Tumour suppressor (TS) genes

1. Tumour suppressor (TS) genes • genes which are involved in normal cell regulation by negatively controlling progression through the cell cycle • identified as genes that prevent the onset of cancer and tumour growth • more specifically, they have all been found to be involved in one or more of the following functions: - inhibition of cell division - DNA repair - apoptosis • many TS genes with various functions have been identified to date including: p53, pRB, INK4a (p16), APC, MYH, NF1, NF2, BRCA1, BRCA2 and PTEN. • they have often been identified by loss of heterozygosity (LOH) at specific chromosomal regions in tumours

2. Tumour suppressor (TS) genes cont’d • both oncogenes and TS genes can cause cancer • however, there are some very important differences between oncogenes and TS genes - oncogenes result from mutations that activate proto-oncogenes (gain of function) whereas TS genes usually cause cancer when they are inactivated (loss of function) - most oncogenes result from acquired mutations of ‘normal’ genes, whereas mutations in TS genes can be inherited as well as acquired • classically, TS genes behave recessively and two mutations (‘hits’) are required for tumour formation • eg a mutation in one of several TS genes results in a susceptibility to cancer, where a second sporadic mutation initiates tumourigenesis • however, there is a very highly variable penetrance for mutations in the various TS genes, also haploinsufficient/dominant negative mutations and epigenetic modifications have now been identified

3. The retinoblastoma (RB) gene • the RB gene is found on chromosome 13 and was the first tumour suppressor gene to be cloned • encodes a 105 kDa protein (pRb) which is part of a small nuclear protein family also including p130 (pRb2) and p107 • these 3 proteins make up the retinoblastoma protein family aka ‘pocket proteins’ as they all contain a conserved ‘pocket’ region, important for protein binding • pRb, pRb2 and p107 all have specific functions and are non-reduntant but there is some overlap • mutations in the RB gene cause retinoblastoma, which is a malignant tumour of developing retinal cells • however, mutations in the RB gene have been implicated in many other cancers eg breast cancer and lung cancer

4. Retinoblastoma • retinoblastoma is an uncommon childhood disease – it very rarely presents in adults as it affects developing retinal cells • 90% of children with retinoblastoma have no recorded family history of the disease • 10% have a known affected family member and inherit the disease in an autosomal dominant manner • however, 40% of patients have actually inherited a germ line RB mutation and usually present with bilateral retinoblastoma (and/or multi focal tumours) at average age 9 months • the 60% ‘sporadic’ cases present at average age around 2 years with unilateral retinoblastoma

5. The “two-hit” hypothesis • in 1971, Knudson used Retinoblastoma as a model for explaining tumourigenesis involving a tumour suppressor gene

6. Cellular functions of pRb

7. Cellular functions of pRb • the E2F transcription factor family regulates expression of genes involved in G1-S phase transition • E2F/DP-mediated transcription is inhibited by the pRb family of proteins • pRB proteins are in turn regulated by the kinases CDK2, CDK4 and CDK6 and their inhibitors • as well as blocking S-phase transition, pRb is also thought to be involved in several other cellular processes including apoptosis, DNA replication, DNA repair, checkpoint control and differentiation • it is not yet clear whether these pRb functions are all mediated through regulation of E2F since pRB also binds many other factors

8. pRb interacting with E2F

9. pRb interacting with E2F • during G0 phase and most of G1, pRb binds to the E2F/DP complex, thus inhibiting its activity for promoting transcription of genes required for the onset of S phase • pRb repression of E2F-mediated transcription is regulated by phosphorylation by the kinases CDK2, CDK4 and CDK6 • during resting phase (G0/G1) when pRb is bound to the E2F/DP complex, it is in a hypophosphorylated state • pRb repression of E2F/DP is believed to be mediated by recruiting chromatin remodelling complexes to the E2F promoter during resting phase (G0/G1) • pRb has been shown to interact with the chromatin remodelling complexes such as SWI/SNF complex, the histone deacteylases, polycomb proteins and methylases

10. pRb interacting with E2F cont’d • from late G1 through to M phase, pRb is hyper-phosphorylated, releasing E2F and allowing it to activate transcription of the required genes • there are at least 6 E2F transcription factors in mammals, known as E2F1-6 • pRb has been shown to interact with E2F1, E2F2 and E2F3 which are the most potent transcriptional activators, and less well to E2F4 • E2F4 and E2F5 are weaker transcriptional activators and have been found to most readily interact with p107 and pRb2 • E2F6 does not appear to interact with any of the pocket proteins and is believed to be a transcriptional repressor

11. p53 • the TP53 gene which encodes p53 resides on the short arm of chromosome 17 • it encodes a 53 kDa phosphoprotein which belongs to a small family of related proteins including p63 and p73 • however, though structurally and functionally similar, p53 appears to have evolved primarily to prevent tumour formation, whereas p63 and p73 have clear roles in normal development • in healthy unstressed cells, p53 is continually produced and then is led by Mdm2 from the nucleus to the cytosol where it undergoes targeted degradation by the ubiquitin pathway • p53 activation is induced in response to many cellular stresses including DNA damage (eg UV exposure), hypoxia and oncogene deregulation

12. The stress response of p53

13. Upstream p53 pathway

14. p53 activation • upon stress, firstly the half-life of p53 increases dramatically, leading to rapid accumulation within the cell • secondly, the N-terminal domain is phosphorylated, resulting in p53 becoming an active transcription regulator • this phosphorylation is carried out by either of two different groups of kinases: - a group of MAP kinases (eg JNK1-3, ERK1&2, p38 MAPK) which are known to be induced by heat shock, oxidative stress etc - cell cycle ‘checkpoint’ proteins (eg Atm, Chk1, Chk2) which are triggered by DNA damage • the N terminal phosphorylation sites are close to or within the Mdm2-binding domain and inhibit p53/Mdm2 association • oncogenes also stimulate p53 activation, mediated by p14(ARF), which in turn inhibits Mdm2, promoting p53 activity

15. p53 cellular function • p53 assists in the inhibition of tumour formation in several ways - when DNA damage occurs, it can block G1-S transition to allow DNA repair to take place - it can also transcriptionally activate DNA repair proteins - it can initiate apoptosis if the DNA damage is irreparable • the best known downstream pathway for p53 is that for cell cycle arrest at the G1-S transition • p53 is known to activate the transcription of the gene encoding p21 which inhibits the CDK4/cyclin D1 and CDK2/cyclin E complexes • these CDK/cyclin complexes are required for progression from G1 to S phase

16. P53 downstream pathways

17. p53 and disease • somatic mutations in TP53 have been identified in at least 50% of all human tumours • however, only one inherited condition is known to be caused by germline TP53 mutations > Li-Fraumeni syndrome • a very rare cancer predisposition syndrome with high penetrance • patients with Li-Fraumeni have an estimated life time risk of cancer of ~90% and have increased risk of multiple primary tumours • only 50-70% of Li-Fraumeni patients have an identifiable TP53 mutation

18. Mutations in TP53 • 95% of p53 mutations have been found in exons 4-9 which encodes the DNA binding domain • neary 30% of mutations affect only six residues: 175, 245, 248, 249, 273 and 282. • over 90% of tumour-associated mutations in p53 are point mutations which result in single amino acid substitutions • this is a different mutation spectrum to many TS genes, where large deletions or frameshift mutations predominate • this results in the cells still having the ability to express the mutant form of p53 • in tumour cells, the mutant protein is usually present at much higher levels than the wild-type

19. Mutations in TP53

20. p53 regulates pRb

21. References Lohmann DR and Gallie BL (2004) Am J Med Genet, 129C 23-28 Classon M and Harlow E (2002) Nat Rev Cancer, 2, p910-7 Steele RJ et al (1998) Br J Surg, 85, p1460-67 Vousden KH and Liu X (2002) Nat Rev Cancer,2, p594-604 Shannon SR et al (2005) Carcinogen, 26, p2031-2045 Leiderman YI et al (2007) Sem Opth, 22, p247-254 http://www.geneclinics.org/ http://p53.free.fr/ Strachan and Read Third Edition, p492-497