Robert A.

Weinberg

The Biology of Cancer

First Edition

Chapter 7: Tumor Suppressor Genes

Copyright Garland Science 2007

Outlines:
-Cell fusion: cancer phenotype is recessive -TSG: Knudsons two-hit theory -Retinoblastoma tumor -Familial vs. sporadic forms -Inactivation of TSGs -LOH -mutations -promoter hypermethylation -TSGs and proteins function in diverse ways -pRB (Chap. 8) -p53 (Chap. 9) -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

Identification of recessive cancer-inducing alleles (tumor suppressor genes)

(non-tumorigenic)

(tumorigenic)

Figure 7.3 The Biology of Cancer ( Garland Science 2007)

Identification of recessive cancer-inducing alleles (tumor suppressor genes)

(non-tumorigenic)

(tumorigenic)

(from non-virus-induced human tumors, or from chemically induced rodent tumord)

(from virus-induced tumors)

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

Familial form
-bilateral and often multi-focal

Figure 7.4The Biology of Cancer ( Garland Science 2007)

Kinetics of Rb: unilateral vs. bilateral

Two-hit hypothesis: (proposed by Alfred Knudson in 1971)
The rate of appearance of Rb: familial tumors: consistent with a single random event sporadic tumors: behaved as if two random events

Figure 7.6 The Biology of Cancer ( Garland Science 2007)

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)

Germ-line vs. Somatic Mutations

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

Knudson proposed that two hits, or mutagenic events, were necessary for retinoblastoma development in all cases.

Figure 7.7 The Biology of Cancer ( Garland Science 2007)

Knudsons Two-Hit Hypothesis

NORMAL HEALTHY INDIVIDUAL HEREDITARY RETINOBLASTOMA NONHEREDITARY RETINOBLASTOMA

inherited mutant Rb1 gene

occasional cell inactivates one of its two good Rb1 genes

occasional cell inactivates one of its two good Rb1 genes

occasional cell inactivates its only good Rb1 gene copy

excessive cell proliferation leading to retinoblastoma excessive cell proliferation leading to retinoblastoma

Autosomal dominant mutation

Autosomal dominant mutation of Huntington disease: Both males and females can be affected, and only one allele is needed to confer the mutant phenotype. If either parent is heterozygous for the mutant allele, their children have a 50% chance of inheriting the mutant allele and getting the disease.

Autosomal recessive mutation

Autosomal recessive mutation of cystic fibrosis: Both males and females can be affected but must carry two mutant alleles of the gene to show mutant phenotype. Both parents must be heterozygous carriers for their children to be at risk of being affected.

Sex-linked recessive mutation

Sex-linked Duchenne muscular dystrophy: Males born to mothers heterozygous for a DMD mutation have a 50% chance of inheriting the mutant allele and being affected. Females born to heterozygous mothers have a 50% chance being carriers.

Somatic dominant transmission pattern of familial Rb

Pedigree of a kindred afflicted with Rb

(Affected individual: green-filled circles or squares) 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.

Chromosomal localization of Rb locus, 13q14

Figure 7.10 The Biology of Cancer ( Garland Science 2007)

Rb gene mutation predisposes to osteosarcoma

Rb

Structurally altered Rb gene was found in osteosarcoma.

Figure 7.12 The Biology of Cancer ( Garland Science 2007)

Tumors are monoclonal in origin

Figure 2.18 The Biology of Cancer ( Garland Science 2007)

esterase D locus serves as a surrogate marker for RB

Zymographic analysis of esterase D isoforms

Loss of heterozygosity:

Figure 7.11 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)

Identifying Lost Tumor Suppressor Genes

Loss of Heterozygosity:

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)

Linkage analysis: association of phenotype to genotype

the usage of polymorphic markers

1 2

hetero-

homo-

Disease Gene Localization

-Loss vs. increased cancer risk -an anti-cancer function -Many familial cancers can be explained by inheritance of mutant TSGs.

Table 7.1 The Biology of Cancer ( Garland Science 2007)

Cancer Formation
Multi-step tumorigenic process
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 deregulation 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.

Cancer stem cells

A small population of undifferentiated cells present in the primary tumors with capabilities of self-renewal and pluripotency is responsible for tumor progression

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

Epigenetic modification

-non-genetic changes -does not alter the nucleotide sequence of the DNA -Inactivation of TSG can be achieved by promoter methylation and/or promoter deacetylation -More than half of the TSGs are found to be silenced in sporadic cancers by promoter methylation. -The promoters of a variety of genes that inhibit tumor formation are also found in methylated status.

Maintenance of DNA methylation

Figure 7.16 The Biology of Cancer ( Garland Science 2007)

Hypermethylation of RASSF1A promoter

RASSF1A encodes a protein similar to the RAS effector proteins. The encoded protein was found to interact with DNA repair protein XPA. The protein was also shown to inhibit the accumulation of cyclin D1, and thus induce cell cycle arrest. Loss or altered expression of this gene is associated with the pathogenesis of a variety of cancers. The inactivation of this gene is correlated with the hypermethylation of its CpG-island promoter region.

Figure 7.17 The Biology of Cancer ( Garland Science 2007)

Table 7.2 The Biology of Cancer ( Garland Science 2007)

ISH: a measurement of DNA methylation

(In situ hybridization using a methylation-specific PCR reaction and in situ hybridization for p16INK4A promoter)
low-grade cervical ca. high-grade

normal breast

normal breast

Figure 7.18 The Biology of Cancer ( Garland Science 2007)

Transcription silencing of TSGs

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, polyps 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)

Genetic mapping using large FAP kindreds

Figure 7.23 The Biology of Cancer ( Garland Science 2007)

APC (adenomatosis polyposis coli)

at chromosome 5q21

Figure 7.25a The Biology of Cancer ( Garland Science 2007)

Canonical Wnt pathway

Wnt pathway off
E-cadherin Wnt Arrow/ LRP5/6 Frizzled Frizzled Dsh Dsh GSK3 Axin conductin GSK3 APC
(destruction complex)

Wnt pathway on
E-cadherin Arrow/ LRP5/6

-catenin -catenin
APC

Axin

-catenin -catenin -catenin

HDAC Groucho

TCF

TCF

Cyclin D1 PPAR C-myc Matrilysin CD44 Tcf-1

-catenin interacts with alternative partner proteins

CytoCyto-

Nuc.

2 3

-catenin

APC

Figure 7.25b The Biology of Cancer ( Garland Science 2007)

Role of -catenin/TCF pathway

IHC of Ki67 in TCF-mice
enterocytes
lumen of intestine

mesenchymal core

bottom of crypts

TCF4-/-

TCF4+/-

IHC of -catenin in Min mouse

Figure 7.24a The Biology of Cancer ( Garland Science 2007)

DAPI stain shows nuclei.

Histopathology of colorectal cancer

Nat Rev Cancer 1: 55 (2001)

Mutation of APC/b-Catenin pathway in human colon tumors

* * * * * * * * * * * *
(n = 16 adenomas and 25 carcinomas)

* *

* * * *

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

Jpn. J. Clin. Oncol. 36:337 (2006)

Nat. Clin. Prac. Urol. (2006)

Ubiquitination and proteasome-mediated degradation

Ubi

Figure 7.26a The Biology of Cancer ( Garland Science 2007)

VHL works as a SCF-like ubiquitin ligase

SCF (Skp1Cdc53F-Box protein)

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)