth
factors, Ras exchanges GDP to GTP, leading to activation of downstream signaling events. The
Ras has intrinsic GTPase activity, terminating the signal transduction events when GTP is
hydrolyzed back to GDP, returning Ras to its inactive state.
(b) Non-receptor tyrosine kinases are another important group of signaling molecules. Genes
that code for (b1) cytoplasmic tyrosine kinases such as SRC, (b2) serine/ threonine kinases
such as RAF and MAPK, and (b3) nuclear kinases such as ABL, can also undergo oncogenic
activation. SLIDE 18
- Intramolecular associations normally regulate c-SRC kinase activity; the SRC SH2 domain
binds a carboxy-terminal phosphorylated tyrosine residue (Tyr530) and results in a
conformation that blocks the SRC kinase active site. Repression of SRC kinase activity can be
relieved by dephosphorylation of Tyr530, or by the binding of the SH2 domain to specific
activated tyrosine kinase receptors. In colon cancer, the protein product of oncogenic src is
characterized by a truncation at Tyr530. This aberrant protein is unable to adopt the inactive
conformation described above and therefore kinase activity is constitutive ('always on').
(4) Transcription factors Transcription is the must be considered as an ultimate target of
oncogene action. Many oncogenes act directly as transcription factors. The transcription factors
are expressed at very low levels at quiescent cells but are quickly turned on when cells receive
signal to divide and are rapidly translocated to the nucleus to mediate gene transcription.
(a) c-myc The transcription factors, such as the proto-oncogene myc, integrate divergent growth-promoting
pathways, and ultimately lead to the production of proteins that allow the cell to advance through the cell cycle.
This gene is a frequent target for integration of viruses and for chromosomal rearrangements, both of which
increase expression of c-myc or lead to its expression at an inappropriate time or site.
(b) c-Fos is a cellular proto-oncogene belonging to the immediate-early family of transcription factors. c-Fos has a
leucine-zipper DNA binding domain, and a transactivation domain. Transcription of c-Fos is upregulated in
response to many extracellular signals, e.g. growth factors. Additionally, phosphory-lation by MAPK, PKA or
PKC alters the activity and stability of c-Fos. Members of the Fos family dimerise with c-Jun (another proto-
oncogene) to form the AP1 (activator protein-1), which upregulates transcription of a diverse range of genes
involved in many processes from proliferation and differentiation to defense against invasion and cell damage.

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(5) Cell cycle regulators Alteration in the normal function of these molecules often results in unchecked cell
growth.
(a) cycline D Increased expression of this regulator of the G1 S transition is commonly found in tumors.
(b) CDK4 This regulator is among the most commonly altered gene in this class of genes.
(c) CDK inhibitors: p16, p21, p27

(6) miRNAs constitute a new class of oncogenes.

MicroRNAs (miRNAs) are an abundant class of small noncoding RNAs that function as negative gene regulators.
miRNA deregulation is involved in the initiation and progression of human cancer; however, the underlying
mechanism and its contributions to genome-wide transcriptional changes in cancer are still largely unknown. A
study of mice altered to produce excess c-myc shows that miRNA has an effect on the development of
cancer. Several other evidence show the role of miRNAs in tumor formation.

TUMOR-SUPPRESSOR GENES
SLIDE 19 The human body has mechanisms exerted by tumor suppressor genes that normally
'police' the processes that regulate cell numbers and ensure that new cells receive DNA that has
been precisely replicated. Many tumor suppressor gene products act as stop signs to
uncontrolled growth and therefore may inhibit the cell cycle, promote differentiation, or trigger
apoptosis. In contrast to the cellular oncogenes, for which a change in one allele will alter
normal function, both alleles of a tumor suppressor gene must lose their function before a
tumor develops. The first event is usually a mutation by base exchange or deletion. The second
event, affecting the other allele (allele 2), may also be a mutation, but the loss of function more
often appears to be from loss of the chromosome after a faulty cell division (mitotic
nondisjunction) or other mechanisms. The two-hit hypothesis explains the early onset at
multiple sites in the body of an inherited form of cancer e.g. hereditary retinoblastoma. The
first mutation in a suppressor gene can either be present in the zygote (germinal mutation, i.e.,
germ cell mutation due to transmission from an affected parent or due to new mutation) or
occur in a single cell of the corresponding tissue (somatic mutation). Loss of function of one
allele predisposes the cell to tumor development. With a germinal mutation, all cells are
predisposed. The tumor arises after loss of function of the second allele. When somatic
mutation occurs in a single cell, loss of function of both alleles rarely affects the same cell. But
with a germ cells mutation, loss of function of the second allele is frequent, since all cells carry
the first mutation, i.e., are predisposed. With somatic mutation, the tumor occurs sporadically
(is not hereditary) and arises unifocally from a single cell. In the hereditary form resulting from
a germ cell mutation, several tumors may arise from different cells (multifocal tumor).

SLIDE 20 (1) Cell surface molecules There are numerous cell surface molecules that
antagonize normal cell growth.
(a) Transforming growth factor (TGF)- receptor mediates its inhibitory effects by stimulating
the production of CDK-Is.
(b) Protein product deleted in colon carcinoma (DCC) regulates cell growth through the
integration of signals from the cellular environment.

SLIDE 21 (2) Intracellular signal transduction proteins These molecules possess an

antagonistic role to the actions of intracellular proto-oncogenes. (a) Neurofibromatosis (NF)-1
protein (neurofibromin) and (b) GTPase-activating proteins (GAP) activate the GTPase
function of Ras, converting GTP to GDP and suppressing the growth-promoting function of
Ras. NF-1 contains a GAP domain, but the GTPase activities of NF-1 and GAP are differently
regulated. NF-1 is an inherited tumor predisposition syndrome in which affected individuals
are prone to the development of optic nerve glioma. (c) Adenomatosis polyposis coli (APC)

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gene product promotes the degradationof -catenin which otherwise normally translocates to
the nucleus to induce cellular proliferation.

(3) Transcription factors Several tumor-suppressor proteins residing in the nucleus encode
proteins that play an important role in the integration of growth-promoting and growth-
inhibiting signals.
SLIDE 22 (a) Retinoblastoma (Rb) is the firstly identified tumor suppressor gene. Children
with hereditary retinoblastoma inherit a single defective copy of the RB gene, sometimes seen
as a small deletion on chromosome 13. They develop retinal tumors early in life and generally
in both eyes. Individuals with sporadic retinoblastoma, in contrast, inherit two
normal RB alleles each of which has undergone a somatic loss-of function mutation in a single
retinal cell. Because this is an unlikely occurrence, sporadic retinoblastoma is rare, develops
late in life, and usually affects only one eye. The retinoblastoma protein (Rb) prevents the cell
from replicating damaged DNA by preventing its progression along the cell cycle through G1
into S. Rb binds and inhibits transcription factors of the E2F family, As long as E2F is
inactivated, the cell remains stalled in the G1 phase. When Rb is bound to E2F, the complex
acts as a growth suppressor and prevents progression through the cell cycle.
SLIDE 23 (b) Tumor protein 53 (p53; encoded by the TP53 gene in human) is induced when
DNA is damaged or in the case of other stresses such as hypoxia and cell cycle abnormalities.
The p53 exerts its effects in two ways. (1) It induces the transcription of p21, which inhibits the
CDK/cyclin-mediated phosphorylation of Rb required for the cell to transition to the S phase.
(2) If DNA damage inflicted upon the cell cannot be successfully repaired, p63 mediated the
transcription of genes implicated in the process of apoptosis. In a normal cell p53 is inactivated
by its negative regulator, mdm2. Upon DNA damage or other stresses, various pathways will
lead to the dissociation of the p53 and mdm2 complex. Once activated, p53 will either induce a
cell cycle arrest to allow repair and survival of the cell or apoptosis to discard the damaged cell.
How p53 makes this choice is currently unknown.
SLIDE 24 P53: fix or kill! The p53 gene is a tumor-suppressor and a pro-apoptotic gene. The
p53 protein prevents a cell from completing the cell cycle if (1) its DNA is damaged or (2) the
cell has suffered other types of damage. When the damage is minor, p53 halts the cell cycle
hence cell division until the damage is repaired. When the damage is major and cannot be
repaired, p53 triggers the cell to commit suicide by apoptosis. These functions make p53 a key
player in protecting us against cancer; that is, an important tumor suppressor gene. More than
half of all human cancers do, in fact, harbor p53 mutations and have no functioning p53 protein.
Traditional anti-tumor techniques (chemotherapy and radiotherapy) do not directly damage
tumor cells, but by causing DNA breaks and thus activating p53 to trigger the apoptosis
pathway.

DIA 25 On the role of p53 in detail

Antioxydant activity The p53 gene was the first tumor suppressor gene to be identified and, since its discovery,
scientists have found that the p53 pathway is altered in most human cancers. Its protein product, p53, is at the
heart of the cell's tumor suppressive mechanism and thus has been nicknamed the 'guardian of the genome'. In the
absence of cellular stress, low levels of p53 induce antioxidant activity which decreases the levels of reactive
oxygen species (ROS) and subsequent DNA damage. Normal cell metabolism produces ROS that can react with
DNA. It has been estimated that endogenous ROS modify approximately 20,000 bases of DNA per day in a single
cell. p53 accomplishes this by upregulating genes whose products have antioxidant functions such as glutathione
peroxidase 1 and sestrins, proteins involved in hydrogen peroxide metabolism. This antioxidant activity guards
against mutation and may help prevent cancer.

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(A) Response to cell stress and DNA damage Many types of 'danger signals', such as cell stress and DNA
damage, can activate p53 and trigger several crucial cellular responses that suppress tumor formation. Upstream
stress activators include radiation-, drug-, or carcinogen-induced DNA damage, oncogenic activation, hypoxia and
low ribonucleotide pools. These conditions may nurture tumor initiation. In response to these stress signals p53
can elicit downstream cellular effects including cell cycle arrest, apoptosis, DNA repair, and inhibition of
angiogenesis. The ability to cause the cell cycle to pause allows for repair of DNA mutations and prevents their
propagation within the genome. Apoptosis is another means of preventing propagation of mutations; cell suicide
benefits the organism as a whole if DNA damage cannot be repaired. Mutated cells are better dead. Apoptosis is
the critical biological function mediating the tumor suppressor function of p53. It should be mentioned that p53
may also play a pro-oxidant role under certain stress conditions that may contribute to the cellular effect of
apoptosis. The overall regulation of the p53 pathway possesses an extraordinary complexity that compels us to try
to unravel each layer.
(B) Domains of the p53 protein and location of mutational hotspots are shown in this slide.
(C) Regulation of p53 protein by MDM2 Normally, the level of p53 protein in a cell is low. The activity of p53
in a cell is regulated at the level of protein degradation, not at the level of expression of the p53 gene. The MDM2
protein, a ubiquitin ligase, is its main regulator. Ubiquitin ligases are enzymes that attach a small peptide called
ubiquitin to proteins, flagging it for proteolysis in proteosomes. MDM2 targets p53 for degradation by
proteosomes in the cytoplasm. In addition, MDM2 modifies the activity of p53 since it binds to and inhibits the
p53 trans-activation domain at the amino-terminal and transports the protein into the cytoplasm, away from
nuclear DNA. Thus the activity of p53 as a transcription factor is out of reach. The binding of MDM2 to p53 is
part of an autoregulatory feedback loop since the MDM2 gene is a transcriptional target of p53. Therefore, p53
stimulates the production of its negative regulator MDM2 that causes the degradation of p53. Small amounts of
p53 will reduce the amount of MDM2 protein and this will result in an increase of p53 activity, thus completing
the loop.
(D) Upstream: molecular pathways of p53 activation The mechanism by which p53 becomes activated depends
on the nature of the stress signal. Stress is 'sensed' by cellular proteins, many of which are kinases that convey the
danger signals to p53 via phosphorylation. Disruption of the p53-MDM2 interaction is fundamental to the activa-
tion of p53 by its upstream factors. The upstream activators of p53 utilize three main independent molecular
pathways to signal cellular distress. DNA damage caused by ionizing radiation is signaled by two protein kinases.
The first kinase, ATM, stimulated by DNA double-strand breaks, phosphorylates and activates a second kinase
Chk2. Both ATM and Chk2 kinases phosphory-late amino-terminal sites of p53 and this phosphorylation
interferes with binding of MDM2. A second molecular pathway that signals cellular distress to p53 is executed by
two different kinases, ATR and casein kinase II. These also phosphorylate p53 and disrupt its interaction with
MDM2. Lastly, activated oncogenes, such as Ras, induce the activity of the protein p14arf, another modulator of
the p53-MDM2 complex. P14arf is one of two translational products of the INK4a/CDKN2A gene (p16, a cyclin
kinase inhibitor, is the other product). P14arf does not bind to the interface of p53-MDM2, but functions by
sequestering MDM2 to the nucleolus of the cell. All three pathways prevent degradation of p53 by MDM2.
(E) Downstream: molecular mechanisms of p53 cellular effects The main mechanism by which p53 exerts its
tumor suppressing effects is by inducing the expression of specific target genes. Let us examine how the resulting
network of proteins triggers these responses. (1) Inhibition of the cell cycle One of the central functions of p53 is
to cause cell cycle arrest in response to DNA damage so that there is an opportunity to repair the damage prior to
the next round of replication; thus damaged DNA will be prevented from being replicated and passed on to
daughter cells and maintenance of the genome will be facilitated. The molecular mechanism responsible for this
cellular response involves the transcriptional induction of the p21 gene. Its product, the p21 protein, inhibits
several cyclin-cdk complexes and causes a pause in the G1 to S (and G2 to M) transition of the cell cycle.
In addition, p21 also binds PCNA (proliferating cell nuclear antigen), a protein that has a role in DNA synthesis
and DNA repair. The interaction with p21 is such that it inhibits PCNA's role in DNA replication but not in DNA
repair. Therefore, p21 is an important part of the molecular mechanism that facilitates the ability of p53 to bring
about a pause in the cell cycle and at the same time allow DNA repair. (2) Apoptosis The expression of several
mediators of apoptosis is transcriptionally regulated directly by p53. The targets include genes that code for
proteins involved in two apoptotic pathways that respond to external and internal signals, respectively. In general,
genes encoding proteins that promote apoptosis, pro-apoptotic proteins, are induced while genes encoding proteins
that antagonize apoptosis, anti-apoptotic proteins, are repressed. The mitochondrial pro-apoptotic proteins NOXA
and PUMA that cause the release of cytochrome c and activate the apoptosome, are induced. Also, p53 tips the
balance regulated by the Bcl-2 protein family towards apoptosis by inducing gene expression of the pro-apoptotic
protein Bax and repressing the expression of anti-apoptotic protein Bcl-2. Fas receptor (FASR) is a
transmembrane receptor that receives extracellular (death) stimuli to stimulate apoptosis. Expression of the Fas
receptor gene is induced by p53. Apoptosis is also triggered when survival signaling is blocked by p53's induction
of IGF-BP3 (insulin-like growth factor-binding protein 3). IGF-BP3 blocks the signaling of IGF-1 to its receptor.

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Activation of these different pathways in concert is required for a full apoptotic response. Transcription-
independent mechanisms for the induction of apoptosis by p53 also exist. (3) DNA repair and angiogenesis For
example, the gene XFC that is involved in nucleotide excision repair is regulated by p53 through a p53 response
element in its promoter. Thrombospondin, an inhibitor of angiogenesis, is also transcriptionally regulated by p53.
This further supports the role of p53 as a transcriptional regulator in different biological responses.
(F) Decision making As the guardian of the genome, p53 prevents damaged DNA from being passed on to
daughter cells either by inhibiting the cell cycle or by inducing apoptosis. Cell cycle inhibition and apoptosis are
two independent effects of p53. The molecular factors that determine the biological outcome of whether inhibition
of the cell cycle or apoptosis takes place are just being elucidated. One model that has been put forth is that
different combinations of transcription factors that act as dimers influence the biological response. Oncogene
activation (e.g. Myc) is an upstream inducer of p53 that triggers apoptosis. The mechanism of this stress signal
acts via the cyclin-cdk inhibitor p21, the main effector of cell cycle inhibition but also an inhibitor of cell death.
The regulation of the p21 gene is a pivotal point in the p53 decision-making process. Both p53 and a transcription
factor called Miz-1 are required for p21 gene expression. Now enter the oncogene, Myc, which competes with p53
for binding with Miz-1. Myc interacts with Miz-1 and inhibits the transcription of p21. Through this mechanism of
preventing expression of p21, Myc not only overrides the p53-regulated block to cell cycle progression but also
blocks the p21-mediated inhibition of apoptosis (Figure 6.8). p53 is not altered and is free to induce the expression
of pro-apoptotic targets. Additional events are also required for full activation of apoptosis, since p53
phosphorylation and apoptotic co-factors are required for the induction of some apoptotic genes. Revealing the
mechanisms behind other modes of upstream stress inducers of p53, such as oxidative stress, requires further
studies. The apoptosis stimulating proteins of p53 (ASPP) family also plays a role in p53 decision making. These
proteins bind to the p53 DNA-binding domain and have been shown specifically to enhance the ability of p53 to
activate genes involved in apoptosis and not cell cycle arrest. The selection of apoptotic genes versus growth arrest
genes could potentially be accomplished by specific promoter sequences that serve to distinguish the functionally
distinct classes of genes. The regulation of ASPP itself requires further study. Mutations in the ASPP binding site
of the p53 gene and epigenetic silencing of the ASPP gene have been identified in tumor cells. These tumor cells
may have been initiated because they escaped from the apoptotic program normally augmented by ASPP. Other
co-activators may also enhance the selectivity of p53 to activate apoptotic genes.
Mutations in the p53 pathway and cancer p53 mutant cells are characterized by genomic instability, since
mutations are more likely to be maintained in dividing cells, providing an environment that is permissive for
tumor initiation. The high frequency of p53 pathway mutations found in tumor cells is most likely to be the result
of selective pressure favoring mutant cells that escape tumor suppression. Over 75% of all p53 mutations are
missense mutations and result in single amino acid substitutions. Many mutant p53 molecules are more stable than
wild-type p53 protein and can accumulate in cells. More than 90% of the missense mutations are located in the
DNA-binding domain (amino acids 102-292) and more than 30% of these affect only six codons and are therefore
referred to as 'hotspots'. In addition to mutation of the p53 gene, there are other ways to interfere with the p53
pathway. Defects in pathways that lead to the activation of p53 in response to stress, such as mutations in the chkl
gene, have been identified in cancer cells that do not contain mutations in the p53 gene. In these cases, the stress
signal would not be transmitted to p53 via phosphorylation as in normal cells (see Figure 6.6). Also, over-
expression of the MDM2 protein has been demonstrated to alter the regulation of p53, leading to a 'p53-
inactivated' phenotype. Inactivation of downstream effectors, such as Bax and FASR, also perturbs the pathway.
Some of these other p53 pathway disruptions may not lead to effects as severe as p53 mutation, since p53 protein
is a central node for receiving and eliciting many stress signals and biological responses, respectively, but may
mimic a partial aspect of p53 inactivation and be permissive for tumor formation. Li-Fraumeni syndrome is
predominantly characterized by a mutation of the p53 gene and leads to a predisposition to a wide range of cancers.
It is an autosomal dominant disease, so an affected individual has a 50% chance of passing the mutation to each
offspring. Patients have a 25-fold increased risk of developing cancer before they are 50 years old compared with
the general population. The young age at which individuals develop cancer and the frequent occurrence of
multiple primary tumors in individuals are characteristic features of the syndrome. The types of cancer seen within
families that carry the mutation include sarcomas, breast cancer, leukemia, and brain tumors. Cancer develops at
an earlier age over several generations. The protein product of the mutated p53 gene does not function to protect
the genome and an accumulation of mutations goes unchallenged and eventually leads to tumor formation.

The mechanism of tumor suppressor genes may be more complex than the two-hit hypothesis suggests. This is
particularly clear for p53. Loss of heterozygosity is not commonly observed in tumors from cells with only one
p53 allele, suggesting that reduced amounts (haploinsufficiency) of p53 can cause transformation. Mutations may
result in varied amounts of tumor suppressor gene expression and therefore tumor suppressor 'dose' may play a
role in the cancer outcome. Recent experiments which generated p53 hypomorphs (animals that exhibit reduced
levels of p53 expression) using RNA interference support this mechanism.Unlike most other tumor suppressor
genes, some p53 mutations do not lead to loss of function. Some missense mutations form an altered protein that

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interacts with the product of a normal p53 allele via dimerization to inactivate its function. This type of effect is
referred to as dominant negative, whereby the mutated gene product dominates to inactivate the wild-type gene
product. In this situation, the autoregulatory loop is affected because p53 fails to induce its inhibitor, MDM2, and
as a result p53 mutant protein accumulates. Other mutations can lead to a newly acquired 'gain-of-function'
phenotype that can accumulate in and transform cells. In this instance, mutant p53 (not wild-type p53) can be
considered an oncogene having an active role in carcinogenesis.

(4) DNA repair genes

SLIDE 26 (a) BRCA 1 (Breast Cancer Type 1 susceptibility protein) and BRCA2 although the
structures of the BRCA1 and BRCA2 genes are very different, at least some functions are
interrelated. The proteins made by both genes are essential for repairing damaged DNA.
Certain variations of the BRCA1 gene lead to an increased risk for breast cancer. Researchers
have identified hundreds of mutations in the BRCA1 gene, many of which are associated with
an increased risk of cancer. Women with an abnormal BRCA1 or BRCA2 gene have up to an
60% risk of developing breast cancer by age 90; increased risk of developing ovarian cancer is
about 55% for women with BRCA1 mutations and about 25% for women with BRCA2
mutations. A mutated BRCA1 gene usually makes a protein that does not function properly
because it is abnormally short. Researchers believe that the defective BRCA1 protein is unable
to help fix mutations that occur in other genes. These defects accumulate and may allow cells
to grow and divide uncontrollably to form a tumor. In addition to breast cancer, mutations in
the BRCA1 gene also increase the risk of ovarian and prostate cancers.
SLIDE 27 (b) Xeroderma pigmentosum (XP) genes. Among eight genes so far identified as
responsible for XP, XPA through XPG are involved in nucleotide excision repair of DNA
damage induced by UV as well as various chemical carcinogens. If tumor suppressor genes (e.g.
p53) are affected, the result may be cancer. XP is characterized by sun sensitivity, ocular
involvement, and greater than 1000-fold increased risk of cutaneous and ocular neoplasms. The
median age of onset of non-melanoma skin cancer is before age ten years.
(6) miRNAs were also shown to act as tumor suppressors.

OTHER CANCER GENES

In addition to oncogenes and tumor suppressor genes, most cancers acquire several other key
mutations that enable cancer to progress. While researchers dont yet know all the mutations
involved, they have organized them in terms of their activities in support of tumor growth and
metastasis. In addition to the contributions of oncogenes and mutated suppressor genes,
additional genomic mutations enable the invasion of neighboring tissue, evasion of immune
system detection, recruitment of a new blood supply, dissemination and targeting of new sites,
and the penetration and reinvasion through new blood and tissue layers. Over time, successful
metastasis occurs.
SLIDE 28 Other cancer-related genes In addition to oncogenes and tumor suppressor genes,
most cancers acquire several other key mutations that enable cancer to progress. While
researchers dont yet know all the mutations involved, they have organized them in terms of
their activities in support of tumor growth and metastasis. In addition to the contributions of
oncogenes and mutated suppressor genes, additional genomic mutations enable the invasion of
neighboring tissue, evasion of immune system detection, recruitment of a new blood supply,
dissemination and targeting of new sites, and the penetration and reinvasion through new blood
and tissue layers. Over time, successful metastasis occurs.

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SLIDE 29 Metastasis Most cells of the body normally remain resident within a particular
tissue or organ (though hematopoietic stem cells are a notable exception). Liver cells remain in
the liver and cannot be found in the lung and vice versa. Organs have well-demarcated
boundaries defined by surrounding basement membranes. Basement membranes are acellular
structures made up of a fabric of extracellular matrix (ECM) proteins: predominantly laminins,
type IV collagen, and proteoglycans. Cancer is distinctly characterized by the spreading of
tumor cells throughout the body. The process by which tumor cells migrate from a primary site
to other parts of the body is called metastasis. Metastasis is the fundamental difference between
a benign and malignant growth and represents the major clinical problem of cancer. A primary
tumor can be surgically removed relatively easily, whereas once hundreds or more metastases
have been established throughout the body they are practically impossible to remove. Sadly,
over 50% of solid tumors have metastasized at the time of diagnosis. The spread of cells
throughout the body results in physical obstruction, competition with normal cells for nutrients
and oxygen, and invasion and interference with organ function. Interestingly, specific cancers
metastasize to particular sites. Many of the preferences observed for the spread of specific
cancers to specific metastatic locations can be explained by the directionality of blood flow.
Since the bloodstream is the predominant means of long-distance transport, organs in close
proximity are likely to be main sites of metastasis for a particular primary tumor. However,
about one-third of the locations of frequent metastases is puzzling in this regard. For example,
breast cancer metastasizes to bone more frequently than anatomy would suggest. One
explanation of this observation was described over 100 years ago in the 'seed and soil' theory.
It described cancer cells as 'seeds' requiring a match with optimal environments or 'soils' to
succeed. The ability of cancer cells to metastasize is dependent on the interactions of their cell
surface molecules with the microenvironment, including neighboring cells and the extracellular
matrix. Recent molecular observations suggest that receptors lining the capillaries in the organs
to which cancer spreads influence the destination of metastasized cells, and these findings
support the 'seed and soil' theory. This theory is also supported by the concept of the
establishment of a pre-metastatic niche*, a site of future metastasis that is altered in
preparation for the arrival of tumor cells. Further studies are needed to discover the factors
needed for a tumor cell to be successful in metastasis. Although cancers are largely successful
in metastasizing in the long run, on the cellular level, only 1 in 10,000 metastasizing cells
survives transport.
Pre-metastatic niche Haematopoietic precursor cells in the bone marrow expressing VEGFR1 (vascular
endothelial growth factor receptor 1) appear to home in on specific sites before the tumour cells get there, paving
the way for wandering metastatic cells by forming niches where they can locate and multiply. The concept of a
pre-metastatic niche, in which non-cancer cells promote future metastasis, is a novel one that raises the possibility
that targeting VEGFR1 and related molecules could have therapeutic value.

SLIDE 30 Steps of metastasis There are several major steps involved in metastasis: migration,
intravasation (the invasion of a cancer through the basal membrane and into blood vessel),
transport, extravasation (cancer cells exiting the capillaries and entering organs), and metastatic
colonization (Figure 9.1). The first step (migration) and the last step (metastatic colonization)
have been demonstrated to be rate limiting and their rate dictates the overall metastatic ability
of the cancer.
Angiogenesis Members of the VEGF (vascular endothelial growth factor) family are specific endothelial cell
growth factors that are key players in angiogenesis.

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EPIGENETIC FACTORS
SLIDE 31 Much remains unknown about the role of epigenetic factors and cancer. Epigenetic
changes are reversible modifications to genes or proteins that occur in the tumor and its
microenvironment. Epigenetic modifier molecules have been observed making tumor-friendly,
nonmutational changes in an already confused biosystem. For example, by heavily methylating
genes or promoter regions, gene activity critical to counteract a tumors drive toward metastasis
gets turned off. Or noncoding ribonucleic acids meddle in epigenetic fashion, interfering with a
cells regulation of growth or attempt to repair damage. Epigenetic factors are mechanisms
outside the gene such as a cells exposure to carcinogens or hormones, or genetic variations
that modify a gene or its protein by methylation, demethylation, phosphorylation, or
dephosphorylation. These factors can alter what is ultimately expressed; they can change a
phenotype. For example, hormone and reproductive factors may influence the penetrance of
certain cancer-linked mutations. Breast and ovarian cancer are more likely to occur in women
with early menarche, late menopause, and a first child after age 30 (or no children at all). These
factors are believed to be linked to a womans exposure to estrogen and progesterone and their
effects on cell differentiation in the breast that occur during pregnancy. In cancer, both the
genotype and the phenotype change over time. Epigenetic factors play a key role in these
changes.

General aspects
SLIDE 32 Same allele, different locus, different phenotype Different mutations in the same gene can result in
different phenotypes. A good example is the RET proto-oncogene. Germline mutations of RET lead to multiple
endocrine neoplasia (MEN) type 2. The disease produced varies depending on where in the RET gene the germline
mutation sits, so the phenotype may be MEN-2A, MEN-2B, or familial medullary thyroid cancer.

SLIDE 33 Different locus, different allele, same phenotype Many cancer susceptibility syndromes are
genetically heterogeneous (a mixture), which means that different mutations (genotypes) can be expressed as the
same phenotype (e.g., cancer). These different mutations may be located within the same gene but at different
locations (locus heterogeneity) or on different genes altogether (allelic heterogeneity). For example, hereditary
breast and ovarian cancer susceptibility has both locus and allelic heterogeneity. More than 500 different
mutations have been identified that can occur in the BRCA1 gene on chromosome 17 and increase a womans risk
for breast cancer. And more than 300 mutations scattered throughout the BRCA2 gene on chromosome 13 are
associated with hereditary breast and ovarian cancer susceptibility.

SLIDE 34 Autosomal Dominant Inheritance Most hereditary cancer syndromes are inherited
in autosomal dominant fashion. Dominant inheritance occurs when only one copy of an allele
is required for a particular trait to be expressed (phenotype). In autosomal dominant
inheritance, multiple generations express the traits, with no skipped generations (assuming
complete penetrance).
SLIDE 35 Autosomal Recessive Inheritance In autosomal recessive inheritance, two copies
of the allele are required for the trait to be expressed. Carriers of one disease allele will not
develop the illness, and several generations may be unaffected, leading to the appearance of
skipped generations. Males and females are equally affected. If both parents carry one copy of
the recessive allele, one in four offspring, on average, will express the trait.
SLIDE 36 Penetrance Sometimes one person with a dominant allele will express a trait, yet
that same genotype in another person will remain silent. This is an example of differences in
penetrance. In classic Mendelian genetics, if an individual carries a dominant allele, the trait
will be expressed (genotype = phenotype). However, if all carriers of a certain dominant allele

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in a population do not express the trait (same genotypes/different phenotypes), the gene is said
to have incomplete penetrance.

SLIDE 37 Prevalence and founder effect Some populations have a higher prevalence of
specific cancer-associated alleles than others. This may result from a founder effect, which
occurs when a population undergoes rapid shrinkage and then expansion in an isolated setting.
In a population that is geographically or reproductively isolated, an individual called a founder
carries or develops a germline mutation that is rare in the general population.

NEW THEORIES
The Order of Disorder The dominant paradigm for 30 years has been that genetic damage to a cell deletes or
disrupts a tumor suppressor gene - RB, p53, BRCA2 and APC are among the best knownthereby suppressing
proteins that normally ensure the integrity of the genome and the process of cell division. Alternatively, a mutation
may increase the activity of an oncogenesuch as HER2/NEU, or c-foswhose proteins then stimulate the cell to
reproduce. Changes to cancer genes free the cell of one or more normal restraints, allowing it to outbreed its
neighbors. The cell passes abnormalities in its DNA sequence on to its descendants, which become a kind of clone
army that grows to the limits of its capacity. Eventually another random mutation to a cancer gene looses another
shackle, initiating another burst of growth. A mutation to just one allele is enough to activate an oncogene
permanently. But it takes two hits to knock out both alleles of a tumor suppressor gene. Four to 10 mutations in
the right genes can transform any cell. Or so the theory goes. The mutant-gene paradigm gained almost
universal acceptance because it explained very well what scientists saw in their experiments on genetically
engineered mice and human cell cultures. But new technologies now allow researchers to survey large fractions of
the genomes of cancerous and precancerous cells taken directly from people. Many recent observations seem to
contradict the idea that mutations to a few specific genes lie at the root of all cancers.

Unexplained Phenomena Many studies have shown that for many common human cancers, just a small fraction
of the cells in a tumor are responsible for its growth and metastasis and so for the illness and death of the
patient. The discovery of such cells, which some began calling cancer stem cells, posed a problem for the
mutant gene theory of cancer. In fact, most tumors are not masses of identical clones. On the contrary, closer
examination has revealed amazing genetic diversity among their cells, some of which are so different from normal
human cells (and from one another) that they might fairly be called new species. In 2006 Vogelstein and a large
team of collaborators used new genetic sequencing technologies to examine more than 13,000 genes in samples
taken from 11 breast tumors and 11 colorectal cancers. They found noninherited mutations in 1,149 (nearly 9 %)
of those genes. Even more startling than the sheer number of genes mutated was the lack of consistency from one
tumor to the nextor even from one cell to another within the same tumor. Not a single gene was mutated in more
than 5 percent of the tumors. Moreover, some of the most commonly altered cancer genes have oddly inconsistent
effects. Vogelsteins group has also reported that the much studied oncogenes c-fos and c-erbB3 are curiously less
active in tumors than they are in nearby normal tissues. The tumor suppressor gene RB was shown to be
hyperactivenot disabledin some colon cancers, and, perversely, it appears to protect those tumors from their
autodestruct mechanisms. The two hit hypothesisthat both alleles of a tumor suppressor gene must be
deactivatedhas also been upended by the discovery of a phenomenon called haplo-insufficiency. In some
cancers, it turns out, tumor suppressors are not mutated at all. Their output is simply reduced, and that seems to be
enough to push cells toward malignancy. This effect has now been seen for more than a dozen tumor suppressor
genes. Searching for the mere presence or absence of a genes protein is too simplistic. Dosage matters.

Beyond Mutation researchers are now looking more closely at other phenomena, besides errors in a genes DNA
sequence, that can dramatically alter the dosage of a protein in a cell. The loss or gain of a chromosome (or part of
one) containing the gene can do this. So can tweaks to regulatory factors that control how often a gene is translated
into protein, as well as epigenetic phenomena that alter gene activity by reversible means. All these changes are
nearly ubiquitous in established cancers. The genome of a typical cancer cell is not merely aneuploid but unstable
as well, changing every few generations. The aneuploidy and massive genomic instability inside tumor cells were
dismissed as side effects of cancer, not prerequisites. But the oncogene/tumor suppressor gene hypothesis has
also failed, despite three decades of effort, to identify a particular set of gene mutations that occurs in every
instance of any of the most common and deadly kinds of human cancer. So now, the question is which comes first:
mutations or aneuploidy? There are at least four competing answers: (1) Increased mutation rate theory, (2) the
early instability theory and (3) the aneuploidy theory. (4) Cancer stem cell theory The newest and most

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controversial theory is that cancers often arise from the very stem cells that give healthy organs their growth and
healing abilities. Encouragingly, all four of these theories seem to be converging as they bend to accommodate
new experimental results.

SLIDE 38 (1) Inreased Mutation rate theory (modified traditional theory) revives an idea proposed in 1974
by Lawrence A. Loeb. He and other geneticists have estimated that, on average, random mutation will affect just
one gene in any given cell over the course of a lifetime. A carcinogen, reactive oxidants, or perhaps a malfunction
in the DNA duplication and repair machinery of the cellmust dramatically accelerate the mutation rate. In 2006
technology advanced to the point that Loeb was able to test his hypothesis by comparing the rate at which a
noncoding portion of the p53 gene suffered small mutations in both normal and malignant human cells. Cancerous
cells, his test revealed, harbored anywhere from 65 to 475 mutations per 100 million nucleotides, whereas normal
cells had four or fewer. Loebs modified dogma thus may add a prologue to the long-accepted life history of
cancer. But the most important plot points in that story would still remain the same: mutations to genes that serve
to increase the reproductive success of cells. Mangled and ever changing chromosomes are, in this narrative, mere
fortuitous by-products. So, this theory is not basically different from that of traditional

SLIDE 39 (2) Early instability theory (Unstable from the Outset) Vogelstein and Lengauer proposed an
alternative theory in which chromosomal instability can occur early on. The genetic flux then combines forces
with natural selection to produce a benign growth that may later be converted to an invasive malignancy and life-
threatening metastases. In their hypothesis, there are several master genes whose function is critical for a cell to
reproduce correctly. If just one of these genes is disabled, either epigenetically or by mutation, the cell stumbles
each time it attempts the carefully regulated cell division, muddling some of the chromosomes into an aneuploid
state. One result is to increase 100,000-fold the rate at which cells randomly lose one of the two alleles of their
genes. For a tumor suppressor gene, a lost allele may effectively put the gene out of commission, either because
the remaining copy is already mutated or because of the haplo-insufficiency effect. Lengauer and Vogelstein still
assume that some cancer genes must be altered before a malignancy can erupt. Some observations do support the
early instability theory. In 2000 Lengauers laboratory examined colon adenomas benign polyps that
occasionally turn malignant and observed that more than 90 percent had extra or missing pieces of at least one
chromosome. More than half had lost the long arm of chromosome 5, home to the APC tumor suppressor gene,
long implicated in the formation of colon cancer. Other researchers have discovered similarly aberrant
chromosomes in precancerous growths taken from the stomach, esophagus and breast. The early instability theory
still has some loose ends, however. How can cells with shifty chromosomes outcompete their stable counterparts?
Under normal conditions, they probably do not. But in a war zone, where a carcinogen or other stressor is
continually inflicting damage to cells, normal cells stop dividing until they have completed repairs to their DNA.
Genetically unstable cells get that way because their DNA repair systems are already broken. So they simply
ignore the damage, keep on proliferating, and thus pull ahead. But what jumbles the chromosomes in the first
place? No genes have yet been conclusively identified as master genes, although several strong suspects have
surfaced.

SLIDE 40 (3) Aneuploidy theory on the other hand, maybe cells can become malignant even before any master
genes, oncogenes or tumor suppressor genes are mutated. Duesberg has put forth a fourth theory: nearly all cancer
cells are aneuploid (leukemia being one exception) because they start that way. Lots of things can interfere with a
dividing cell so that one of its daughter cells is cheated of its normal complement of 46 chromosomes and the
other daughter is endowed with a bonus. Asbestos fibers can physically disrupt the process. Most aneuploid cells
are stillborn or growth-retarded. But in the rare survivor, he suggests, the dosage of thousands of genes is altered.
That corrupts teams of enzymes that synthesize and maintain DNA. Breaks appear in the double helix,
destabilizing the genome further. The more aneuploid the cell is, the more unstable it is, and the more likely it will
produce new combinations of chromosomes that will allow it to grow anywhere. Unlike the other theories, the
aneuploidy hypothesis predicts that the emergence and progress of a tumor are more closely connected to the
assortment of chromosomes in its cells than to the mutations in the genes on those chromosomes. Some
observations do seem to corroborate the idea. Every single case of [nonhereditary] colorectal cancer, for example,
has gains of chromosomes 7, 8, 13 or 20or a loss of 18. In cervical cancer, aneuploidy of chromosome 3
happens very early, and those cells seem to have a selective advantage.

SLIDE 41 (4) Cancer stem cells theory Conventional wisdom has long held that every tumor cells have equal
capability for spreading in body. Current treatments therefore focus on killing the great number of cancer cells.
Howevet, it is now clear that that only a tiny percentage of tumor cells have the power to produce new cancerous
tissue and that targeting these specific cells for destruction may be a far more effective way to eliminate the
disease. Because thaty are the engines driving the malignancy itself, these cells are called cancer stem cells. The
existence of CSCs is a source of debate within medical research There is also debate on the cell of origin of CSCs

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- whether they originate from stem cells that have lost the ability to regulate proliferation, or from more
differentiated population of progenitor cells that have acquired abilities to self-renew.

SLIDE 42 (5) The cell of origin and evolution of a cancer stem cell. Normal cellular hierarchy comprising
stem cells that progressively generate common and more restricted progenitor cells, yielding all the mature cell
types that constitute a particular tissue. Although the cell of origin for a particular tumour could be an early
precursor cell such as a common progenitor, the accumulation of further epigenetic mutations by a cell within the
aberrant population (in this case expanded) during neoplastic progression may result in the emergence of a CSC.
In this model, only the CSCs (and not other tumour cells) are capable of sustaining tumorigenesis. Thus, the cell of
origin, in which tumorigenesis is initiated, may be distinct from the CSC, which propagates the tumour.

SLIDE 43 Differences in tumors (a), In the genetic (and epigenetic) mutation model, mutations primarily
determine the phenotype of the tumor, such that different mutations result in different tumor morphology. (b) In
the cell-of-origin model, different cell populations in the lineage hierarchy serve as cells of origin for the different
cancer subtypes arising within that organ or tissue

SLIDE 44 Microarray results Novel studies show that tumors are heterogonous even within the same body. It
has been demonstrated that tumors classified to the same group by traditional methods contain different mutant
genes. Only very few mutant genes were common the different tumor, most of the mutations were unique in each
tumors.

SLIDE 45 Driver and passenger mutations Mutations which participated in the causation of tumors are called
driver mutations, while passenger mutations are only by-products of tumorigenesis.

Parasite tumor cells

SLIDE 46 Cancer is not a contagious disease, although, infectious agents like viruses can contribute to the
origin of cancer. This paradigm appears to be not true in canine cancer called Stickers sarcoma. This tumor can
be transmitted both through sex and by licking or touching a tumor. Once established in a new host, it can produce
tumors that grow to the size of grapefruits before gradually disappearing. Many scientists once thought that the
disease, like cervical cancer, was spread by viruses. Now they know that the cancer cells themselves move from
dog to dog and have been spreading this way for centuries.

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