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 lossof-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 caretaker gene allele
Mutation of a 2nd caretaker-gene allele leads to genetic instability
Mutation of a gatekeepergene allele
Mutation of 2nd gatekeeper-gene allele leads to 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 Budding yeast
Cyclin B Clb1, 2, 3, 4 Cdk1 (Cdc2)
Cyclin D Cdk4, 6 Cln3
Clb5, 6 Cyclin A 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 domain
N
I
Sequencespecific DNA binding domain
II
III
IV
Tetramerization domain
Basic
V NLS
Phosphorylation
N Activation, stabilization C DNA binding
Acetylation DNA binding
NLS
NLS
C
Stressinduced cell cycle checkpoints DNA damage
Replication block
Rad17, Rad9, Rad1, Hus1 ATM + ATR CHK1
CHK2
Cdc25 Cdk
Cell cycle arrest
p21 1433 σ
MDM2
p53 Bax, AIP1 PUMA
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 control
PRIMA
PRIMA-1
His-273
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 gene expression is temporally and spatially regulated - developing kidney, spleen, gonads
WT1 protein
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 HereditaryCowden disease Bannayan-Zonana Syndrome
A.
SporadicGlioma Endometrial carcinoma Melanoma
B.
Dual specificity phosphatasePIP3, 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
Brca2
17q
13q
20-30% of hereditary
10-20% of hereditary
Hotspot mut.185delAG,
No hotspot mut.
5382insC Ovarian cancer
less ovarian cancer
-/- mice, embryonic lethal
embryonic lethal (some are viable)
-/- cells hypersensitive to IR
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 tissuespecific 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 vs.
Creating mice with loxP 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, McGrawHill 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