Tumor Suppressor Genes

“…in every normal cell there is a specific arrangement

for inhibiting (growth), which allows the process of
division only when the inhibition has been overcome by a
special stimulus. To assume the presence of definite
chromosomes which inhibit division, would harmonize
best with my fundamental idea…(that) cells of tumors
with unlimited growth would arise if those ‘inhibiting
chromosomes’ were eliminated…”

Theodor Boveri, 1911 “The Origin of Malignant Tumors”

   
Nat. Rev. Cancer 1:157-170, 2001

   
 Karyotype analysis

   
 Array Comparative Genomic Hybridization (aCGH)

Cancer Informatics 2: 48-58, 2006

   
 Knudson’s Two-Hit Model

Tumor

   
 Tumor suppressor genes -- Definition

“ a gene that sustains unequivocal, biallelic loss-

of-function mutations resulting in the
development of cancer”

1. biallelic, inactivating mutation

2. involved in both hereditary and sporadic
cancers (exception: Brca1)
3. suppress tumor growth (exceptions:
hMLH1, hMSH2)

   
 Gatekeepers vs. Caretakers
Gatekeeper pathway

Caretaker pathway

Mutation of a 2nd Mutation of 2nd

Mutation of Mutation of a
caretaker-gene gatekeeper- gatekeeper-gene
a caretaker allele leads to allele leads to
gene allele gene allele
genetic instability tumor initiation

Gatekeepers: genes that directly regulate the growth of tumors by inhibiting

growth or promoting death (Rb, APC, p53, PTEN, p16, NF1 etc.)
Caretakers: genes that involve in DNA repair and maintaining genome stability
(hMLH1, hMSH2, p53, ATM, Brca etc)
Landscapers: mutations that occur in the stromal cells surrounding the tumor, not
in the tumor cells themselves (VHL, PTEN)
   
 Tumor Suppressor Genes and associated cancers

   
 Cont’d

   
 Cell Cycle
Vertebrates
Cyclin B Clb1, 2, 3, 4
Budding yeast
Cdk1 (Cdc2)

Cyclin D
Clb5, 6 Cdk4, 6
Cyclin A Cln3
Cdk2

Start
Cyclin E, Cdk2
  Cln1, 2 
 Cdk inhibitor proteins (CKI)

   
 Cell Cycle Checkpoints
Replication checkpoint
DNA damage checkpoint
G1/S checkpoint
Intra-S checkpoint
G2/M checkpoint
Mitotic spindle assembly checkpoint

   
 Structure domain of RB (Retinoblastoma)

Majority of tumor derived mutations occur in the pocket

domain, and disrupt E2F binding.

   
 Pocket proteins and E2Fs

Oncogene 24: 2796, 2005

   
 RB and G1 to S transition

   
Oncogene 24: 2796, 2005
 RB and E2Fs - Cyc E promoter

   
Oncogene 24: 2796, 2005
 Rb and epigenetic control

    Oncogene 20: 3128-3133, 2001

 Mutations in the G1 to S checkpoint
proteins in human cancers

   
 p16 and ARF- two products from the
INK4a locus

   
 p53 - the guardian of genome

   
 p53 domain structure and modification

Activation Sequence­specific DNA  Tetramerization

domain binding domain domain

N I II III IV V Basic C
NLS NLS NLS

Phosphorylation
N ­­ Activation, stabilization
C ­­ DNA binding

Acetylation
DNA binding

   
Stress­induced cell cycle checkpoints
DNA damage Replication block

Rad17, Rad9, Rad1, Hus1

ATM + ATR

CHK1 CHK2 MDM2 p53

p21
Cdc25 Bax, AIP1
14­3­3 σ
PUMA

Cdk
Cell cycle arrest Apoptosis
 p53 and MDM2

p53 activates the

expression of MDM2
gene

MDM2 downregulates
p53 through binding the
N-teminus of p53 --
blocks p53 mediated
transactivation and
promotes p53
degradation

   
 p53 target genes

   
 p53 isofroms

    Mol. Cell 19: 719, 2005

 Chemotherapeutic approach - using
p53 to kill cancer cells

Foster et al., Phamacological rescue of mutant p53

conformation and function. Science 286: 207, 1999.
-- stabilization of p53 DNA binding domain
Bykov et al., Restorationof the tumor suppressor funciton to
mutant p53 by a low-molecular-weight compound. Nat. Med. 8:
282, 2002.
-- PRIMA-1 restores DNA binding to mutant p53
Friedler et al., A peptide that binds and stabilizes p53 core
domain: chaperone strategy for rescue of oncogenic mutants.
PNAS 99: 937, 2002.
-- CDB3 binds the p53 core domain, acts as a
“chaperon”, and maintains p53 in active conformation

   
 Science 286: 2507 (1999) mAb1620 binding

   
 Nat. Medicine 8: 282 (2002)

P53-null His-273

control

PRIMA
PRIMA-1

   
 Nat. Medicine 8: 282 (2002) – Cont’d

SKOV-His273

control

PRIMA

   
Viral proteins that interact with RB and p53

p53 RB
Adenovirus E1B 55 kD Adenovirus E1A
HPV E6 HPV E7
SV40 large T Ag SV40 large T Ag
HBV HBx JCV large T Ag
EBV BZLF1 BKV large T Ag
EBV EBNA-5

   
 WT1 and Wilms Tumor
Wilms tumor: nephroblastoma, childhood kidney cancer

WT1 protein
WT1 gene expression is
temporally and spatially
regulated - developing kidney,
spleen, gonads

   
Non-classical TSG -
Haploinsufficiency (-/+)
Epigenetic modification
Insufficient protein level
Dominant negative effects of the mutant protein
Transcriptional silencing of the wild type allele

Hemizygote in WT1 gene: genitourinary defect

(RB hemizygote is normal)
Other examples: p53, Arf, PTEN

   
 PTEN
Hereditary-
A. Cowden disease
Bannayan-Zonana
Syndrome

Sporadic-
Glioma
Endometrial carcinoma
Melanoma

B. Dual specificity
phosphatase-
PIP3, P-Tyr

   
 PI3K pathways regulated by PTEN

   
 PTEN and p53

Cancer Cell 3: 97-99 (2003)

   
 NF1- Neurofibromin

Mutations cause
neurofibromas and
malignancies of the
central and peripheral
nervous system

   
 Neurofibromin as Ras-GAP

   
 von Hippel-Lindau (VHL)-1
Hereditary cancer syndrome
Bilateral, multifocal clear-cell renal carcinoma
Bilateral, multifocal renal cysts
Cerebellar and spinal hemangioblastoma
Endolymphatic sac tumors
Retinal angioma
Panceratic cysts, microcystic adenomas, islet-cell tumors
Pheochromocytoma
Epididymal cystadenoma

   
 von Hippel-Lindau (VHL)-2
E3 Ubiquitin ligase compexes

SCF - Skp1, Cul1, F-box

VCB-Cul2 - VHL, Elongin B & C, Cul2

   
 von Hippel-Lindau (VHL)-3
Normoxia Hypoxia

   
 Colorectal Cancers

Hereditary nonpolyposis colorectal cancer

(HNPCC) –hMLH1, hMSH2

Familiar adenomatous polyposis (FAP)-APC

   
 Multiple hits to colorectal cancer

Nuclear β-catenin and chromosomal instability

Nat. Rev. Cancer 5: 184-198, 2005

   
 APC and the Wnt signaling pathway

   
 APC (Adenomatous Polyposis Coli)

1 2843

   
 Chromosome instability (CIN)
associated with APC mutations

Localization of APC in
kinetochores and centrosomes

APC null cells have unattached

kinetochores and supernumerary
centrosomes

   
Loss of C-terminus creates imbalance of functions?

   
Ann. Rev. Cell Dev. Biol. 20: 337, 2004
 Familial Breast Cancers

Brca1 and Brca2

   
Features of Brca1 and Brca2 proteins

   
 Brca1
17q
20-30% of hereditary
Hotspot mut.185delAG,
5382insC
Ovarian cancer
-/- mice, embryonic lethal
-/- cells hypersensitive to IR
Brca2
13q
10-20% of hereditary
No hotspot mut.
less ovarian cancer
embryonic lethal (some
are viable)
hypersensitive to IR
 DSB repair in Brca-deficient cells

NHEJ: non-homologous end joining

SSA: single-strand annealing
HR: homologous recombination

   
 Putative roles of Brca1

   
 Proposed model for Brca2 function in HR

   
 Some molecular mechanisms underlying genetic
specificity in cancer

 Expression of cancer genes might be tissue-

specific
 Proteins could function differently depending on
the cell type or/and developmental stage
 Mutations might have cell-type-specific toxic
effects
 Functional redundancy in certain tissues
 Synergistic interaction in different cancer genes
 The tumorigenic effect depends on extrinsic
stimuli
Nat. Rev. Cancer 5: 649-655, 2005
   
 Modeling cancer in mice

Resource: http://emice.nci.nih.gov

   
Colorectal Adenoma initiated by conditional
targeting of the APC gene
Science 278: 120, 1997

Site-specific recombination system

- bacteriphage derived Cre-loxP

Cre recombinase
Creating mice with loxP
vs. sequence inserted in the intron
region of the APC gene

Cre introduced by adenoviral

infection of the colon

   
 Conditional biallelic Nf2 mutation in mouse
promotes Schwannomas
Genes & Dev. 14: 1617, 2000
NF2 (Merlin) -mutation causes schwannoma in human and
osteosarcoma in mouse
Nf2/loxP mouse X P0Cre (P0: schwann cell-specific promoter)

Conditional mutation of Brca1 and mammary

tumors
Nature Genet. 22: 37, 1999
Brca1Ko/Co Wap-Cre or Brca1Ko/Co MMTV-Cre
Brca1Ko/Co MMTV-Cre Trp53+/-
A p53 mutant allele accelerates mammary gland tumor formation

   
 Temporal dissection of p53 function in vitro
and in vivo
Nat. Genet. 37: 718, 2005
Knock-in model – the endogenous p53 gene is substituted
by one encoding p53ERTAM, a p53 fusion whose function can
be induced by 4-hydroxytamoxifen.
Advantage – specific, rapid and reversible perturbation

   
 Colony growth
suppression assay

Flow cytometric
analysis of cell cycle

   
References:

The Genetic Basis of Human Cancer, 2nd Ed. by Vogelstein and Kinzler, McGraw-
Hill pub. 2002

Tumor Suppressor Genes, Vol. I and Vol. II, by El-Deiry, Humana Press, 2003

Nature Rev. Cancer 1: 157, 2001

Cell 116: 235-246, 2004

Rb
Cancer Cell 2: 103, 2002
Oncogene 24: 2796, 2005

P53
Nature Rev. Cancer 4:793, 2004

INK4a/ARF
Mut. Res. 576: 22, 2005

APC and β-catenin

Annu. Rev. Cell Dev. Biol. 20: 337, 2004

   
 Ref. Cont’d
PTEN
Nat. Rev. Cancer 6: 184-192, 2006

NF1
Curr. Opin. Genet. Dev. 13: 20, 2003

Brca1 & Brca2

Nat. Rev. Cancer 4: 266, 2004

WT1
Nat. Rev. Cancer 5: 699-712, 2005

Mouse Modeling
Oncogene 21: 5504, 2002