Download Slide 1

Book reading chapter 1st December 2006
Overview
•
Definition
•
The first two tumor-suppressor genes identified: Rb (Retinoblastoma and
the “two-hit hypothesis”) and p53
•
Mechanisms of inactivation of tumor suppressor genes
•
Other examples (NF-1 and VHL)
•
Key points and criteria to define a tumor suppressor gene
•
Anti-oncogenes
•
The genes affected by mutations in cancer are often divided into two classes: genes
that have gain-of-function (activating) mutations in cancer are known as oncogenes;
and genes for which both alleles have loss-of-function (inactivating) mutations in
cancer are known as tumor suppressor genes.
•
The unifying feature of tumor suppressor genes is the fact that: normally they function
to reduce the likelihood of cancer development (Weinberg)
•
They are called Gatekeeper genes to distinguish from Caretaker genes
•
Tumor suppressor genes are recessive at the cellular level, since complete loss of
function is needed to reveal phenotype
•
Germline mutations of tumor suppressor genes function dominantly at the organismic
level, predisposing the carrier to early onset of disease.
The normal role is to suppress increases in cell number either by
suppressing proliferation or by triggering apoptosis.
Table of tumor suppressor genes
Name
Chrom.
location
Familial cancer
syndrome
Sporadic cancer
Function of the protein
RUNX3
1q36
-
Gastric carcinoma
TF co-factor
FHIT
3p14.2
-
Many types
Hydrolase
RASSF1A
3p21.3
-
Many types
Multiple
TGFBR2
3p2.2
HNPCC
Colon, gastric, pancreatic
carcinomas
TGF-β receptor
VHL
3p25
von Hippel-Lindau
Renal cell carcinoma
Ubiquitination of HIFα
APC
5p21
Familial adenomatous
polyposis coli
Colorectal, pancreatic, stomach
and prostate carcinomas
β- catenin degradation
NKX3.1
8p21
-
Prostate carcinoma
Homeobox TF
p16ink4a
9p21
Familial melanoma
Many types
CDK inhibitor
p14ARF
9p21
-
All types
p53 stabilizer
PTEN
9q34
Cowden’s disease, breast
and gastrointestinal
carcinomas
Glioblastoma; prostate, breast and
thyroid carcinomas
PIP3 phosphatase
WT1
10q23.3
Wilms tumor
Wilms tumor
TF
RB
11p13
Retinoblastoma,
osteosarcoma
Retinoblastoma; sarcomas;
bladder, breast and lung
carcinomas
Transcriptional
repression; control E2F
TP53
17p13.1
Li-Fraumeni syndrome
Many types
TF
NF-1
17q11.2
Neurofibromatosis type 1
Colon carcinoma, astrocytoma,
breast, ovarian, prostate
Ras-GAP
By The biology of cancer, Weinberg
Knudson and the “two-hit hypothesis”
•
•
•
•
•
An introduction to genetic analysis, Griffith et al.
Rare, aggressive chilhood tumor of
retina
60% of cases are sporadic and
unilateral, whereas the remaining 40%
are familial bilateral
In 1971 Knudson proposed the “two-hit
hypothesis”. This mean that 2
successive mutation (hits) are needed
to turn a normal cells into a tumor one
In
hereditary
or
familial
retinoblastoma, one parent transmits
a mutated allele. The child is
heterozygose for this gene. Then a
second mutation occur in the second
allele of the same gene leading to LOH
(Loss Of Heterozygosity, loss of the
second allele) and cancer.
In non-hereditary or sporadic
retinoblastoma, allele inherited by both
parents are wt. Then a mutation occur
in one copy of the gene (first hit),
which is followed by the mutation or
loss of the second allele (second hit),
leading to LOH.
The “two-hit hypothesis”
In both forms of Retinobalstoma, tumor development requires that both
copies of the gene must be inactivated for cancer to occur.
2 HITS
• Tumor suppressor genes are recessive at the cellular level, since
complete loss of function (both copies inactivated) is needed to develop the
tumor.
• Germline mutations of tumor suppressor genes function dominantly at the
organismic level, predisposing to early and more serious tumor
development.
Mechanisms for eliminating the wt copies of tumor suppressor
genes
•
Mutations (frequency about 10-6 per cell generation)
•
Mitotic recombination (reciprocal exchange of information between paired
homologous chromosomes one of which carries the mutant allele)  LOH if
1 allele of the tumor suppressor gene is already mutated
Frequency about 10-4-10-5 per cell generation
•
Gene conversion (during DNA replication the DNA pol initially begins to use
a strand as a template for synthesis of a daughter strand of DNA, after DNA
pol continue the replication by jumping to the homologous chromosome,
and then jump again to the first one). If the first template is mutated  LOH
Non reciprocal exchange.
•
Breaking off (deletion) an entire chromosome region  Hemizygosity (one
copy left)
•
Promoter methylation (epigenetic mechanism)
Mitotic recombination
Gene conversion
DNA replication
Promoter methylation as a mechanism for tumor suppressor gene
inactivation
Methylation is the covalent attachment of methyl groups to cystoine bases.
• In mammalian methylation occurs at the CpG site.
• When methylation occurs in the proximity of a gene promoter, it can cause
repression of transcription of the associated gene.
• Is reversible.
Examples:
• In sporadic retinoblastoma Rb promoter is found to be methylated.
• Runx3 (implicated in stomach cancer development) is found to be methylated in
45-60% of these cancer but it’s never inactivated by mutation.
• RARβ2 in breast cancer.
Rb
• Rb family includes Rb, p107 and p130.
• pRb family proteins negatively regulates
the cell proliferation by repressing the
transcription of the genes for cell cycle
progression.
• Rb phosphorylation fluctuates throughout
the cell cycle:
• In G0and early G1 pRB is
underphosphorylated and in complex
with E2F TFs and their partner
proteins DP, repressing transcription.
E
• Upon phosphorylation (on Ser and
Thr) by cyclinD-cdk4-6 and cyclin Ecdk2 complexes, pRb release E2FDP complexes that induce the
transcription of genes required for the
entry in S phase.
• In mitosis pRb is dephosphorylated
by PP1α2
Modern genetic analysis, Griffith et al.
The Rb signaling pathway
The Rb signaling in human cancers
IINK4A loss %
CycD1 and CDK4
overexpression
RB loss %
Small cell lung
15
5% cycD1
80
Non-small cell lung
58
Pancreatic
80
Breast
31
50% cycD1
Glioblastoma
60
40% cdk4
T-ALL
75
Cancer types
Mantle cell lymphomas
20-30
90% cycD1
Table 7.2 of Pelegaris book
INK4A (p16), INK4B (p15), KIP1 (p27), RB
Tumor suppressor genes
CDK4, cyclin D1
Oncogenes
p53
•
•
•
•
•
•
•
•
TAD
SH3 BD
DNA binding core domain
NLSoligo NRD
The guardian of the genome
Second tumor suppressor genes identified Its inactivation is found in more than a half
of tumors
Transcription factor
Sensor of multiple cellular stresses (e.g., UV, X-ray, carcinogens, oncogenic stresses)
Regulates: cell cycle arrest, apoptosis and DNA repair.
Mdm2 is the negative regulator of p53 (E3 ligase activity so involved in p53
ubiquitination)
Li-Fraumeni syndrome is characterized by the inheritance of a p53 mutated allele
Inactivation occur through:
mis-sense mut
deletion
binding to viral proteins (T-antigen from SV-40 or E6 from HPV)
degradation by Mdm2 (often over-expressed in tumors)
Link: http:///www.iarc.fr/p53
the database of p53 mutations
The current version of the database is R11, released in October
2006.
The R11 release contains 23,544 somatic mutations, 376 germline
mutations and functional data on more than 2300 mutant proteins.
The p53 signaling pathway
Giono and Manfredi, J. of Cellular Phys. (2006)
NF-1
•
•
•
Neurofibromin
Neurofibromatosis type 1, which is a familial cancer syndrome associated
with mutation in the NF-1 gene
NF-1 is a Ras-GAP (GTPase –activating protein), forces Ras to convert
itself from the activated form (GTP-bound) to its inactive form (GDPbound)
Molecular biology of the cell, Alberts et al.
pVHL
•
•
Associated with von Hippel-Lindau syndrome (vascularization of retina,
hemangioblastoma,...)
Main function of pVHL protein is the inhibition of HIF-1 (Hipoxia-inducible
factor-1)
Futile cycle
Targeted therapy for metastatic renal cell carcinoma , Patel et al., BJC (2006)
Tumor suppressor genes: Key points
1.
Tumor suppressor genes’ loss usually affects cell phenotype only when
both copies are lost in a cell (recessive at the cellular level).
Haploinsufficiency can occur.
2.
The loss can occur either through genetic mutation or the epigenetic
silencing via promoter methylation.
3.
Inactivation (by mutation or methylation) of one copy may be followed by
other mechanisms that facilitate loss of the other gene copy (LOH
through mitotic recombination, deletion of a chromosomal region, gene
conversion)
4.
Tumor suppressor genes regulate cell proliferation through different
mechanisms (very different biological activities).
Tumor suppressor genes: Key points
5. The unifying theme is the fact that the loss of each one of these increases
the likelihood that a cell will undergo neoplastic transformation.
6. When mutant (defective) copies of tumor suppressor genes are inherited in
the germ line, there’s often increased susceptibility to cancers (Hereditary
retinoblastoma and Li-Fraumeni syndrome) dominant effect at the
organismic level.
7. Discovery of tumor suppressors helps to understand familial cancers since
oncogenes function in a dominant way at the cellular level, mutant alleles are
likely to perturb the embryo development.
8. Tumor suppressor genes are called Gatekeepers, since they are involved in
the regulation of cell proliferation. Do not confuse with Caretaker genes
involved in maintenance of genome integrity.
Candidate tumor suppressor genes: Criteria
•
Expression reduced or absent in cancer cells.
Maybe it reflects only the actions of normal differentiation or maybe it is only a
consequence of cancer and not a cause.
Restoration of expression of tumor suppressor gene may lead to reversion of the
phenotype only because of a too high level of expression.
Normal counterpart is very important and also difficult to define!!
•
Genetic criteria.
LOH in many tumor cell genomes and the derived protein should have clear
inactivating mutations.
•
Promoter methylation
There are cases in which LOH doesn’t occur (ex Runx3).
•
•
Knock out of a candidate in the mouse germline (tool for validation)
Functional criterion
THE VERY EXISTENCE OF A TUMOR SUPPRESSOR GENE BECOMES
APPARENT ONLY WHEN IT IS ABSENT