Download Biology of Cancer

Robert A. Weinberg
The Biology of Cancer
Second Edition
CHAPTER 7
Tumor Suppressor Genes
Copyright © Garland Science 2014
Tumor suppressor genes
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Tumor suppressor genes are targeted by the loss of function
mutations in cancer.
The DNA repair genes are often affected by the LOH; a subset of TSG,
“ Mutator” phenotypes; increase the mutations on genes involving in
affecting cell proliferation, survival etc
Identification of TSG has proven more difficult compared to that of
oncogenes
Knudson’s epigenetic studies leads to propose : “two-hit hypothesis”
that two inactivating mutations were necessary for retinoblastoma
development
More than 20 TSG have been identified by cytogenetic studies,
linkage analysis to localize gene that predispose to cancer, LOH,
allelic loss studies between normal and cancer tissues
The authentic of a TSG is established by the identification if
inactivating germ line mutations that segregate with cancer
predisposition, coupled with the identification of somatic mutations
in cancer also.
Somatic cell studies
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Identification of oncogenic allele in tumor; identification
of retroviral oncogenes, molecular cloning of
oncogenic DNA seq at chromosomal breakpoints, or
DNA tranfection assay with DNA derived from cancers
But identification of the TSG are far more difficult.
Why ?
Somatic cell studies
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Method; “Cell Fusion Technique” Hybrid
cell fusion of malignant cells with
nonmalignant cells, or one
chromosome,--reverted the
tumoregenicity, the malignancy was
suppressed in hybrid cells or one
chromosome; loss of specific
chromosome or genes
Cell fusion experiments shows that Wt
genes antagonize the cancer
phenotypes and this indicates that the
cancer phenotypes is “recessive”.
Recessive means loss of both alleles of
TSG
Loss of tumor suppressor protein
function by mutation is lot more easier
than hyperactive mutation of
oncogenes induced by mutation.
An overly broad definition of TSG;
transferred genes suppressed at least
some of phenotypic properties in
cancer cells. “ not always become TSG”
Pediatric Retinonlastoma
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Retinoblastoma is the most
common intraocular malignancy
in children (worldwide incidence
between 1 in 13,500 and 1 in
25,000 live births)
Tumor arises from an
oligopoteintial stem cell
precursor of multiple retinal cell
types
-> late onset retinoblastoma,
13q-deletion syndrome,
retinoma, bilateral
retinoblastoma, and second-site
primary tumors (osteosarcoma,
Ewing sarcoma, leukemia,
lymphoma)
Figure 7.4a The Biology of Cancer (© Garland Science 2007)
Unilateral verus bilateral Rb
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Bilateral Retinblastoma showed other
types of cancer osteosarcoma, Ewing
sarcoma, leukemia, lymphoma
Unilateral Rb showed no other tumors.
Sporadic tumors Why?
Pedigree shows multiple generations of
a kindred afflicted with familial Rb
- confirmation of involvement of
genetic alteration cytogenetic analysis.
microscopically visible deletion of one
chromosome 13, band q14
-> autosomal dominant hereditary form
of retinoblastoma : large deletions of
chromosome 13
-> germ-line mutation in hereditary
retinoblastoma / somatic genetic
alteration of the RB1 locus in a retinal
cell in nonhereditary retinoblastoma
Figure 7.5b The Biology of Cancer (© Garland Science 2007)
Knudson’ “Two-hit hypothesis”
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The rate of familiar tumors was consistent
with a single random event, while the
sporadis tumors behaved as if two
random events were required for their
formation
Predicted that one of allele of the Rb
genes in familial Rb carries mutant form
The significances; illustrate the
mechanism that inherited and somatic
changes may collaborate in tumor
formation, liked the notion of recessive
genetic determinants for human cancer to
the somatic cell genetic studies
10-6 mutation rate/ cell division
10-6 /cell division
Figure 7.6 The Biology of Cancer (© Garland Science 2007)
10-12 /cell division
Loss of second allele of Rb is likely due to another mechanism
rather than random mutation: 10-4 - 10-5 cell division.
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How does cell eliminate the wild-type of genes ?
mitotic recombination between the RB1 locus encoding the mutant allele
and the centromere -> heterozygosity at loci in the proximal region and
homozygosity throughout the rest of the chromosome
Figure 7.8 The Biology of Cancer (© Garland Science 2007)
Loss of second allele of Rb is likely due to another mechanism
rather than random mutation: 10-4 - 10-5 cell division.
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several other more regionalized events such as gene conversion, deletion or
mutation
mitotic non-disjunction with loss of the wild-type chromosome ->hemizygosity at all
loci on chromosome 13
mitotic nondisjunction with duplication of the mutant chromosome
Figure 7.8 The Biology of Cancer (© Garland Science 2007)
Loss of heterozygousity (LOH)
Loss of both alleles of Rb: Esterase D isoform linked to Rb,
Restriction fragment length polymorphisms, PCR method
Loss of Rb genes in tumors
Figure 7.11 The Biology of Cancer (© Garland Science 2007)
PCR is able to map tumor suppressor genes
rapidly
Figure 7.15 The Biology of Cancer (© Garland Science 2007)
Approaches to identify TSGs
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Cytogenetic studies to identify constitutional chromosomal
alterations in cancer patients; deletion of specific chromosome or
specific region.
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But small portion of cancer cells; Difficulty
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Ex) Familial Rb; Ch13q 14 in blood sample or skin fibroblasts, Wilms
tumor; 11q13, adenomatous intestinal polyps; 5q
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Linkage analysis; trace the genetic markers from implicated
chromosomal region co-segregated with the inheritance of the
disease phenotypes; pinpoint the location of the TSG
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LOH studies; ex) Rb-esterase D; family with Rb(+/-) showed
decreased level of Esterase D. True in somatic patient.
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13q12; BRAC2, 18q21.1; DPC4
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Positional cloning of the TSGs
Loss of Heterozygousity of
chromosomal arms in colon cancers
Figure 7.14 The Biology of Cancer (© Garland Science 2007)
Measurement of deleted chromosomal segments
carrying TSGs
Tumor suppressor can be also inactivated by
methylation of its promoter region (CpG sites)
Epigenetic
Figure 7.16 The Biology of Cancer (© Garland Science 2007)
Expression of the DNMT3B enzyme in colon cancer
Methylation of the promoter of
p16 INK4A In Situ hybridization)
Figure 7.18 The Biology of Cancer (© Garland Science 2007)
Table 7.2 The Biology of Cancer (© Garland Science 2007)
Methylation of multiple genes within tumor cell
genomes
Figure 7.19 The Biology of Cancer (© Garland Science 2007)
NF1 (Neurofibromatosis type 1)
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A common autosomal
dominant disoder(1/3000
person)
Also develop Glioblastoma
(astrocyte lineage tumor),
pheochromocytomas(adrenal
glands), and myeolgenous
leukemia(CML)
Showed Café au lait spots:
hyperpigmentation, Lisch
nodules, distinctive boney
lesions etc
Chromsome 17q11.2
MW. 2818 amino acids,
structural and functional
similarity to a family of RasGAP, down-regulate the rasGTP
Figure 7.20a The Biology of Cancer (© Garland Science 2007)
NF1 acts as an negative regulator of Ras
signaling
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Function as Ras-GAP protein
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After growth factor stimuli, NF1
rapidly is degraded, but NF1
levels return back to normal to
shut down further the Rassignaling: Negative feedback
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NF1+/- cells, elevated the level
of Ras-GTP , substantially
increasing the level of Rassignaling:
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“Haploinsufficiency” : p27,
smad4, Pten etc, but not Rb,
p53
Figure 7.21 The Biology of Cancer (© Garland Science 2007)
Colorectal cancer
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a series ranging from single crypt lesions(aberrant crypt foci)through
small benign tumors (adenomatous polyps) to malignant
cancers(carcinomas)
inherited factor : HNPCC(hereditary nonpolyposis colorectal cancer)
and FAP( familial adenomatous polyposis)
activation of RAS oncogenes (50 % of colorectal cancers, c-Kis-RAS,
N-RAS )and inactivation tumor suppressor genes on ch 5q, 17p (DCC,
SAMD4/DPC4, SMAD2), 18q
mutation of APC-tumor suppressor gene on ch 5q, increased βcatenin/Tcf-mediated transcription
several inherited predispositions can result from inheritance of a single
defective gene.
-caretakers (DNA MMR gene in HNPCC),
- gatekeepers(APC),
definition of a model for colorectal tumor development
- mutations of APC→K-RAS→18q suppressor and p53
accumulation of genetic changes is facilitated by a chromosomal
instability
. Inherited Predisposition
1. presence of polyposis(FAP)
• develop hundreds to thousands of adenomatous polyps during their
lifetime
• colorectal cancer develop at median age of about 40
• increased risk for thyroid, small intestine, stomach, and brain
• GS(Gardner syndrome), and Turcot syndrom are variants of FAP
• - germ-line mutations of the APC gene
2. absence of polyposis (HNPCC)
• - develop at median age of about 42 years
• - defect in DNA MMR genes
• - at increased risk for uterus, ovary, brain and other cancers
• Other genetic Factors and environmental factors(diets) are
associated with increased risk for colorectal cancer
Familiar Adenomatous polyposis(FAP)
Positional cloning of APC in heritable cancers in the
Mormon populations
Figure 7.22 The Biology of Cancer (© Garland Science 2007)
Chromosome 5q : The APC gene
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expression increasing as cells migrate to the top of the crypt
β-catenin, γ-catenin, GSK-3β, AXIN family proteins, EB-1 and hDLG bind to the
C-terminus of APC
→ all APC mutations result in loss of the C-terminus of APC → no interaction.
β-catenin binding to APC : two cellular processes
cellular adhesion : binding of β-catenin to cadherins or to APC is mutually
exclusive.
Wg/Wnt pathway
- Wnt signaing inhibits ZW3/GSK3β protein kinase
- ZW3/GSK3β promote β-catenin binding APC
→ ubiquitin-dependent proteosomal degradation of β-catenin (inhibit βcatenin/Tcf mediated transcription )
① mutation of APC : prevent its inhibition of β-catenin
② dominant activating β-catenin mutations that render the protein insensitive to
APC/GSK-3β-mediated degradation
- increased β-catenin/Tcf mediated transcription results in expression of genes
that promote cell growth of inhibit cell death (ex. c-MYC)
- APC binds to tubulin and stabilize the microtubule, possibly involving the
chromosomal stability
- APC homologue(APCL) shares APC’s ability to interact with β-catenin is
redundancy
APC and beta catenin in intestinal lumen formation
Figure 7.24a The Biology of Cancer (© Garland Science 2007)
Figure 7.25b The Biology of Cancer (© Garland Science 2007)
Von Hippel –Lindau diseases: pVHL modulates the
hypoxic response
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VHL: develops many caner types
(kidney, blood vessel tumor etc)
Inactivated in many kidney
carcinoma
Figure 7.25c The Biology of Cancer (© Garland Science 2007)
VEGF mRNA
Insights into rate-limiting mutations.
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Rate limiting step; mutations that cause both
significant expansion of a variety clone—increased
the proliferation and survival. But Most of mutations
are deterimental.
Rate-limiting mutations; expanding populations of
precancerous cells
Ex) Rb germ line mutation of one copy; not
associated with any cancerous effect, but mutation
of second copy(LOH); an early rate-limiting event in
the cancer formation.
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Lung cancer; Rb mutation is late event
Tissue specific effects of germ line
mutations.
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Germ line mutations of TSGs are predisposed to a
limited spectrum of cancer types; Puzzle ?
Ex) Rb, BRCA1, colon cancer
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Rb; Rb heterozygous develop the retinoblastoma,
some osteocarsinoma, but mutations are found in
many cancer types
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Rb may be a controller in retinoblast growth, but less
critical controller in other tissues.
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Rb mutations may contribute the tumor progression
other than initiate tumor formation; Rb mutations
without other mutations lead to cell death.
Candidate tumor suppressor genes
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Inactivated in somatic cancer cells, not found in familial cancers,
need to be determine the function of these genes (initiation
progression of cancers).
Ch1p, 3p, 8p,10q, 17q, 22q regions have a putative tumor
suppressor
ex) 18q- allelic loss in many cancers (gastric, colorectal, pancreatic
etc); 18q21.1 – 20-25 % in pancrearic cancers; DPC4 genestranscription factor in TGF signaling-, mutation was found in
juvenile polyposis syndrome(JPS).
Several KO mice displaced increased spontaneous cancers; tumor
suppressor; true in human cancers ?
Difference between human and mice ex) BRCA1 +/-; no tumor
Gene Knockout in mice; 마우스에 유전적 돌연변이를 유도하는 기술
ICM
Injection
E1
Replacement vector
Implantation
x
Homologous
recombination
ES cells
neo
E4
TK
Targeting vector
x
Genomic locus
E1 E2 E3
E1
neo
E4
E4
Targeted locus
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Chimeric
mice
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x
x
Select for drug
resistance
Cre-LoxP Tissue specific or conditional targeting vector
TK
Neo
Homologous
recombination
X
Cre
Tet on/Off
promoter
X
Cre
F1(+/-)
E1
E2 E3 E4
X
Neo
Targeted ES cells
(+/-)
F2 (-/-)
형질변화 분석
Tissue specific
promoter
Cre expression &
recombination
Cre-TG
Cre –TG
KO mice
Retina
Rb WT
Rb -/-
H3p
Postmitotic differentiated cells
Cerebellum
Outer layer
Inner layer
Figure 7.32 The Biology of Cancer (© Garland Science 2007)