Aging and Cancer: the Double-edged sword of cellular senescence And

1. Lawrence Berkeley National Laboratory and Buck Institute for Age Research Aging and Cancer: the Double-edged sword of cellular senescence And How to teach an old cell new tricks!

2. INCIDENCE AGE Cancer Rises Exponentially with Age Age is largest single risk factor Incidence vs mortality

3. What Causes Cancer? Mutations, mutations, mutations … AND A permissive tissue

4. Tumor Suppressor Mechanisms Organisms with renewable tissues had to evolve mechanisms to prevent cancer

5. CARETAKERS Prevent or repair genomic damage GATEKEEPERS Control cellular responses to damage Tumor Suppressor Mechanisms/Genes (prevent mutations) (eliminates or arrests potential cancer cells) Apoptosis (programmed cell death) Cellular senescence

6. Caretaker tumor suppressor genes are longevity assurance genes Gatekeeper tumor suppressor genes are antagonistically pleiotropic

7. Antagonistic Pleiotropy What’s good for you when you are young, can be bad for you when you are old.

8. Aging before cell phones …….. "Protected" Environment (climate control, biomedical intervention etc.) 100% SURVIVORS "Natural" Environment (hazards, predators, infection, etc.) HUMANS: MICE: 4 mos 40 yrs 80 and > 3-4 AGE

9. Aging before cell phones …….. "Protected" Environment (climate control, biomedical intervention etc.) 100% "Natural" Environment (hazards, predators, infection, etc.) SURVIVORS AGE Mutation Accumulation Antagonistic Pleiotropy

10. Cellular Senescence: A Gatekeeper Tumor Suppressor Induced by potentially oncogenic stimuli Most tumor cells acquire mutations that abrogate the senescence response Controlled by p53 and pRB -- tumor suppressors inactivated in most tumors Mouse models/human cancer-prone syndromes

11. Cellular Senescence: Induced by Potentially Cancer-Causing Events Stress/ Signals Chromatin Instability Irreversible arrest of cell proliferation DNA Damage Oncogenes Short/dysfunctional telomeres (REPLICATIVE SENESCENCE)

12. The senescence response is not simply an arrest of cell growth

13. The Senescent Phenotype Irreversible Growth Arrest Altered Differentiated Functions Resistance to Apoptosis

14. Resistance to Apoptosis Altered Differentiated Functions Cellular Senescence and Antagonistic Pleiotropy Selected/Unselected (deleterious) Traits Irreversible Growth Arrest Unselected traits of little consequence, unless senescent cells accumulate to appreciable levels

15. Senescent Cells Accumulate In Vivo With Increasing Age Skin Retina Liver At Sites of Age-Related Pathology Venous ulcers Atherosclerotic plaques Benign prostatic hyperplasia Preneoplastic hepatic lesions

16. Senescent Cells May Contribute to Aging Phenotypes/Diseases ……. Including Cancer

17. Ana Krtolica Simona Parrinello Steve Lockett - LBNL Imaging Group Pierre Desprez - CPMC Proc. Natl Acad. Sci USA 98:12072-12077 (2001)

18. Senescent Fibroblasts Stimulate the Proliferation of Premalignant Epithelial Cells Fibroblasts: Presenescent Senescent Epithelial Fluorescence HaCAT SCp2 S1 Premalignant Epithelial Cells

19. Senescent Fibroblasts Do NOT Stimulate Normal Epithelial Cells Fibroblasts: Presenescent Senescent Epithelial Fluorescence Adult HMEC Adult NHEK Neonatal NHEK Genetically Normal Human Epithelial Cells

20. Senescent Fibroblasts Stimulate Tumorigenesis of Premalignant Epithelial Cells In Vivo 100 SCp2 cells alone 0 200 + Presenescent Fibroblasts 100 Tumor size (mm3 x 10) 0 200 + Senescent Fibroblasts 100 0 Days 40 80 120

22. Christian Beausejour Ana Krtolica Francesco Galimi (Verma lab, Salk Institute) Masasha Narita, Scott Lowe (CSH) Paul Yaswen (LBNL) EMBO J 22:4212-4222 (2003)

23. p16 ARF MDM2 CDK4 Tx Changes (downstream effectors) Growth Arrest + Senescent Gene Expression Senescence Response, Controlled by p53 and pRB Pathways p53 pRB

24. p16 ARF MDM2 CDK4 pRB p53 Lentiviruses for high-efficiency expression of genes in senescent cells Lenti-GSE (inactivates p53) Lenti-CDK4m (inactivates pRB) Lenti-p16 (activates pRB) Lenti-p16(RNAi) (inactivates pRB)

25. 20 Doublings No proliferation Senescent WI-38 (fetal lung fb) Senescent BJ (foreskin fb) + Lenti-GSE (inactivate p53)

26. % LN S-WI LgT GFP GSE hTERT CDK4 LgTK1 GSE+LgT CDK4+GSE % GROWTH: 0 0 <1 <1 <1 <1 0 <1 S-BJ rescued GSE % LN S-BJ Population doubling LgT, LgT[K1] LgT GFP GSE CDK4 LgTK1 hTERT GSE+LgT CDK4m % GROWTH: 0 0 >90 60 40 >90 20* Days post infection

27. p53 inactivation can reverse the senescent growth arrest of BJ, but not WI-38, cells What distinguishes reversibly from irreversibly senescent cells??

28. p16 BJ WI38 P S P S p16 actin CDK4 pRB Fibroblasts differ in expression of p16 at senescence: BJ = low p16 WI38 = high p16

29. Do differences in p16 expression explain differences in reversibility of the senescence arrest?

30. 100 80 % growth 40 0 GFP p16 p16 + GSE p16 + LgT LgT + GFP LgT + p16 Presenescent BJ fibroblasts (low p16): 1) + lenti-p16 2) + lenti-GSE DNA synthesis, but no proliferation

31. 100 80 % growth 40 0 GFP p16 RNAi + Sn p16 RNAi + Sn + GSE Sn + GFP + LgT Sn + p16 RNA + LgT p16 RNAi + Sn + LgT Presenescent WI-38 fibroblasts (high p16): 1) + lenti-p16-RNAi ----> Senescence 2) + lenti-GSE DNA synthesis + proliferation

32. p53 maintains the senescent state; p16 maintains a dominant barrier to reversal The senescent phenotype is reversible upon p53 inactivation …… Providing the p16/pRB pathway has not been engaged Why does p16 render the senescence growth arrest irreversible?

33. HYPOTHESIS: p16 enables pRB to establish an “irreversibly” repressive chromatin state that, once established, is independent of p16 or pRB Senescent cells form RB-dependent heterochromatic domains that repress positive acting cell cycle genes Lowe and colleagues, Cell, 2003 Once cells express high levels of p16, they no longer require p16 or active pRB to maintain the senescence growth arrest

34. 100 80 100 % growth 80 40 % growth 0 40 GFP p16 RNAi + Sn p16 RNAi + Sn + GSE Sn + GFP + LgT Sn + p16 RNAi + LgT p16 RNAi + Sn + LgT 0 GFP p16 p16 + GSE p16 + LgT LgT + GFP LgT + p16 p16 renders senescence irreversible PBJ + p16 PWI + P16-RNAi

35. Senescence is not necessarily irreversible in human cells Hint: ask about mouse cells! p53 inactivation is not a recommended therapy (but p53 modifiers, such as SIR2, may be!) What determines the extent to which cells express p16? How can we reverse the p16/pRB-initiated chromatin?

36. Aging and Tumor Suppression Cancer Aging Phenotypes Tumor suppressors Can tumor suppression and aging be uncoupled??