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)
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
(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).
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
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.
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.
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.
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
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.
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
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
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
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.