Download The Biology of Cancer

Robert A. Weinberg
The Biology of Cancer
First Edition
Chapter 7:
Tumor Suppressor Genes
Copyright © Garland Science 2007
張久瑗
中央研究院
生物醫學研究所
副研究員
1. Cancer Genomics
2. Tumor and
Tel. (02)2789-9046
Molecular Biology
Fax. (02)2785-8594
[email protected]
Outlines:
-Cell fusion: indicating that the cancer phenotype is recessive
-The retinoblastoma tumor
-Familial vs. sporadic forms
-Knudson’s two-hit theory
-Inactivation of TSGs
-LOH
-mutations
-promoter hypermethylation
-TSGs and proteins function in diverse ways
-pRB (Chap. 8) and cell cycle control
-p53 (Chap. 9) and apoptosis
-NF1 protein acts as a negative regulator of Ras signaling
-Apc facilitates egress of cells from colonic crypts
-Von Hippel-Lindau disease: pVHL modulates the hypoxic response
Hallmarks of Cancer
The Hallmarks of Cancer Cell (2000) 100:57
Rules for Making Human Tumor Cells N Engl J Med (2002) 14:1593
Normal vs. Transformed Cells
Normal 3T3 cells:
Elongated
Aligned and packed in an ordered fashion
RSV-transformed Cells:
more rounded and covered by
hair-like processes
Lost side-by-side organization
Cell fusion
Fusion of mouse 3T3 cells with monkey kidney cells
(PEG or inactivated
Sendai virus)
heterokaryon
(cultured under selection)
(a polykaryon containing 9 nuclei)
Figure 7.1 The Biology of Cancer (© Garland Science 2007)
Identification of recessive cancer-inducing alleles
(tumor suppressor genes)
(non-tumorigenic)
(from virus-induced tumors)
(tumorigenic)
(fom non-virus-induced human tumors, or
from chemically induced rodent tumord)
If the cancer cell had originally arisen without the involvement of a tumor virus, then its
malignant phenotype was recessive when this cell was fused with a normal cell.
Figure 7.3 The Biology of Cancer (© Garland Science 2007)
Pediatric retinoblastoma tumor
Sporadic form
-unilateral and unifocal
-once the tumor is eliminated, no further risk
Familiar 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
Pedigree of a kindred afflicted with Rb
(Affected individual: green-filled circles or squares)
-Conforms somatic dominant transmission pattern
Figure 7.4The Biology of Cancer (© Garland Science 2007)
Kinetics of Rb: unilateral vs. bilateral
Two-hit hypothesis:
Alfred Knudson concluded in 1971 that the rate
of appearance of familial tumors was consistent
with a single random event, while the sporadic
tumors behaved as if two random events were
required for their formation.
Figure 7.6 The Biology of Cancer (© Garland Science 2007)
Dynamics of retinoblastoma formation
Figure 7.7 The Biology of Cancer (© Garland Science 2007)
Germ-line vs. Somatic Mutations
-bilateral
-multi-focal
-unilateral
-uni-focal
Knudson proposed that two “hits”, or mutagenic events, were necessary for
retinoblastoma development in all cases.
Incidence of developing non-retinal tumors
of Rb patients
-Among the 1601 Rb patients diagnosed between 1914-1984, those cured of bilateral
tumors have a dramatically higher risk of developing second tumors
Figure 7.4The Biology of Cancer (© Garland Science 2007)
Knudson’s Two-Hit Hypothesis
NORMAL HEALTHY INDIVIDUAL
HEREDITARY RETINOBLASTOMA
inherited
mutant
Rb1 gene
occasional cell
inactivates one of its two
good Rb1 genes
occasional cell
inactivates its
only good Rb1
gene copy
NONHEREDITARY RETINOBLASTOMA
occasional cell
inactivates one of its
two good Rb1 genes
excessive cell
proliferation leading
to retinoblastoma
excessive cell
proliferation leading
to retinoblastoma
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).
Figure 7.8 The Biology of Cancer (© Garland Science 2007)
Mutant Rb genes are both dominant and recessive.
An individual who inherits a mutant, defective allele of Rb is almost certain to
develop retinoblastoma at some point in childhood. However, a cell carrying a
mutant and a wild-type Rb gene copy would behave normally.
The mutant Rb allele acts dominantly at the organismic level and recessively at
cellular level.
Gene conversion: another possible mechanism
to eliminate the wild-type copy of Rb gene?
Figure 7.9 The Biology of Cancer (© Garland Science 2007)
Chromosomal localization of Rb locus, 13q14
Figure 7.10 The Biology of Cancer (© Garland Science 2007)
Loss of heterozygosity:
esterase D locus serves as a surrogate marker for RB
Zymographic analysis of esterase D isoforms
Figure 7.11 The Biology of Cancer (© Garland Science 2007)
Mutations of Rb gene
Inactivation of Rb gene is achieved by the deletion of the wild-type
allele accompanied with the mutations in the second allele.
Figure 7.12 The Biology of Cancer (© Garland Science 2007)
Genetic Markers
(To qualify as a genetic marker, a locus must be polymorphic.)
RFLP (Restriction Fragment Length Polymorphisms):
-single-base or insertion/deletion polymorphisms lying within restriction enzyme recognition sites
-detected as variations in the lengths of restriction fragments
VNTR (Variable Number of Tandem Repeats)
-VNTRs of a common motif of 16 nucleotides or greater
Microsatellite polymorphisms (also called STRP, Short Tandem Repeat
Polymorphisms)
-Short tandem repeat motifs, term “microsatellites“
-arise from variation in the number of di-, tri-, or a larger number of nucleotides.
-have a large number of alleles (and are thus highly polymorphic)
-Normally occur once every 30 kb.
-More than 8900 microsatellite markers that span the entire human genome have been identified,
characterized, and mapped.
SNP (Single Nucleotide Polymorphisms)
-polymorphisms corresponding to differences at a single nucleotide position (e.g., substitutions,
deletions, and insertions)
-biallelic polymorphisms, generally less informative than STRPs.)
-In human genome, an SNP with a hetrozygosity greater than 30% occurs, on average, approximately
every 1.3 kb.
-powerful tools for searching for complex disease genes by association study or linkage
disequilibrium analysis
Restriction fragment length polymorphism
(RFLP)
Figure 7.13 The Biology of Cancer (© Garland Science 2007)
Loss of Heterozygosity:
Identifying Lost Tumor Suppressor Genes
Single nucleatide polymorphism (SNP)
Allele-specific primer extension reaction
Figure 7.15 The Biology of Cancer (© Garland Science 2007)
LOH of chromosomal arms in CRC:
High frequency of LOH allows the detection of putative TSG
Figure 7.14 The Biology of Cancer (© Garland Science 2007)
-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 TSGs creats an
enormous risk of
contracting one or
another specific type of
cancer. In some cases,
mutant germline alleles of
these genes lead to
susceptibility to multiple
cancer types.
Table 7.1 The Biology of Cancer (© Garland Science 2007)
Cancer Formation
• Clonal Expansion
-Tumor formation is driven by a multi-step process of genetic alterations.
-Each event confers growth advantage to the expanded cell population.
-Genetic alterations promote clonal evolution include activation of protooncogenes, inactivation of tumor suppressor genes, and de-regulation
of cell cycle regulators, etc.
• Mutator Phenotype
-Pre-existing mutations in genes involved in the maintenance of
genomic integrity drives cancer formation.
-Increasing genetic instability is observed in the process of tumor
progression.
-Events contributing to mutator phenotype include errors in DNA
replication, deficits in DNA repair, disregulation of checkpoint control,
and abnormalities in chromosomal segregation, etc.
Gatekeeper vs. Caretaker
Gatekeeper are TSGs that directly control the biology of cells by affecting proliferation,
differentiation, and apoptosis, and therefore.
The name of gatekeeper indicate their role in allowing or disallowing cells to progress
through cell cycles of growth and division.
Inactivation of these genes is rate-limiting for the initiation of tumor formation, and both
copies must be altered for tumor development.
Caretaker are DNA maintenance genes that affect cell biology indirectly by controlling the rate
at which cells accumulate mutant genes.
The name of Caretaker reflects their role in the meintenance of cellular genomes.
Inactivation of TSG:
Genetic and epigenetic alterations
Hypermethylation of RASSF1A promoter
Figure 7.17 The Biology of Cancer (© Garland Science 2007)
Table 7.2 The Biology of Cancer (© Garland Science 2007)
Transcription silencing of TSGs in tumors
Figure 7.19 The Biology of Cancer (© Garland Science 2007)
NF1: a negative regulator of Ras signaling
Figure 7.21 The Biology of Cancer (© Garland Science 2007)
Familial adenomatous polyposis (FAP)
the wall of colon from a
FAP patient
polyps
the wall of a normal colon
Adenomatous growths, polys in colon
-Pedunculated polys are shown as stalk-like growths.
Sessil polys appearing as flat thickenings of colonic
wall are not shown.
Figure 7.23 The Biology of Cancer (© Garland Science 2007)
Disease Gene Localization
Genetic mapping using large FAP kindreds
Figure 7.23 The Biology of Cancer (© Garland Science 2007)
Linkage analysis: association of phenotype to genotype
the usage of polymorphic markers
APC (adenomatosis polyposis coli)
at chromosome 5q21
Figure 7.25a The Biology of Cancer (© Garland Science 2007)
β-catenin interacts with alternative partner
proteins
APC
β-catenin
Figure 7.25b The Biology of Cancer (© Garland Science 2007)
Canonical Wnt pathway
Wnt pathway off
Wnt pathway on
E-cadherin
E-cadherin
Wnt
Arrow/
LRP5/6
Arrow/
LRP5/6
Frizzled
Frizzled
β-catenin
Dsh
Axin
Dsh
β-catenin
GSK3
Axin conductin
GSK3 APC
β-catenin
APC
β-catenin
β-catenin
HDAC Groucho
TCF
TCF
Cyclin D1
PPARδ
C-myc
Matrilysin
CD44
Tcf-1
Role of β-catenin/TCF pathway
IHC of Ki67
enterocytes
lumen of intestine
mesenchymal core
bottom of crypts
TCF4+/-
Figure 7.24a The Biology of Cancer (© Garland Science 2007)
Role of β-catenin/TCF pathway
IHC of β-catenin in Min mouse
DAPI stains nuclei.
Figure 7.24-25 The Biology of Cancer (© Garland Science 2007)
IHC of Ki67 in TCF-mice
Histopathology of colorectal cancer
Nat Rev Cancer 1: 55 (2001)
Mutation of APC/β-Catenin pathway in human colon tumors
Mitotic spindle in relation to APC and CIN
APC accumulates at the
kinetochore through interaction with
the microtubule-associated protein
EB, where it may facilitate the
binding of spindle microtubules to
kinetochore.
Interaction of kinetochores and
spindle microtubule is disrupted,
leading to chromosomal instability.
Von Hippel-Lindau (VHL): LOH of 3q25
Nat Rev Cancer 2: 673 (2002)
Ubiquitination and proteasome-mediated degradation
Ubi
Figure 7.26a The Biology of Cancer (© Garland Science 2007)
Regulation of HIF-1 by pVHL
angiogenesis
pVHL is a component of an E3 ubiquitin ligase that targets the a subunits of HIF (hypoxia-inducible
factor) transcription factor for destruction in the presence of oxygen.
Figure 7.28a The Biology of Cancer (© Garland Science 2007)
Table 7.3 The Biology of Cancer (© Garland Science 2007)