Louise Stanley Newcastle 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. Louise Stanley Newcastle

Keywords Tumour suppressor (TS) – a gene whose normal function is to inhibit/control cell division or to maintain integrity of the genome Loss of function Cell Cycle Control RB and p53

Cell Cycle S phase: When DNA replication occurs M (Mitosis) Phase: cell division resulting in two daughter cells G1: Follows from mitosis and cell is responsive to both positive and negative growth signals G0 (quiescence): cell may reversibly exit from G1 if it is deprived of the appropriate growth-promoting signals. G2: gap after S phase, cell prepares for entry into mitosis Cell cycle progression in clockwise direction Garrett (2001), Current Science, 81 515-522

Cell Cycle - Checkpoints Three main checkpoints during cell cycle: G1-S: Replication blocked when there is unrepaired DNA damage – irreparable damage and cell is induced to undergo apoptosis. Additional damage checkpoint during S phase. G2-M: Cells block from entering M phase unless all DNA damage corrected. Spindle checkpoint: Prevent chromosome segregation at mitosis unless all chromosomes correctly attached to spindle fibres.

Retinoblastoma and RB Malignant tumour of the eye originating from developing cells of the retina Incidence ~ 1 in 15,000 to 1 in 20,000 live births Diagnosis usually under 5 years of age ~10% cases familial Patients with familial retinoblastoma have increased risk of osteogenic sarcoma, soft tissue sarcoma and malignant melanoma Presenting sign white papillary reflex http://www.eyecancermd.org/retinoblastoma/diagnosis.html leukocoria, a whitening of the pupil that looks like a "cat's eye".

Knudson’s Two Hit Hypothesis (1971) Model to explain genetic mechanism underlying hereditary and non-hereditary retinoblastoma. Both occur as a result of two mutations (recessive at cellular level). Hereditary retinoblastoma arises when first mutation is inherited via the germinal cells (most are de novo). Tumour foci are initiated by the second “hit” mutation in somatic retinal cells – unilateral/bilateral. Arise with kinetics consistent with one hit. Nonhereditary form of retinoblastoma two mutations occur in the same somatic retinal cell – only affects one eye – arose with two-hit kinetics RB first tumour suppressor gene cloned (13q14)

Knudson’s Two Hit Hypothesis (1971) Second Hit A somatic mutation within the normal allele Loss of heterozygosity (LOH) – this can occur by a variety of methods including mitotic recombination, loss of the chromosomal region that harbours the gene, inappropriate chromosomal segregation (nondisjunction) and loss (of normal allele) and reduplication of the mutated allele. Promoter methylation http://www.garlandscience.com/textbooks/0815340788/pdf/ch07.pdf

RB Role and Loss of Function Cells over expressing RB undergo arrest in G1 phase Cells deficient show accelerated G1 transition RB role in controlling cell cycle is through inhibition of E2F mediated gene expression. Rb also recruits chromatin-remodelling complexes to promoter regions causing chromatin condensation and subsequent inhibition of transcription Ability to repress E2F mediated gene expression is regulated by phosphorylation of RB by cyclin dependent kinases (CDK2/CDK4/CDK6 and their inhibitors). Phosphorylated RB is unable to bind and repress E2F mediated transcription E2F-mediated gene expression required for onset of S phase

RB Role and Loss of Function As well as ability to regulate E2F transcription RB is thought to regulate variety of processes such as apoptosis, DNA replication, DNA repair, checkpoint control and differentiation. Some functional overlap may be provided by RB-family member proteins p107 and p130 (“protein pocket” family). Loss of RB means E2F-mediated gene expression is not controlled/regulated RB loss observed in 80% of small cell lung cancer Mutations affecting RB pathway generally occur in a mutually exclusive fashion – mutation in p16 is unaccompanied by others (e.g. RB mutation or cyclin D-Cdk overexpression) Cyclin D1 overexpression observed > 50% breast cancer Member of the INK4 proteins

RB Role and Loss of Function Cells lacking RB or p16 (INK4a), or those overexpressing cyclin D1 do not divide at an accelerated rate However, blunted responses to extracellular growth-inhibitory signals may prevent cells from differentiating or undergoing senescence Disruption of RB pathway initiates compensatory p53-dependent transcriptional program – reinforces cell cycle exit or apoptosis – subsequent failure in p53 function would allow cells to remain in cycle, abnormally extend cellular lifespan INK4 proteins cyclin D-dependent kinases RB family of proteins

p53 Inherited predisposition to Li-Fraumeni syndrome mutations in p53 Suffer multiple primary tumours including soft tissue sarcomas, breast, brain and leukaemia – patients have poor prognosis with very high lifetime risk of cancer Referred to as “Guardian of the genome” Loss of p53 function in cancer is very common (~50% of all tumours) but occurs in late stages of malignancy Loss of function can occur by variety of methods including: Lesions that prevent activation of p53 Mutations in p53 itself Mutations in downstream mediators of p53 function Mutations in p53 itself are predominantly point mutations with cells expressing mutant p53. May act via a dominant negative manner as p53 forms tetramers

p53 and MDM2 Levels of p53 tightly regulated in cells - level of protein kept low Achieved through interaction with MDM2 (E3 ubiquitin ligase) which together with p300 protein mediate ubiquitinylation and proteasome-dependent degradation of p53 MDM2 also binds to transactivation domain within N-terminus of p53 – blocks interaction of p53 with transcriptional apparatus MDM2 can also mediate translocation of p53 to cytoplasm removing from site of action MDM2 is itself a p53 target and therefore p53 and MDM2 function within an autoregulatory loop – p53 positively regulates MDM2, MDM2 negatively regulates p53

p53 Response to DNA damage Cellular stress sensors (damage sensors as well) lead to phosphorylation and stabilisation of p53. Response to DNA damage most understood. Activation of p53 (to DNA damage) results in Stabilisation and accumulation of p53 in nucleus Activation of biochemical functions encompassed within p53 protein Stabilisation occurs through inhibition of MDM2 mediated degradation – achieved by multi-site phosphorylation of both p53 and MDM2 P-p53 leads to transcription of p53 dependent genes – stimulates recruitment of transcription factors including p300, CBP and P/CAF – increase transcription from p53 responsive promoters and also promote acetylation of certain residues in p53, preventing ubiquitination and subsequent degradation. Different stresses (including levels) result in different modifications (both qualitative and quantitative) to p53 – modulates response

p53 Response to DNA damage Many targets of p53 activated transcription - divided into groups inhibition of cell growth DNA repair activation of apoptosis Angiogenesis Cell must sense whether DNA damage is repairable and either promote growth arrest or promote apoptosis Complex pathways involving multiple genes p21WAF1/CIP1 – inhibits cell cycling by binding to Cdk2/cyclin E causing cell cycle arrest at the G1/S transition PUMA and PIG3 – control apoptosis.

RB and p53 responses integrated Summary DNA Damage Irreparable damage – trigger apoptosis Repairable damage: pause for repair CDK 4/6 Cyclin D Strachan and Read

TS gene Different to RB and p53 Inherited predisposition in an autosomal dominant manner (likewise for APC, BRCA1/2, MLH1/MSH2 etc mutations) – others are inherited in an autosomal recessive manner – e.g. MutYH (MYH-associated polyposis), BLM (Bloom’s Syndrome), Many examples of genes involved in maintenance of the stability of the human genome rather than cell cycle control – DNA repair e.g. MLH1/MSH2, BRCA1/2 – discussed in other talks

References: Garrett, M. (2001) Current Science, 81, 515-522: Cell cycle control and cancer Classon, M and Harlow, E (2002) Nature Reviews Cancer, 2, 910-917: The retinoblastoma tumour suppressor in development and cancer Lohmann, D.R. and Gallie, B. L. (2004) American Journal of Medical Genetics Part C, 129C, 23-29: Retinoblastoma – Revisiting the Model Prototype of Inherited Cancer Vousden, K.H. and Lu, X. (2002) Nature Review Cancer, 2, 594-604: Live or Let Die: The Cell’s response to p53 Meek, D. (2004) DNA Repair, 3, 1049-1056: the p53 response to DNA damage Sherr and McCormick (2002) Cancer Cell, 2, 103-112: The RB and p53 pathways in cancer Garber, J.E. and Offit, K. (2005) Journal of Clinical Oncology, 23, 276-292: Hereditary Cancer Predisposition Syndromes Kim, E., Giese, A. and Deppert, W. (2009) Biochemical Pharmacology, 77, 11-20: Wild-type p53 cancer cells: When a guardian turns into a blackguard