MRCPath Self Help Course 12 th January 2010

1. MRCPath Self Help Course 12th January 2010 Mai M Abd El-Aziz Describe the mechanism by which mutations in tumour suppressor genes can lead to the development of cancer?? Concentrate primarily on the RB and p53 genes?? but give examples of other genes if you come across significant differences in the way in which they act??

2. Tumour Suppressor genes(TS) • One of three major classes of genes tumorigenesis • Inactivation of TS development of cancer • Inactivation arise from • Missense mutations • Nonesense mutations • Deletions • Insertions • Epigenetic silencing • Mutations in M & P alleles are needed  selective advantage to the cell • Exceptions: inactivation of one allele can also exert the same action on the cell through haploinsufficiency

3. How TS genes operate? • Mutations in TS genes drive the neoplastic process by increasing tumour cell number through • Stimulation of cell birth • Inhibition of cell death/cell cycle arrest • Mutations in TS can be either • Germlinehereditary predisposition to cancer • Somatic sporadic tumours • The first mutation clonal expansion & initiate the neoplastic process • Subsequent mutations  additional rounds of clonal expansion  tumour progression

4. TS Genes and Pathways

5. pRb and p53 pathways • Loss of function of both p53 and the pRB pathways play a significant role in the development of most human cancers • pRb regulates apoptosis during development, and its loss results in deregulated cell proliferation and apoptosis • p53, however guards against genomic instability and oncogene expression by inducing both arrest of the cell cycle and apoptosis hence its loss will desensitise cells to checkpoint signals, including apoptosis • All DNA tumor viruses that cause tumours in experimental animals or humans encode proteins that inactivate both pRb and p53

6. The retinoblastoma paradigm • Knudson in 1971 proposed that all retinoblastomas involved two hit mechanisms • In the familial cases one hit was inherited • In the sporadic cases both hits occurred somatically to inactivate the RB gene Complications of the RP paradigm • BRCA1 is inactivated in only 10-15%, when inactivation happens is not by chromosomal mechanisms but by DNA methylation • Some genes lose function of one allele in tumours, but the retained allele appears fully functional • Cases with three hits, eg, APC certain germline mutations are weak and hence two somatic mutations in addition to the inherited one are needed

7. The RB pathway • pRb functions as a gatekeeper • -vely regulates progression through the G1 phase of the cell cycle During the G1 phase pRb is hypo(P) (Active)binds E2F  repression of E2F-mediated transcription In late G1 and through the M phase pRb is (P) i.e. inactivated by CDK E2F is released  promote the expression of genes required for cell division. • P16 inhibits CDK4/6 kinase Loss of P16 function loss of pRb function  inappropriate cell cycling

8. The RB pathway cont. • pRb repression of E2F is believed to be mediated by recruiting chromatin remodelling complexes to the E2F promoter during the resting phase (G0/G1) • SWI/SNF complex, histone deacteylases, polycomb proteins and methylases are examples of chromatin remodelling complexes that interacts with pRb • pRb is a member of the pocket proteins which includes p107& p130 • pRb interacts with E2F1-4 whereas p107 interacts with E2F4, p130 interacts with E2F5

9. Potential functions of RB • Besides cell cycle regulation • DNA damage responses • DNA repair • DNA replication • Protection against apoptosis • Differentiation • Rb-/- mice die between days 13-15 gest. • P170 or p130-null mice develop normally • Rb+/-  predisposition to pitut & thyroid tumours • Rb+/-&p53 delretinal dysplasia

10. TP53 • The TP53 gene encodes a 53 kDa phosphoprotein which belongs to small family of related proteins (p63 & p73) • 95% of the mutations were found to occur in the central DNA binding core of p53 • Nearly 28% of mutations affect only six residues (shown) • Over 90% of the mutations lead to single amino acid substitutions

11. p53 pathway • Normally, levels of p53 are low due to the short half-life of the protein • Stress signals (DNA-damaging agents, such as ultraviolet or γ irradiation and chemotherapeutic drugs) can induce the stabilisation and activation of p53 • This is by covalent modification involving phosphorylation of the transactivation domain, and acetylation and phosphorylation of the basic allosteric control region

12. p53 activation Can be activated by both external and internal aberrations ATM kinase (UV) (IR) A) Stress Phosphorylate p53 ATR kinase Activation of p53 B) Deregulation of cellular oncogenes Expression of P14 (ARF) Blocking the degradation of P53 Deactivation of MDM2 Activation of P53

13. How P53 prevents tumour formation ?? • First, it arrests the G1/S transition to allow DNA repair to happen • Second, it can transcriptionally activate DNA repair proteins • Last, if DNA repair fails then it will induce apoptosis

14. Arresting the G1/S transition phase • By activating the transcription of the gene encoding P21 • Inhibition of CDK4/cyclinD1 & CDK2/cyclin E complexes  • Blocking of the G1/S transition phase

15. P53 apoptosis pathway

16. APC pathway & FAP • Absence of WNT signal  phosphorylation of -catenin at multiple residues • This is dependent on a multiprotein complex made of APC, Axin and GSK3 • (P)-catenin will be degraded by ubiquitin and proteosome pathway • WNT sig.  stabiliz. of -catenin & its accumul. in the cytoplasm  move to the nucleus where it leads to transcription of target genes • Mut. Of APC   -catenin   transcrip. Of genes & TCF APC downregulate the WNT pathway by acting as a shuttle for -catenin

17. HIF1 Pathway & VHL • VHL in presence of oxygen  degrade HIF1 • In absence of O2 or if VHL is mutated  HIF1 will be stabilized  • Expression of VEGF  stim. of angiogenesis

18. Implication of oncogenes and TS genes in the process of angiogenesis

19. References • Retinoblastoma: revisiting the model prototype of inherited cancer. Lohmann DR and Gallie BL (2004) Am J Med Genet, 129C 23-28 • The retinoblastoma tumour suppressor in development and cancer. Classon M and Harlow E (2002) Nat Rev Cancer, 2, 910-7 • The p53 tumour suppressor gene.Steele RJ at al (1998) Br J Surg, 85, 1460-67 • Live or let die: the cell's response to p53.Vousden KH and Liu X (2002) Nat Rev Cancer,2, 594-604 • The role of p53 and pRB in apoptosis and cancer. Hickman ES, Moroni MC, Helin K. Curr Opin Genet Dev. 2002 Feb;12(1):60-6. Review • Apoptosis - the p53 network. Haupt S, Berger M, Goldberg Z, Haupt Y. J Cell Sci. 2003 Oct 15;116(Pt 20):4077-85. • Tumour suppressor genes: Pathways and isolate strategies by Wafik S. El-Deiry • Vogelstein B and Kinzler KW (2004) Nat Med, 10, 789-799