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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! INCIDENCE Cancer Rises Exponentially with Age AGE Age is largest single risk factor Incidence vs mortality What Causes Cancer? Mutations, mutations, mutations … AND A permissive tissue Organisms with renewable tissues had to evolve mechanisms to prevent cancer Tumor Suppressor Mechanisms Tumor Suppressor Mechanisms/Genes CARETAKERS Prevent or repair genomic damage (prevent mutations) GATEKEEPERS Control cellular responses to damage (eliminates or arrests potential cancer cells) Apoptosis (programmed cell death) Cellular senescence Caretaker tumor suppressor genes are longevity assurance genes Gatekeeper tumor suppressor genes are antagonistically pleiotropic Antagonistic Pleiotropy What’s good for you when you are young, can be bad for you when you are old. Aging before cell phones …….. "Protected" Environment (climate control, biomedical intervention etc.) 100% "Natural" Environment (hazards, predators, infection, etc.) HUMANS: 40 yrs MICE: 4 mos 80 and > 3-4 Aging before cell phones …….. 100% "Natural" Environment (hazards, predators, infection, etc.) "Protected" Environment (climate control, biomedical intervention etc.) Mutation Accumulation Antagonistic Pleiotropy 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 Cellular Senescence: Induced by Potentially Cancer-Causing Events Chromatin Instability Irreversible arrest of cell proliferation DNA Damage Stress/ Signals Oncogenes Short/dysfunctional telomeres (REPLICATIVE SENESCENCE) The senescence response is not simply an arrest of cell growth The Senescent Phenotype Irreversible Growth Arrest Resistance to Apoptosis Altered Differentiated Functions Cellular Senescence and Antagonistic Pleiotropy Selected/Unselected (deleterious) Traits Irreversible Growth Arrest Resistance to Apoptosis Altered Differentiated Functions Unselected traits of little consequence, unless senescent cells accumulate to appreciable levels 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 Senescent Cells May Contribute to Aging Phenotypes/Diseases ……. Including Cancer Ana Krtolica Simona Parrinello Steve Lockett - LBNL Imaging Group Pierre Desprez - CPMC Proc. Natl Acad. Sci USA 98:12072-12077 (2001) Senescent Fibroblasts Stimulate the Proliferation of Premalignant Epithelial Cells Epithelial Fluorescence Fibroblasts: Presenescent Senescent HaCAT SCp2 S1 Premalignant Epithelial Cells Epithelial Fluorescence Senescent Fibroblasts Do NOT Stimulate Normal Epithelial Cells Fibroblasts: Presenescent Senescent Adult NHEK Neonatal NHEK Adult HMEC Genetically Normal Human Epithelial Cells Senescent Fibroblasts Stimulate Tumorigenesis of Premalignant Epithelial Cells In Vivo 100 SCp2 cells alone Tumor size (mm3 x 10) 0 200 100 + Presenescent Fibroblasts 0 200 + Senescent Fibroblasts 100 0 40 80 120 Days Christian Beausejour Ana Krtolica Francesco Galimi (Verma lab, Salk Institute) Masasha Narita, Scott Lowe (CSH) Paul Yaswen (LBNL) EMBO J 22:4212-4222 (2003) Senescence Response, Controlled by p53 and pRB Pathways p16 ARF MDM2 p53 CDK4 pRB Tx Changes (downstream effectors) Growth Arrest + Senescent Gene Expression 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) ARF p16 MDM2 p53 CDK4 pRB Senescent WI-38 Senescent BJ (fetal lung fb) (foreskin fb) + Lenti-GSE (inactivate p53) No proliferation 20 Doublings % LN 100 S-WI 80 60 40 % GROWTH: 0 0 <1 <1 <1 <1 CDK4+GSE CDK4 LgTK1 GSE+LgT GSE LgT 0 GFP hTERT 20 0 <1 S-BJ rescued 60 40 20 LgTK1 GSE+LgT CDK4 0 GFP hTERT GSE LgT S-BJ 80 Population doubling % LN 100 25 LgT 20 LgTK1 CDK4 15 LgT, LgT[K1] 10 5 CDK4m 0 0 % GROWTH: 0 0 >90 60 40 >90 20* GSE GSE 20 40 60 80 100 Days post infection p53 inactivation can reverse the senescent growth arrest of BJ, but not WI-38, cells What distinguishes reversibly from irreversibly senescent cells?? Fibroblasts differ in expression of p16 at senescence: BJ = low p16 WI38 = high p16 BJ P S p16 WI38 P S p16 actin CDK4 pRB Do differences in p16 expression explain differences in reversibility of the senescence arrest? Presenescent BJ fibroblasts (low p16): 1) + lenti-p16 2) + lenti-GSE DNA synthesis, but no proliferation % growth 100 80 40 0 GFP p16 p16 + GSE p16 + LgT LgT LgT + + GFP p16 Presenescent WI-38 fibroblasts (high p16): 1) + lenti-p16-RNAi ----> Senescence 2) + lenti-GSE DNA synthesis + proliferation % growth 100 80 40 0 GFP p16 p16 RNAi RNAi + Sn + Sn + GSE Sn RNAi + + GFP Sn + + LgT LgT p16 Sn + p16 RNA + LgT 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? 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 PWI + P16-RNAi 100 80 40 0 GFPp16 p16 p16 + + GSE LgT % growth PBJ + p16 % growth p16 renders senescence irreversible LgT LgT + + GFP p16 100 80 40 0 GFPp16 p16 p16 Sn Sn RNAi + + + + + GFP p16 Sn Sn Sn + RNAi + + LgT + GSE LgT LgT RNAi RNAi 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? Aging and Tumor Suppression Aging Phenotypes Cancer Tumor suppressors Can tumor suppression and aging be uncoupled??