Dialogue calcique et Sénescence David Bernard « Senescence Escape Mechanisms » lab Tumoral Escape Department What is cellular senescence ? -A stable form of cell cycle arrest (Hayflick et al, Exp Cell Res, 1965) young -Morphological alterations (Hayflick et al, Exp Cell Res,1965) -β β galactosidase activity at pH6 (SA b Gal) (Dimri et al, PNAS, 1995) -Secretory phenotype (SASP) (Coppe et al, PLos Biol, 2008) n s senescence Senescence: a stable proliferation arrest -where it occurs : in normal (replicative and premature) but also in cancer cells (premature) -how is it induced: by various stresses (short telomeres, oncogenes,oxidative stress, genotoxic drugs…) -how is it recognized : SAbGal activity, strongly released cytokines (SASP), may display nuclear heterochromatin dots (SAHF) and others markers have been proposed Physiopathological functions of cellular senescence Senescence Timely: -tumor suppressive -embryonic development -wound healing … Chronic: -tumor promoter -progeria/aging -aging related deseases … Physiopathological functions of cellular senescence Senescence Timely: -tumor suppressive -embryonic development -wound healing … Chronic: -tumor promoter -progeria/aging -aging related deseases … Tissue and organism homeostasis Proliferative disorder Cancer Degenerative disorder Aging Senescence Cellular senescence promotes aging: From old concept to recent (partial) demonstration proliferating cells normal human cell in culture non proliferating cells Replicative senescence = replicative limit 1) Cells derived from old human have a decreased replicative limit when compared to young ones Cellular senescence promotes aging: From old concept to recent (partial) demonstration Skin young (38) donor old (73) donor From Dimri et al, PNAS, 1995 2) Old tissues display more senescent cells in vivo (allowed by the discovery of the first senescence marker (SA-b-Gal activity)) Cellular senescence promotes aging: From old concept to recent (partial) demonstration From Baker et al, Nature, 2011 3) Eliminating senescent cells improve progeroid syndromes in mouse Cellular senescence promotes aging: From old concept to recent (partial) demonstration Senescent cells Limit renewal capacity of organs (proliferation arrest) Alter the tissue organization and function by its secretome (inflammatory mediators, MMP…) Cellular senescence inhibits tumorigenesis: From old concept to demonstration stress senescence 1) Cancer being a proliferative disease if cells stop to grow they cannot form tumors Cellular senescence inhibits tumorigenesis: From old concept to demonstration From Serrano et al, Cell, 1997 2) Oncogenes, which are the engine of the tumorigenesis process, induce normal human cell senescence Cellular senescence inhibits tumorigenesis: From old concept to demonstration From Collado et al, Nature, 2005 3) Senescence escape occurs in malignant tumors Cellular senescence inhibits tumorigenesis: From old concept to demonstration RasV12 From Xue et al, Nature, 2007 4) Senescence induction blocks tumorigenesis process Cellular senescence inhibits tumorigenesis: From old concept to demonstration Senescent cells the proliferation is blocked Secreted factors will activate the immune system Understand the mechanisms regulating cellular senescence should allow to better understand aging and cancer processes and might give rise to new tools to improve healthy aging as well as to fight cancer. Functional genetic screen to identify new regulators of senescence 1) Infection with the whole genome shRNA library lentivirus 3) Identification of the shRNA 2) Induction of senescence Model of senescence Immortalized mammary human epithelial cells (HMEC) upon oncogenic stress HMEC-Tert-MEK:ER (or RAF:ER) 120 4-OHT P-ERK PS10-H3 - + 100 80 60 40 20 ACTB Relative DcR2 mRNA level Cyclin A 7 6 5 4 3 2 1 0 0 - 4-OHT SA-β β-Gal Relative Dec1 mRNA level + + - + - + 3.5 3 Relative IL8 mRNA level - Relative Sprouty-2 mRNA level 4-OHT senescence marker 2.5 3 2.5 2 1.5 1 0.5 1 0.5 0 0 4-OHT 2 1.5 - + Identified Senescence Regulators -KCNA1 (Lallet-Daher et al, Cancer Research, 2013) -ABCC3 (Wiel et al, Oncogene, in press) -CASP2 (Gitenay et al, Oncotarget, 2014) -ITPR2 and MCU (Wiel et al, Nature Com, 2014) mito ER -Is calcium flow an important regulators of senescence ? -Is there a calcic dialog between ER and mitochondria during senescence ? -How mtCa2+ participate to senescence ? ITPR2 knockdown allows OIS escape ctrl ctrl shITPR2_2 shITPR2_3 ER ITPR2 100 80 60 40 20 0 + + 4OHT + + IL8 mRNA relative expression % of SA-β β -Gal positive cells Senescence markers 4OHT - 25 20 15 10 5 0 + 4OHT MCU knockdown allows OIS escape + + 60 50 40 30 20 10 0 +4OHT + +4OHT 4 3 2 1 0 mito MCU + IL8 mRNA relative epxression - pRS/shMCU_1pRS/shMCU_2pRS/shMCU_3 % of SA-β β -Gal positive cells pRS Senescence markers 4OHT pRS Calcium accumulates in the mitochondria during OIS Mitochondrial calcium genetic reporter ITPR2 MCU proliferating senescent Calcium accumulation in the mitochondria during OIS is inhibited by ITPR2 or MCU knockdown ITPR2 MCU 0.4 0.6 Fluorescence/area 0.5 - 4OHT 0.4 0.3 0.3 + 4OHT 0.2 0.2 0.1 0.1 0 0.0 red ? MCU JC-1 Red/Green ratio green 3.5 2.5 3.0 2.0 2.5 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 merge Ratiometric JC1 probe mCa2+ accumulation during OIS leads to mitochondrial depolarization ITPR2 proliferating senescent + 4OHT + 4OHT mCa2+ accumulation during OIS leads to ROS production ITPR2 ? 100 MCU 11 80 pRS count pRS + 4-OHT 18 60 17 pRS/shITPR2_2 + 4OHT 40 pRS/shITPR2_3 + 4OHT 33 20 0 100 101 102 103 Fluorescence intensity (ROS) 104 Model ITPR2 ∆ψ(M) ∆ψ senescence MCU ROS Wiel et al, Nature Com, 2014 Ca2+ in the mitochondria Normal level of Ca2+ High level of Ca2+ Required for mitochondrial activity Induce cell death or senescence To know more about the role of mitochondria in cellular senescence Senescence Escape Mechanism lab Nadine Martin (CR INSERM) David Vindrieux (MCF) Audrey Griveau (Postdoc) Marine Warnier (Postdoc) Xingjie Ma (PhD student) Mylène Ferrand (AI) Past members: Hélène Simonnet (MCF) Clotilde Wiel (PhD student) Arnaud Augert (PhD student) Hélène Lallet-Daher (Postdoc) Delphine Gitenay (Postdoc) Benjamin Le Calvé (Postdoc) collaborator N Prevarskaya (Lille)