Tumor Suppressors / Cell cycle Eldad Zacksenaus MGY-425 March 1 st , 3 rd , 2010

1. Tumor Suppressors / Cell cycle Eldad Zacksenaus MGY-425 March 1st, 3rd, 2010

2. The Hallmarks of Cancer Principles of cancer therapy: oncogene and non-oncogene addiction Luo J, Solimini NL, Elledge SJ. Cell. 2009 Mar 6;136(5):823-37.

3. Oncogenes are activated in human cancer and promote hallmarks of cancer Tumor suppressors (TS) are lost in human cancer and inhibit hallmarks of cancer

4. For example, evasion of apoptosis: • • Bcl-2 is a survival factor (It inhibits mitochondrial outer • membrane permeabilization (MOMP) and the release of • cytochrome c, which is required for caspase activation and cell death). • - Bcl-2 is amplified in cancer, hence an oncogene • • p53 is an anti-survival factor (among other functions, it transcriptionally • activates Bax which induces MOMP and cell death) • p53 is lost in cancer hence a TS (however most mutations in p53 are • not null but dominant negative mutations that also inhibit p63 - hence • oncogenic) • • The TS RB inhibits cell proliferation - but also apoptosis. Its loss leads • to ectopic proliferation (oncogenic) but also so apoptosis (tumor • suppression).

5. Tumor suppressor Hallmark Function Evading apoptosis p53 Induces apoptotic genes (Bax) Self-sufficiency in Pten Dephosphorylates PIP3, counteracting PI3K growth signal pRb Inhibits E2F Insensitivity to anti- SMAD4 Induces CDK4/6 inhibitor p16ink4a in response to TGF growth signals p16ink4a Inhibits cyclins DNA damage stress ATM Induces p53 and DNA repair machinery Mitotic stress LATS2 Inhibits MDM2 (activates p53) Metabolic stress/hypoxia VHL E3 ligase for HIF-1

6. Signaling, Oncogenes, tumor-suppressors and non-oncogene addiction signal + Oncogene Tumor suppressor - Negative regulator that is not mutated/lost in cancer - Positive regulator that is not activated in cancer Non-oncogenic addiction +

7. For example: The phosphatidylinositol 3-kinase (PI3K) signaling cascade G protein couple receptor Receptor tyrosine kinase RTK, +, onc Ras, +, onc PI3K, +, onc Pten, - , TS Akt, +, onc MDM2, +, onc P53, - , TS/onc GSK3, - B-cat, + Myc, +, onc CyclinD1, +, onc TSC, -, TS + cyclin D1 -catenin myc Courtney, K. D. et al. J Clin Oncol; 28:1075-1083 2010

8. The landscape of somatic copy-number alteration across human cancers High-resolution Affimetric analysis (250K array containing probes for 238,270 single nucleotode polymorphisms (SNPs) for somatic copy-number alterations (SCNAs) from 3,131 cancer specimens Ink4-Arf Beroukhim R., et al Nature463, 899-905 (18 February 2010)

9. Notes: • On average: 24 gains and 18 losses • Most frequent - Myc amplification and Cdkn2A/B (p16ink4a and Arf) deletion. • At least 10 known tumor suppressors not identified by this analysis - Brca2, Fbxw7, Nf2, Ptch1, Smarcb1, Stk11, Sufu, Vhl, Wt1, and Wtx - Some of these are specific to cancer types (e.g. Nf2, Wt1) - Other primarily suffer arm-level deletions (e.g. Brca2) - Some TS genes undergo point mutations not deletions

10. To cycle or not R.A. Weinberg Biology of Cancer 2006. Figure 8.1 The Biology of Cancer (© Garland Science 2007)

11. Examples of checkpoints in the cell cycle Figure 8.4 The Biology of Cancer (© Garland Science 2007)

12. The restriction point Figure 8.6 The Biology of Cancer (© Garland Science 2007)

13. Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation Stages during the cell cycle when pRb in under-phosphorylated Chen PL, Scully P, Shew JY, Wang JY, Lee WH. Cell. 1989 Sep 22;58(6):1193-8. Karen Buchkovich, Linda A. Duffy and Ed Harlow Cell. 1989 Sep 22;58(6):1097-105. James A. DeCaprio, John W. Ludlow, Dennis Lynch, Yusuke Furukawa, James Griffin, Helen Piwnica-Worms, Chun-Ming Huang and David M. Livingston 1989 Cell ;58(6):1085-95.

14. The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Micro-injection of Rb protein during this period blocks entry into S phase Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH.Cell. 1991 18;67(2):293-302. Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH.Cell. 1991 67(2):293-302.

15. D and E type cyclins, which are active in early and late G1, sequentially phosphorylate pRb (Cyclin A/B maintain pRb phosphorylation later on) Figure 8.8 The Biology of Cancer (© Garland Science 2007)

16. Cell cycle-dependent fluctuations in cyclin B levels Figure 8.9 The Biology of Cancer (© Garland Science 2007)

17. Fluctuation of cyclin levels during the cell cycle Figure 8.10 The Biology of Cancer (© Garland Science 2007)

18. Cell cycle-dependent phosphorylation of pRb

19. Control of cyclin levels during the cell cycle CDC2 = Cdk1 is sufficient to drive the mammalian cell cycle - CDK4/6/3/2 are not essential for cell proliferation and early embryogenesis - till midgestation) Santamar D, Barrie C, Cerqueira A, Hunt S, Tardy C, Newton K, Ceres JF, Dubus P, Malumbres M, Barbacid M. Nature. 2007 Aug 16;448(7155):811-5. Figure 8.12 The Biology of Cancer (© Garland Science 2007)

20. Schematic structure of pRb and CDK phospho-acceptor sites Amino acid S S T T T T S S S S S S S S T T Location 224 243 246 350 364 367 561 601 605 773 781 788 800 804 814 819 S S T T T T S S S SSSSSTT pRb N A B C Spacer Exon23 788 800/804 814/819 G1 to S transition G0 to G1 transition Binding to LxCxE containing factors + Constitutive interaction with E2F1 suppression of Rat1 cell proliferation

21. Two-dimensional tryptic phosphopeptide analysis of pRB phosphorylated by different CDKs. Zarkowska T , Mittnacht S J. Biol. Chem. 1997;272:12738-12746 ©1997 by American Society for Biochemistry and Molecular Biology

22. Localization and assignment of CDK2-cyclin A phosphorylated residues using phosphoamino acid analysis and phosphorylation of pRB subfragments. Zarkowska T , Mittnacht S J. Biol. Chem. 1997;272:12738-12746 ©1997 by American Society for Biochemistry and Molecular Biology

23. Identification of a CDK4-cyclin D1 specific phosphorylation site by mutagenesis. Zarkowska T , Mittnacht S J. Biol. Chem. 1997;272:12738-12746 ©1997 by American Society for Biochemistry and Molecular Biology

24. Summary of pRB residues phosphorylated by individual CDKs

25. E2F1, 2 and 3a are major targets of pRb Figure 8.23d The Biology of Cancer (© Garland Science 2007)

26. Figure 8.23a The Biology of Cancer (© Garland Science 2007)

27. LxCxE binding LxCxE motif Figure 8.24a The Biology of Cancer (© Garland Science 2007)

28. Figure 8.24b The Biology of Cancer (© Garland Science 2007)

29. Actions of CDK inhibitors Figure 8.13a The Biology of Cancer (© Garland Science 2007)

30. Control of cell cycle progression by TGF- Figure 8.14a The Biology of Cancer (© Garland Science 2007)

31. Figure 8.15a The Biology of Cancer (© Garland Science 2007)

32. Regulation of p21 localization Figure 8.15b The Biology of Cancer (© Garland Science 2007)

33. Regulation of p27 localization AKT phosphorylates p27 on T157, causing it to localize to the cytoplasm Nat Med 2002;8:1145–52; Nat Med 2002;8:1136–44 Figure 8.15c The Biology of Cancer (© Garland Science 2007)

34. Nuclear p27 accumulation in tumors inversely correlates with pAKT Figure 8.16a The Biology of Cancer (© Garland Science 2007)