1. Tumor Suppressor Genes
Robert A. Weinberg
The Biology of Cancer
First Edition
Chapter 7:
Tumor Suppressor Genes
Copyright © Garland Science 2007

2. Tumor suppressor genes
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 oncogenens
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.

3. Somatic cell studies
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 ?

4. Somatic cell studies
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”

5. Pediatric Retinonlastoma
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)

6. Unilateral verus bilateral Rb
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)

7. Knudson’ “Two-hit hypothesis”
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
10-12 /cell division
Figure 7.6 The Biology of Cancer (© Garland Science 2007)

8. Loss of second allele of Rb is likey due to another mechanism rather than random mutation: cell division.
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
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)

9. Figure 7.10 The Biology of Cancer (© Garland Science 2007)

10. Loss of heterozygousity (LOH)

11. 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 The Biology of Cancer (© Garland Science 2007)

12. PCR is able to map tumor suppressor genes rapidly
Figure The Biology of Cancer (© Garland Science 2007)

13. Approaches to identify TSGs
Cytogenetic studies to identify constitutional chromosomal alterations in cancer patients; deletion of specific chromosome or specific region.
But small portion of cancer cells; Difficulty
Ex) Familial Rb; Ch13q 14 in blood sample or skin fibroblasts, Wilms tumor; 11q13, adenomatous intestinal polyps; 5q
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
LOH studies; ex) Rb-esterase D; family with Rb(+/-) showed decreased level of Esterase D. True in somatic patient.
13q12; BRAC2, 18q21.1; DPC4
Positional cloning of the TSGs

14. Loss of Heterozygousity of chromosomal arms in colon cancers
Figure The Biology of Cancer (© Garland Science 2007)

15. Table 7.1 part 1 of 2 The Biology of Cancer (© Garland Science 2007)

16. Table 7.1 part 2 of 2 The Biology of Cancer (© Garland Science 2007)

17. Tumor suppressor can be also inactivated by methylation of its promoter region (CpG sites) Epigenetic
Figure The Biology of Cancer (© Garland Science 2007)

18. Methylation of the promoter of p16 INK4A In Situ hybridization)
Figure The Biology of Cancer (© Garland Science 2007)

19. Table 7.2 The Biology of Cancer (© Garland Science 2007)

20. Methylation of multiple genes within tumor cell genomes
Figure The Biology of Cancer (© Garland Science 2007)

21. NF1 (Neurofibromatosis type 1)
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: hypergimentation, Lisch nodules, distinctive boney lesions etc
Chromsome 17q11.2
MW amino acids, structural and functional similarity to a family of Ras-GAP, down-regulate the ras-GTP
Figure 7.20a The Biology of Cancer (© Garland Science 2007)

22. NF1 acts as an negative regulator of Ras signaling
Function as Ras-GAP protein
After growth factor stimuli, NF1 rapidly is degraded, but NF1 levels return back to normal to shut down further the Ras-signaling: Negative feedback
NF1+/- cells, elevated the level of Ras-GTP , substantially increasing the level of Ras-signaling:
“Haploinsufficiency” : p27, smad4, Pten etc, but not Rb, p53
Figure The Biology of Cancer (© Garland Science 2007)

23. Colorectal cancer
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 β-catenin/Tcf-mediated transcription
several inherited predispositions can result from inheritance of a single defective gene.
-caretakers (DMA 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

24. . 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), CHRPE, 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

25. Familiar Adenomatous polyposis(FAP) Positional cloning of APC in heritable cancers in the Mormon populations
Figure The Biology of Cancer (© Garland Science 2007)

26. Chromosome 5q : The APC gene
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 β-catenin/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

27. APC and beta catenin in intestinal lumen formation
Figure 7.24a The Biology of Cancer (© Garland Science 2007)

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

29. Von Hippel –Lindau diseases: pVHL modulates the hypoxic response
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)

30. VEGF mRNA

31. Insights into rate-limiting mutations.
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.
Lung cancer; Rb mutation is late event

32. Tissue specific effects of germ line mutations.
Germ line mutations of TSGs are predisposed to a limited spectrum of cancer types; Puzzle ?
Ex) Rb, BRCA1, colon cancer
Rb; Rb heterozygous develop the retinoblastoma, some osteocarsinoma, but mutations are found in many cancer types
Rb may be a controller in retinoblast growth, but less critical controller in other tissues.
Rb mutations may contribute the tumor progression other than initiate tumor formation; Rb mutations without other mutations lead to cell death.

33. Candidate tumor suppressor genes
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 – % in pancrearic cancers; DPC4 genes- transcription 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

34. Gene Knockout in mice; 마우스에 유전적 돌연변이를 유도하는 기술
E E4
E E4
E E4
Replacement vector
Replacement vector
Replacement vector
neo
neo
neo TK TK TK
Targeting vector
Targeting vector
Targeting vector x x x x x x
Homologous
Homologous
Homologous
Genomic locus
Genomic locus
Genomic locus
recombination
recombination
recombination
E1 E2 E3 E4
E1 E2 E3 E4
E1 E2 E3 E4
E E4
E E4
E E4
E E4
neo
neo
neo
neo
Targeted locus
Targeted locus
Targeted locus
Cre
Cre - -
LoxP
LoxP
Tissue specific or conditional targeting vector
Tissue specific or conditional targeting vector
Tissue specific
Tissue specific
Neo
Neo TK TK
promoter
promoter
Cre
Cre
Homologous
Homologous X X X X
Tet
Tet
on/Off
on/Off
recombination
recombination
promoter
promoter
Cre
Cre
E E2 E3 E4
E E2 E3 E4
Cre
Cre - - TG TG X X
Neo
Neo
Cre
Cre
expression
expression
&
&
recombination
recombination
Cre
Cre – – TG TG
KO mice
KO mice

35. X LoxP Tissue specific or conditional targeting vector – KO mice Cre -TG –
KO mice
Tissue specific
promoter
Tet
on/Off
LoxP
Tissue specific or conditional targeting vector TK
Neo
Homologous
recombination
expression
&
E E2 E3 E4

36. Postmitotic differentiated cells
Retina
Rb WT
Rb -/-
H3p
Postmitotic differentiated cells
Cerebellum
Outer layer
Inner layer
Figure The Biology of Cancer (© Garland Science 2007)