Download p16INK4A tumor suppressor gene

The genetic basis for the
development of cancer
Molecular Biology of Cancer
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Cancers arise through a multistage process
in which inherited and somatic mutations of
cellular genes lead to clonal selection of
variant progeny with the most robust and
aggressive growth properties.
Two classes of genes are targets for the
mutations:
Protooncogenes
tumor-suppressor genes
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The vast majority of the mutations that contribute
to the development and behavior of cancer cells
are somatic (ie, arising during tumor development)
present only in the neoplastic cells of the patient
A small fraction of all mutations in cancer cells
are constitutional
present in all somatic cells of affected individuals
such mutations not only predispose to cancer, but can
also be passed on to future generations.
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Tumor Suppressor Genes
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A large number of tumor suppressor genes
have been hypothesized to exist
Thus far, approximately 20 tumorsuppressor genes have been identified and
definitively implicated in cancer
development.
The cellular functions of the tumorsuppressor genes appear to be diverse
Molecular Biology of Cancer
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Cancer-inducing genes, specifically viral
oncogenes, act in a dominant fashion
Viral oncogenes dictate cellular behavior in
spite of the continued presence and expression
of opposing cellular genes within the virusinfected cell that usually functioned to ensure
normal cell proliferation.
The viral genes were able to induce a dominant
phenotype—they were bringing about a cell
transformation.
Most human cancers do not seem to arise as
consequences of tumor virus infections
Molecular Biology of Cancer
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Cell fusion experiments indicate that
the cancer phenotype is recessive
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fusion of a cancer cell with a wild-type
cell
 The resulting hybrid cells have
lost the ability to form tumors
when these hybrid cells were
injected into appropriate host
animals.
 This, unexpectedly, mean that the
malignant cell phenotype is
recessive to the phenotype of
normal, wild-type growth.
 Exception: when the
transformed parental cell had
been transformed by tumor
virus infection.
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Tumor suppressor genes (TSGs)
Hypothesis
 Normal cells carry genes that constrain or suppress
their proliferation.
During the development of a tumor, the evolving cancer
cells inactivate one or more of these genes.
Once these growth-suppressing genes are lost, the
proliferation of the cancer cells accelerates.
 As long as the cancer cell lacks these genes, it
continues to proliferate in a malignant fashion.
When wild-type, intact versions of these genes operate
once again within the cancer cell (by cell fusion) it will
loose its ability to proliferate or to form tumors.
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The retinoblastoma tumor is the first
example of tumor suppressor genes
Sporadic form:
unilateral and unifocal
once the tumor is eliminated, no further
risk
Familial form
bilateral and often multi-focal
curing the eye tumor does not protect
the children from a greatly increased
risk to bone cancers and other
cancers
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Familial form
The familial form of
retinoblastoma is
passed from one
generation to the next
in a fashion that
conforms to the
behavior of a Mendelian
dominant allele.
Molecular
Figure 7.5b The Biology of Cancer (© Garland Science
2007)
Biology of Cancer
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Kinetics of Rb: familial (bilateral) vs.
sporadic (unilateral)
the rate of appearance of
familial tumors was consistent
with a single random event
(mutation)
the sporadic tumors behaved
as if two random events were
required
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Two-hit hypothesis
Two “hits” or mutagenic events were necessary
for retinoblastoma development
In an individual with the inherited form:
the first hit is present in the germ line, and thus in all
cells of the body.
a second somatic mutation was hypothesized to be
necessary for promoting tumor formation.
 The second mutation explain the behavior of a Mendelian
dominant allele.
In the nonhereditary form:
both mutations were proposed to arise somatically
within the same cell.
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Two-hit hypothesis
 Each of the two hits
could theoretically be
in different genes
 Subsequent studies led
to the conclusion that
both hits were at the
same genetic locus,
ultimately inactivating
both alleles of the
retinoblastoma (RB1)
susceptibility gene
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Loss of Rb heterozygosoty (LOH)
Mitotic recombination: a possible mechanism to eliminate
the wild-type copy of Rb gene
The probability of inactivating a single gene copy
by mutation is on the order of 10-6 per cell
generation
The probability of silencing both copies is on the
order of 10-12 per cell generation.
It seems highly unlikely that both copies of the Rb
gene could be eliminated through two recessive
mutational event in the relatively small target cell
populations in the developing retina (about 106
cells).
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Loss of Rb heterozygosoty (LOH)
Mitotic recombination: a possible mechanism to eliminate
the wild-type copy of Rb gene
This mitotic recombination was found to occur at
a frequency of 10–5 to 10–4 per cell generation
easier way for a cell to rid itself of the remaining wildtype copy of the Rb gene than mutational inactivation of
this gene copy, which, as mentioned above, was known
to occur at a frequency of about 10–6 per cell generation.
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Loss of Rb heterozygosoty (LOH)
Gene conversion: another possible mechanism to
eliminate the wild-type copy of Rb gene
• When gene conversion involve copying of an already
inactivated Rb allele, for example, then once again LOH will
have occurred in this chromosomal region.
• known to occur
even more
frequently per cell
generation than
does mitotic
recombination.
Molecular Biology of Cancer
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The Rb gene often undergoes loss of
heterozygosity in tumors
In a small number of
retinal tumors, careful
karyotypic revealed
interstitial deletions
within the long (“q”) arm
of Chromosome 13.
All of these deletions
caused the loss of
chromosomal material in
the 4th band of the 1st
region of this chromosomal
arm (13q14).
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A number of genes (including Rb) in this region
had been lost simultaneously by the developing
retinal tumor cells.
This is precisely the outcome predicted by the
tumor suppressor gene theory.
LOH achieved by simply breaking off and
discarding an entire chromosomal region without
replacing it with a copy duplicated from the other,
homologous chromosome is called hemizygosity
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Mutations of the Rb gene
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LOH of chromosomal arms in CRC
High frequency of LOH allows the detection of putative TSG
Molecular Biology of Cancer
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Molecular Biology of Cancer
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Molecular
Biology
Table 7.1 part 2 of 2 The Biology of Cancer (© Garland
Science 2007)
of Cancer
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Many familial cancers can be explained by
inheritance of mutant tumor suppressor genes
These genes specify a diverse array of proteins
that operate in many different intracellular sites
to reduce the risk of cancer.
An anti-cancer function is the only property that is
shared by these otherwise unrelated genes.
Many familial cancers can be explained by
inheritance of mutant TSGs.
Inheritance of defective copies of most TSGs
creates an enormously increased risk for cancer
Often a type of relatively rare tumors
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There are two distinct classes of
familial cancer genes
Gatekeepers:
Tumor suppressor genes that function to directly control
the biology of cells (proliferation, differentiation, or
apoptosis)
Caretakers:
The DNA maintenance genes affect cell biology only
indirectly by controlling the rate at which cells
accumulate mutant genes
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Promoter methylation represents an important
mechanism for inactivating tumor suppressor
genes
DNA molecules can be altered covalently by the
attachment of methyl groups to cytosine bases.
This modification of the genomic DNA is as important as
mutation in shutting down tumor suppressor genes.
In mammalian cells, this methylation is found only
when these bases are located in a position that is
5’ to guanosines, that is, in the sequence CpG.
Such methylation can affect the functioning of the
DNA in this region of the chromosome.
When CpG methylation occurs in the vicinity of a
gene promoter, it can cause repression of
transcription of the associated gene.
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• Analyses of five DNA samples from tumor 232 indicate methylation at almost all
CpG sites in the RASSF1A CpG island
• Adjacent, ostensibly normal tissue is unmethylated in most but not all analyses of
this CpG island.
• Analyses of control DNA from a normal individual indicate the absence of any
methylation of the CpGs in this CpG island.
Molecular Biology of Cancer
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More than half of the tumor suppressor
genes that are involved in familial cancer
syndromes because of germ-line mutation
have been found to be silenced in sporadic
cancers by promoter methylation.
Ex. Rb germ line mutations  familial
retinoblastoma.
Rb somatic mutations or promoter methylation
 sporadic retinoblastomas.
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The elimination of tumor suppressor gene
function by promoter methylation
1. One copy might be
methylated and the second
might then be lost through
a loss of heterozygosity
(LOH) accompanied by a
duplication of the alreadymethylated tumor
suppressor gene copy
2. Both copies of a tumor
suppressor gene might be
methylated independently
of one another
Molecular Biology of Cancer
1st hit
Methylation
LOH
Methylation
(2nd hit)
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Example:
p16INK4A tumor suppressor gene
 In a study of the normal bronchial (large airway)
epithelia of the lungs:
p16INK4A methylation:
 in 44% of (ostensibly normal) bronchial epithelial cells cultured
from current and former smokers
 not at all in the comparable cells prepared from those who had
never smoked.
LOH in this chromosomal region:
 in 71 to 73% of the two smoking populations
 in 1.5 to 1.7% of never-smokers.
 Conclusion: methylation of critical growthcontrolling genes often occurs early in the complex,
multi-step process of tumor formation, long before
histological changes are apparent in a tissue
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Molecular Biology of Cancer
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Silencing of genes through promoter methylation
can also involve the “caretaker” genes
Example: the BRCA1 gene:
Its product  maintaining the chromosomal
DNA in ways that are still poorly understood.
Inheritance of mutant alleles of BRCA1  a high
lifetime risk of familial breast and, to a lesser
extent, ovarian cancer syndrome.
10 to 15% of sporadic breast carcinomas carry
inactive BRCA1 gene copies that have been
silenced through promoter methylation.
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• Promoter methylation is common during the development of
a wide variety of human cancers
• the frequency of methylation of a specific gene varies
dramatically from one type of tumor to the next.
• Tumor suppressor genes as well as caretaker genes
undergo hypermethylation.
• Perhaps the
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The (Neurofibromin ) NF1
protein acts as a negative
regulator of Ras signaling
• When cells stimulated by
growth factor, they may
degrade NF1, enabling Ras
signaling to proceed without
interference by NF1.
• After 60 to 90 minutes, NF1
levels return to normal, and
the NF1 protein that
accumulates helps to shut
down further Ras signaling in
a form of negative-feedback
control
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 Neurofibromatosis type 1 is a relatively common
familial cancer syndrome, with 1 in 3500 individuals
affected on average worldwide.
 The primary feature of this disease is the
development of benign neurofibromas of the cell
sheaths around nerves in the peripheral nervous
system
 On occasion, a subclass of these neurofibromas,
progress to malignant tumors termed
neurofibrosarcomas.
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 In neuroectodermal cells lacking NF1 function, Ras
proteins are predicted to exist in their activated,
GTP-bound state for longer than- normal periods of
times.
 In fact, in the cells of neurofibromas, which are
genetically NF1–/–, elevated levels of activated Ras
and Ras effector proteins can be found
 Consequently, the loss of NF1 function in a cell can
mimic functionally the activated Ras proteins that
are created by mutant ras oncogenes.
Molecular Biology of Cancer
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