1. 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

2. 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

3. 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,

4. 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.

5. 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
leukocoria, a whitening of the pupil that looks like a "cat's eye".

6. 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)

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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.

15. 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

16. 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

17. References:
Garrett, M. (2001) Current Science, 81, : Cell cycle control and cancer
Classon, M and Harlow, E (2002) Nature Reviews Cancer, 2, : 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, : Live or Let Die: The Cell’s response to p53
Meek, D. (2004) DNA Repair, 3, : the p53 response to DNA damage
Sherr and McCormick (2002) Cancer Cell, 2, : The RB and p53 pathways in cancer
Garber, J.E. and Offit, K. (2005) Journal of Clinical Oncology, 23, : 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