Cancer DEREGULATION OF CELL CYCLE CONTROL IN ONCOGENESIS. D. Kardassis Division of Basic Sciences University of Crete Medical School and IMBB-FORTH

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1 E : 2007: Molecular Biology of Cancer DEREGULATION OF CELL CYCLE CONTROL IN ONCOGENESIS D. Kardassis Division of Basic Sciences University of Crete Medical School and IMBB-FORTH

2 Literature * Schwartz, G.K. And Shah, M.A. (2005) Targeting the cell cycle: A new approach to cancer therapy. J. Clin. Oncology 23: * Malumbres, M. and Barbacid, M. (2001) To cycle or not to cycle: a critical decision in cancer. Nature Rev Cancer 1: * Vermeulen, K., Van Bockstaele, D.R. and Berneman, Z.N. (2003) The cell cycle: a review of regulation, deregulation and therapeutic targets in cancer. Cell Prolif. 36, Weiss, R.H. (2003) p21 WAF1/Cip1 as a therapeutic target in breast and other cancers. Cancer Cell 4, Vidal, A. and Koff, A. (2000) Cell cycle inhibitors: three families united by a common cause. Gene 247:1-15. Sherr, C.J. (2000) The Pezcoller Lecture: Cancer cell cycles revisited. Cancer Res. 60: Coqueret, O. (2002) Linking cyclins to transcriptional control. Gene 299: * Required reading

3 Topics to be discussed Cell cycle phases and molecular l players Activation and degradation of cyclins Major CDK regulatory mechanisms Structure, function and regulation of cdk inhibitors Regulation of the cell cycle by subcellular localization of critical components Signaling pathways regulating the cell cycle: The paradigm of TGFβ Checkpoints in the cell cycle Cell cycle regulators and cancer CDK regulation and opportunities for therapeutic intervention

4 Summary of cell cycle and its regulation Mitogenic, growth signals

5 The Restriction point (R)

6 The key molecular players in cell cycle control Cyclin-dependent kinases (cdks) Cyclins CDK substrates (pocket family: Rb, p107,p130) Regulators of CDKs (CAK, wee, cdc25) Cyclin-dependent kinase inhibitors (CKIs) Regulators of CKIs

7 Summary of cell cycle and its regulation Mitogenic, growth signals

8 Coordination between cell cycle progression and cell growth through the regulation of cyclin D expression. translational regulation (mrna export) Inhibition of ubiquitination

9 Degradation of cyclins Cyclins A and B contain a Destruction ti Box and cyclins D and E contain a PEST sequence [segment rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues]. These are protein sequences required for efficient ubiquitin-mediated cyclin proteolysis at the end of a cell cycle phase

10 The ubiquitination system for protein degradation

11 Ubiquitination of cyclins

12 The Cyclin-CDK complexes

13 Major CDK regulatory mechanisms

14 Regulation of CDK activity by phosphorylation- dephosphorylation DNA damage ATM,ATR inactive Phosphorylation by CAK (Cdk Activating Cyclin A/B Kinase) which consists of Cyclin H and Chk1/2 P P cdk7: Conform. changes, enhance binding of CDK1 cyclins Phosphorylation by Wee/Myt1 dual specificity kinases at consecutive Thr-Tyr residues: Inactivation of cdk De-phosphorylation p by cdc25 dual specificity phosphatases at consecutive Thr-Tyr residues: Re-activation of cdk P cdc25 Cyclin H CDK7 P Cyclin A/B CDK1 active P Wee1/Myt1

15 Cyclin-dependent kinase inhibitors

16 Regulation of CDK activity by CKIs

17 Domain structure of the Cip/Kips p

18 Cip/Kip proteins regulate cyclin CDK complexes and their activity Cip/Kip proteins ti act as assembly factors for cycd/cdk complexes in the cytoplasm (in contrast to INK4 CKIs which dissociate cyc/cdk complexes) Cip/Kip p proteins enhance the nuclear import of cycd/cdk complexes High Cip/Kip levels Low Cip/Kip levels

19 Mechanism of inhibition of CDK activity by Cip/Kip proteins

20 p21 is an essential mediator of the tumor suppressor activity of p53

21 The role of transcription factor Sp1 in the regulation of the human p21 WAF1/Cip1 gene by tumor suppressor pathways UV, drugs DNA damage p53 p53 human p21 promoter Mithramycin A Sp1 Sp1 Sp1 Sp1 myc Miz1 p21 Koutsodontis et al, JBC, 2001; Koutsodontis et al, Biochemistry, 2002; Koutsodontis and Kardassis, Oncogene, 2004

22 Cytoplasmic Cip/Kip relocalization and cell transformation p27 cytoplasmic localization is found in 40% of breast cancer cells or up to 35% of colon cancer cells and correlates with poor survival rates in Barrett s associated adenocarcinoma. By contrast, nuclear expression of p27 in cancer cells gives a more favorable prognosis.

23 What are the consequences of Cip/Kip relocalization in tumor cells? Nuclear localization of Cip/Kip proteins enhances their cytostatic properties p The cytoplasmic distribution of Cip/Kips is utilized by tumour cells for their advantage i.e. Inhibition of apoptosis p Increased cytoskeletal reorganization (cell migration)

24 The role of p21/waf1 in normal and cancer cells Normal cells Breast cancer cells

25 Signaling pathways regulating the cell cycle: The example of TGFβ

26 Genes that are up- or down- regulated by TGF-β β in epithelial l cells Reference: Massague, J. and Gomis (2006) FEBS Lett. 580,

27 The cytostatic program of TGFβ TGFβ Receptors Smad3/4 FoxO, Sp1, C/EBPβ Primary repression E2F4,5 p107, C/EBPβ Miz1 Induction of a repressor ATF3 p21 Cip1 c-myc Id1 p15 INK4b CDKs M G1 Cell cycle S Mutations that disrupt the cytostatic program FoxO: Mutated in glioblastomas C/EBPβ: mutated in breast cancer G2

28 Regulation of cell cycle progression by the intracellular localization of cell cycle regulatory yproteins During DNA damage, or prior to prophase, cyclin B and cdc25 phosphatase are exported from the nucleus by σ The Wee and Myt1 CDK inactivating kinases are located in the nucleus and Golgi respectively and protect cells from premature mitosis

29 Regulation of G1 and the G1/S transition

30 Animal studies Models of redundant genetic relationships between cell-cycle inhibitors. Pure redundancy Absence of severe developmental defects in most of the KO mouse strains (of CKIs or pocket proteins) suggests: Redundant roles between different family memebrs Compens. redundancy Compensatory mechanisms Phenotypic redundancy Vidal and Koff (2000) Gene 247, 1-15

31 An nima al st tudie es Mouse strain Cip/Kip p21 / p27 / p57 / p21 / p57 / p27 / p57 / INK4 p16 / p15 / p18 / p15 / / p18 / / Pocket proteins Rb / p130 / p107 / p130 / p107 / Phenotypic consequences No evident phenotype. MEFs show defective G1 checkpoint Gigantism and organomegalia. Infertile females. Pituitary hyperplasia Some embryonic and neonatal lethality. Altered cell proliferation and apoptosis in several tissues. Altered lung development. Enhanced skeletal abnormalities. Skeletal muscle differentiation failure Increased embryonic lethality. More severe lens alterations. Placental defects Normal development. General tumor predisposition Viable. No tumor predisposition Gigantism and organomegalia. Pituitary hyperplasia Infertile animals. Embryonic lethality between d and Defects in neurogenesis and hematopoiesis Viable and fertile. Viable and fertile. Neonatal lethality. Impaired chondrocyte differentiation Vidal and Koff (2000) Gene 247, 1-15

32 Cell cycle has checkpoints Checkpoint controls (also known as surveillance mechanisms) ensure the dependency of cell-cycle transitions on the completion of earlier events. They consist of three distinct sets of functions: sensors (which look out for defects and emit a signal); signal-transduction cascades (checkpoint signals need to be transmitted throughout the nucleus or cell); and effectors (a target is regulated to delay cell-cycle progression) The DNA damage checkpoint The mitotic spindle assembly checkpoint

33 The DNA damage checkpoints DNA damage checkpoint

34 The mitotic spindle assembly checkpoint APC: Anaphase Promoting Complex. An E3 ubiquitin ligase that regulates degradation of Securin Ub: Ubiquitin Separase: Caspase-like protease needed for the separation of the sister chromatids BUB (Budding Uninhibited by Benomyl), MAD2 (Mitotic Arrest Deficient 2): Proteins participating in the spindle assembly checkpoint by recruiting cdc20 PLK cdc20: Positive regulator of the APC PLK: Polo-Like Kinase

35 CELL CYCLE DEREGULATION IN CANCER: MUTATIONS IN PROTEINS IMPORTANT AT DIFFERENT LEVELS OF THE CELL CYCLE

36 Cell cycle regulators and cancer Inactivation Checkpoint proteins (p53) Cyclins Overexression, Amplification translocations CDK modulators (CKIs) CDKs CDK targets (Rb) Mutational inactivation, Intracellular localization (promoter methylation) Overexression, Amplification, Mutations in CKI binding Mutational Inactivation (promoter methylation) ti

37 Cell cycle regulators and cancer : good prognostic value : characterized genetic or epigenetic alterations : no mechanistic explanation

38 Gene therapy, Demethylating agents Phosphorylation Proteasomal inhibitors Small molecule Inhibitors peptidomimetics Gene transfer Txnal upregulation sirnas sirnas Phosphorylation CDK regulat tion an nd opp portun ities fo or therap peutic interv vention n. sirnas

39 Small molecule CDK inhibitors

40 Flavopiridol: an anti-cancer drugs that targets the cell cycle

41 Gene therapy, Demethylating agents Phosphorylation Proteasomal inhibitors Small molecule Inhibitors peptidomimetics Gene transfer Txnal upregulation sirnas sirnas Phosphorylation CDK regulat tion an nd opp portun ities fo or therap peutic interv vention n. sirnas

42 The p53 response Stress Oncogenes Microtubule inhibitors Ribonucleotide depletion Chemotherapy DNA damage Hypoxia Loss of survival signals Telomere erosion Response Cell cycle arrest DNA repair Reparable damage Nucleus Cyclin/cdk PCNA p53 activation p21 Cip1 Irreparable damage Cytoplasm p21 KO, p21 sirna p21antisense, c-myc Apoptosis

43 Gene therapy, Demethylating agents Phosphorylation Proteasomal inhibitors Small molecule Inhibitors peptidomimetics Gene transfer Txnal upregulation sirnas sirnas Phosphorylation CDK regulat tion an nd opp portun ities fo or therap peutic interv vention n. sirnas