MINIREVIEW ERK and cell death: Mechanisms of ERK-induced cell death – apoptosis, autophagy and senescence Sebastien Cagnol1 and Jean-Claude Chambard2 Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Canada Institute of Developmental Biology and Cancer, University of Nice, France Keywords apoptosis; autophagy; DUSP; ERK; ROS; senescence Correspondence S Cagnol, Department of Anatomy and Cellular Biology, Faculty of Medicine and Health Sciences, University of Sherbrooke, Sherbrooke, Quebec, Canada Fax: +1 819 564 5320 Tel: +1 819 820 6868 ext 15715 E-mail: sebcagnol@yahoo.fr (Received 18 June 2009, revised 26 August 2009, accepted September 2009) The Ras ⁄ Raf ⁄ extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in almost all cell functions and therefore requires exquisite control of its spatiotemporal activity Depending on the cell type and stimulus, ERK activity will mediate different antiproliferative events, such as apoptosis, autophagy and senescence in vitro and in vivo ERK activity can promote either intrinsic or extrinsic apoptotic pathways by induction of mitochondrial cytochrome c release or caspase-8 activation, permanent cell cycle arrest or autophagic vacuolization These unusual effects require sustained ERK activity in specific subcellular compartments and could depend on the presence of reactive oxygen species We will summarize the mechanisms involved in Ras ⁄ Raf ⁄ ERK antiproliferative functions doi:10.1111/j.1742-4658.2009.07366.x Ras ⁄ Raf ⁄ ERK, the pathway ERK2 ⁄ ERK1 (also known as p42 ⁄ p44MAPK, respectively, and officially named MAPK and 3) are two isoforms of extracellular signal-regulated kinase (ERK) that belong to the family of mitogen-activated protein kinases (MAPKs), which include ERK5, the c-JunNH2-terminal kinases (JNK1 ⁄ ⁄ 3) and the p38 MAP kinases (p38 a,b,d,c) These enzymes are activated through a sequential phosphorylation cascade that amplifies and transduces signals from the cell membrane to the nucleus Upon receptor activation, membrane-bound GTP-loaded Ras recruits one of the Raf kinases, A-Raf, B-Raf and C-Raf (or Raf1), into a complex where it becomes activated Then, Raf phosphorylates two serine residues on the kinase mitogen protein kinase kinase and (MEK1 ⁄ 2; also known as MAP2K1 and MAP2K2, respectively), which in turn activate ERK1 ⁄ by tandem phosphorylation of threonine and tyrosine residues on the dual-specificity motif (T-E-Y) Finally, active ERKs regulate by phosphorylation many cytoplasmic and nuclear targets that perform important biological functions [1] Depending on the duration, the magnitude and its subcellular localization, ERK activation controls various cell responses, such as proliferation, migration, differentiation and death [2] Protein phosphatases play an important role as negative regulators by controlling the Ras ⁄ Raf ⁄ ERK signaling pathway at different levels Phosphotyrosine phosphatases target the tyrosine kinase Abbreviations ATA, aurintricarboxylic acid; cPLA2, cytosolic phospholipase A2; DAPK, death-associated protein kinase; DUSP, dual-specificity phosphatase; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; FADD, Fas-associated death domain; GAIP, G-interacting protein; IGF, insulin-like growth factor; JNK, c-JunNH2-terminal kinase; MAPK, mitogen-activated protein kinase; MEK1/2, mitogen protein kinase kinase and (also known as MAP2K1 and MAP2K2, respectively); MEKCA, constitutively activated forms of MEK; MKP, mitogen-activated protein kinase phosphatase; MOS, v-mos Moloney murine sarcoma viral oncogene homolog; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS S Cagnol and J.-C Chambard receptors, whereas phosphoserine ⁄ phosphothreonine phosphatases target the adapter protein Shc and MEK1 ⁄ Dual-specificity phosphatases [DUSP; also called MAPK phosphatases (MKP)], are able to dephosphorylate both of the threonine and tyrosine residues within the activation loop of MAPK Specific DUSPs tightly regulate subcellular ERK activity DUSP1 ⁄ MKP-1, DUSP2 ⁄ PAC-1, DUSP4 ⁄ MKP-2 and DUSP5 are mainly nuclear, whereas DUSP6 ⁄ MKP-3, DUSP7 ⁄ MKP-X and DUSP9 ⁄ MKP-4 are cytoplasmic Moreover, the expression of DUSP1, -2, -4 and -6 is increased following ERK activation [3,4], taking part in a negative feedback loop aimed at terminating Ras ⁄ Raf ⁄ ERK signaling pathway stimulation The Ras ⁄ Raf ⁄ ERK pathway is frequently deregulated in tumors as a result of activating mutations in Ras or B-Raf, observed particularly in malignant melanoma, pancreas intestine and thyroid tumors (cosmic database: http://www.sanger.ac.uk/genetics/CGP/ cosmic) Many studies associate its oncogenic potential to increased cell survival, mainly by promoting the activity of antiapoptotic proteins, such as Bcl-2, Bcl-XL, Mcl-1, IAP, and repressing proapoptotic proteins, such as Bad and Bim [5] Paradoxically, a growing number of studies also suggest that in certain conditions, aberrant ERK activation can promote cell death This review will summarize the different cellular models in which the Ras ⁄ Raf ⁄ ERK pathway plays an antiproliferative role The specific pro-death function of ERK activity in neurons [6] and lymphocytes [7] and its role in cadmium toxicity [8] will also be discussed in this minireview series Ras ⁄ Raf ⁄ ERK pathway induces apoptosis Programmed cell death by apoptosis is a cell-autonomous mechanism that relies on pathway-controlled activation of caspases and nucleases leading to the death of the injured cells without affecting neighboring cells The intrinsic pathway of apoptosis regulates the activity of the Bcl-2 family proteins that control the integrity of the mitochondrial membrane The release of proapoptotic factors from the mitochondria, such as cytochrome c, into the cytoplasm promotes the activation of initiator caspase-9, which in turn activates effector caspases such as caspase-3 or -7 The extrinsic pathway relies on the activation of death receptors from the tumor necrosis factor (TNF) receptor family that promote the recruitment and activation of initiator caspase-8 via adaptor proteins such as Fas-associated death domain (FADD) or TNFRSF1A-associated via death domain (TRADD) ERK and cell death Strong caspase-8 activity may directly activate effector caspases; it may also require signal amplification through induction of the intrinsic pathway via cleavage of the Bcl-2 family protein Bid [9] Early reports of a proapoptotic function of the Ras ⁄ Raf ⁄ ERK pathway appeared in 1996 Depletion of Raf by the benzoquinone ansamycin geldanamycin was shown to protect MCF-7 cells from apoptosis induced by the antitumor compound taxol [10], whereas MEK antisense cDNA expression prevented bufalin-induced apoptosis in U937 leukemic cells [11] A growing number of studies using MEK inhibitors (PD98059, U0126) and expression of dominant negative or constitutively active forms of Ras, Raf, MEK or ERK have confirmed the implication of the Ras ⁄ Raf ⁄ ERK pathway in the induction of apoptosis (see Table for details) The proapoptotic function of the Ras ⁄ Raf ⁄ ERK pathway is well documented for apoptosis induced by DNA-damaging agents, such as etoposide [12–15], doxorubicin [13,16–20], UV [13] and gamma irradiation [21] ERK activity has been particularly implicated in cisplatin-mediated apoptosis in renal cells [15,22–32] (Table 1) ERK activity has also been involved in cell death induced by various antitumor compounds, such as resveratrol [33], quercetin [34], phenethyl isothiocyanate [35], betulinic acid [36], apigenin [37], oridonin [38], miltefosine [39], shikonin [40] or taxol [10,41] (Table 1) Most of these drugs induce the intrinsic apoptotic pathway However, ERK activity has also been involved in activation of the extrinsic pathway by death receptor ligands such as TNF-related apoptosisinducing ligand (TRAIL) [34,42–46], TNFa [47–49], Fas [50,51] or CD40 ligand [52,53] Cell death induced by other death pathways that occur in response to zinc [54,55], oxidation [56–59], especially in response to ONOO) [60], H2O2 [61–64] or NO treatment [65–67], toxic compounds such as cadmium, [17,68,69], benzo[a]pyrene [70], asbestos [71] or arsenic [17,72], also require ERK activity Many death stimuli, such as estradiol [73] or its antagonist tamoxifen [74], interferon-a [75], cephalosporin [76], the calcium mobilizer calcimycin [77], epidermal growth factor (EGF) deprivation [78], leptin [79], bufalin [11], bacterial infection [80,81], chelerythrine [82] and the dominant negative form of Rac and Cdc42 [83,84], are sensitive to inhibition of the Ras ⁄ Raf ⁄ ERK pathway (Table 1) Conversely, constitutive activation of ERK by dominant active Raf1 combined with c-Myc expression [85] or p53 induction [86], or constitutively active MEK combined with death-associated protein kinase FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS ERK and cell death S Cagnol and J.-C Chambard Table Models of ERK-mediated apoptosis and autophagy LDH, lactate dehydrogenase; MDC, monodansylcadaverine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; ND, not determined; TGHQ, 2,3,5-tris-(glutathione-S-yl)hydroquinone; TPA, 12-O-tetradecanoylphorbol-13-acetate Characteristics of cell death Evidence implicating MEK-ERK in cell death Reference 24 h Caspase-3 PD98059 [12] Etoposide UV Doxorubicin Etoposide 24 h DNA degradation PD98059 [13] 24 h PD98059 DN ERK [14] Doxorubicin ND DNA condensation Caspase-3 PARP cleavage PARP cleavage PD98059 [16] TPA ArsenicCadmium Doxorubicin Doxorubicin 24 h DNA fragmentation DNA condensation U0126 PD98059 [17] 12 h Cell viability U0126 [18] Doxorubicin 24 h DNA fragmentation PARP cleavage PD98059 DN ERK [19] Doxorubicin 48 h nuclear U0126 [20] NIH 3T3 c irradiation 48 h Cisplatin 20 h PD98059 DN ERK U0126 PD98059 [21] Human cervix adenocarcinoma HeLa Human lung carcinoma A549 Human ovarian adenocarcinoma A2780 Human osteosarcoma Saos-2 Cisplatin ND DNA fragmentation Caspase-9, -3 PARP cleavage Membrane integrity Annexin V DNA condensation Caspase-3 PARP cleavage Cytochrome c release DNA fragmentation PD98059 [23] Cisplatin 24 h Cisplatin 24 h Cisplatin 72 h U0126 PD98059 U0126 PD98059 U0126 [24] Rabbit primary renal proximal tubular cells Mouse immortalized proximal tubule cell line (TKPTS) Human carcinoma NCCIT and NTERA In vivo mouse kidney Cell viability DNA fragmentation DNA condensation Caspase-3 Caspase-3 Cisplatin 24 h 72 h U0126 PD98059 U0126 [27] Cisplatin injection Opossum immortalized kidney cells OK cells Cisplatin 48 h DNA condensation Caspase-8, -9, -3 DNA fragmentation Caspase-8, -3 DNA degradation Caspase-3 Human cervix adenocarcinoma HeLa Human papillary thyroid carcinoma BHP 2–7 and BHP 18–21 Human prostate adenocarcinoma PC3 Cisplatin 18 h Resveratrol 24 h nuclear Phenethyl isothiocyanate 24 h In vivo ⁄ cellular model Transformed mouse fibroblast NIH 3T3 Human keratinocytes HaCaT Human hepatocellular carcinoma HepG2 and Huh-7 Human promonocytic leukemia Human breast adenocarcinoma MCF-7 NIH 3T3, human immortalized keratinocytes HaCaT Rat immortalized cardiomyocytes H9c2 Stimuli inducing cell death Duration of ERK activation promoting cell death Etoposide Caspase-9 PARP cleavage DNA degradation Annexin V ROS production [22] [25] [26] [28] U0126 PD98059 DN MEK U0126 [29] [30] PD98059 [33] PD98059 [35] FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS S Cagnol and J.-C Chambard ERK and cell death Table Continued Stimuli inducing cell death Duration of ERK activation promoting cell death Human melanoma C8161,WM164 Mel Juso Betulinic acid 96 h Human cervix adenocarcinoma HeLa Human melanoma A375-S2 Human glioblastoma T98G and U87MG Human cervix adenocarcinoma HeLa Human breast adenocarcinoma MCF-7 Normal human embryonic kidney HEK Human primary fibroblast BJ Human colorectal HT29 cells Apigenin 8h Oridonin 12 h Miltefosine 12 h DNA fragmentation Cytochrome c release Cell viability Shikonin 12 h Caspase-8, -3 PD98059 [40] Taxol 24 h DNA fragmentation PD98059 [41] TRAIL Constitutive ND U0126 PD98059 U0126 [42] TRAIL DNA fragmentation Caspase-8 cell viability TRAIL 5h PD98059 [44] Human prostate cancer LNCaP Human prostate tumor DU-145 Human neuroblastoma SHEP-1 Huaman neuroblastoma SHEP Rat primary Sertoli cells Human acute T leukemia Jurkat Diffuse large B-cell lymphoma Primary human cholangiocytes Pig renal tubular epithelial cells LLCỈPK1 In vivo ⁄ isolated rat renal cortical slices Pig renal tubular epithelial cells LLCỈPK1 Rabbit primary renal proximal tubular cells Human primary retinal pigmented epithelial ARPE19 cells TRAIL 4h U0126 [45] Quercetin TRAIL TRAIL H2O2 FasL 24 h Membrane integrity Nuclear condensation PARP cleavage Annexin V Caspase-3 Membrane integrity PD98059 [34] ND Membrane integrity PD98059 [46] 30 DNA condensation DN MEK1 [50] Fas (CH11) Membrane integrity DNA fragmentation PD98059 [51] CD40 ligation 3h 24 h U0126 PD98059 PD98059 [52] CD40 ligation Zinc 24 h nuclear Membrane integrity DNA fragmentation DNA condensation Caspase-3 activity Cell viabilityROS production U0126 [54] ZnCl2 injection 90 nuclear Cell viability U0126 [55] TGHQ 5h Cell viability PD98059 [56] tert-butylhydroperoxide 8h Annexin V U0126 PD98059 [58] tert-butylhydroperoxide 6h Cell viability Caspase-9 DNA fragmentation U0126 [59] In vivo ⁄ cellular model FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS Characteristics of cell death DNA fragmentation DNA condensation PARP cleavage Cell viability Evidence implicating MEK-ERK in cell death Reference U0126 [36] U0126 PD98059 PD98059 [37] [38] U0126 [39] [43] [53] ERK and cell death S Cagnol and J.-C Chambard Table Continued Stimuli inducing cell death Duration of ERK activation promoting cell death Hyperoxia 4h Caspase-9, -3 Cytochrome c release PD98059 [57] Hyperoxia 72 h PD98059 [57] Primary rat pulmonary myofibroblasts Immortalized mouse fibroblast L929 Mouse immortalized osteoblast Rabbit primary renal proximal tubular cells Rabbit primary renal proximal tubular cells ONOO) 30 DNA fragmentation Caspase-3 Cell viability PD98059 [60] H2O2 3h Biphasic 12 h PD98059 DN ERK PD98059 [61] H2O2 H2O2 h constitutive DNA fragmentation Annexin V Cell viability Membrane integrity DNA condensation Caspase-3 [63] H2O2 2h Membrane integrity Annexin V Human transformed bronchial epithelial cell line BEAS-2B Human melanoma HEK293 NO donor 24 h Cell viability U0126 PD98059 MEKCA U0126 PD98059 MEKCA DN MEK PD98059 NO donor Cadmium ND Biphasic 96 h U0126 U0126 [67] [68] Human hepatocellular carcinoma HepG2 Rat primary pleural mesothelial cells In vivo Caenorhabditis elegans Murin bone marrow-derived primary osteoclasts and murine long bone-derived osteocytes MLO-Y4 Human breast adenocarcinoma MCF-7 Human myeloma cell line U266-1984 and RHEK-1 Isolated rat renal cortical slices Immortalized rabbit lens epithelial cells N ⁄ N1003A Benzo[a]pyrene 48 h PARP cleavage Caspase-8, -3 PARP Cell viability PD98059 [70] Crocidotite Asbestos Arsenic 48 h PD98059 [71] ND DNA condensation ROS production DNA fragmentation [72] Estradiol 24 h Membrane integrity mek-2 (n1989) mpk-1 (ku1) U0126 Tamoxifen 1h Membrane integrity PD98059 [74] Interferon-a 16 h Annexin V Caspase-3 U0126 [75] Cephaloridine 90 nuclear 10 h U0126 PD98059 U0126 DN Raf DN ERK [76] Calcimycin Primary mouse kidney proximal tubular epithelial cells Primary human bone marrow stromal cells EGF deprivation 120 h Cell viability ROS production Membrane integrity DNA fragmentation Annexin V Cytochrome c release Caspase-3 Cell viability U0126 PD98059 [78] Leptin 12 h Cell viability Cytochrome c release Caspase-3 U0126 PD98059 [79] In vivo ⁄ cellular model Murine transformed lung epithelial MLE12 In vivo mouse lung Characteristics of cell death Evidence implicating MEK-ERK in cell death Reference [62] [64] [65] [73] [77] FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS S Cagnol and J.-C Chambard ERK and cell death Table Continued In vivo ⁄ cellular model Human leukemia U937 Pig renal tubular epithelial cells LLCỈPK1 Mouse monocyte ⁄ macrophage J774.2 Stimuli inducing cell death Duration of ERK activation promoting cell death Evidence implicating MEK-ERK in cell death Reference DNA fragmentation AnnexinV PARP cleavage Annexin V DNA fragmentation MEK antisense PD98059 [11] [80] U0126 PD98059 [81] MEKCA U0126 PD98059 DN Ras PD98059 PD98059 DN DUSP6 RAF-CAAX MEKCA [82] U0126 Raf CA DRAF1 [86] DRaf1 HrasV12T35S [89] U0126 MEK siRNA [90] DRaf1:ER U0126 [91] U0126 PD98059 [48] U0126 PD98059 U0126 [49] PD98059 [69] PD98059 [114] RasV12S35 [115] U0126 [116] Characteristics of cell death Bufalin Escherichia coli toxin Francisella tularensis infection Chelerythrine 12 h 44 h Mouse embryonary fibroblast Mouse embryonary fibroblast RacN17 Cdc42N17 RacN17 Cdc42N17 ND 24 h DNA fragmentation Caspase-8, -9, -7 PARP cleavage Membrane integrity Annexin V Rat fibroblast Rat1 Mouse immortalized fibroblast NIH 3T3 Murine erythroleukemia DP16.1 ⁄ p53ts cells Human breast adenocarcinoma MCF-7 Human primary osteoblast RAF-CAAX DAPK Constitutive Constitutive DNA condensation Cell viability P53 induction Constitutive Annexin V DRAF1 Constitutive DRaf1 HrasV12 T35S Constitutive HEK293T IGF-I receptor 48 h HEK293 DRaf1:ER Anti-Fas(CH11) Constitutive Murine fibrosarcoma cells L929 TNFa Human breast adenocarcinoma MCF-7 Mouse RAW264.7 macrophages TNFa 10 h NO 2h constitutive Transformed mouse mesengial MES-13 cells Human colon adenocarcinoma HT29 Cadmium 3h Amino acid depletion 4h Human colon adenocarcinoma HT29 Amino acid depletion ATA RasV12S35 Soyaponin 4h constitutive DNA fragmentation Vacuolization Cell viability Membrane integrity Annexin V DNA fragmentation Membrane integrity Caspase-3 Vacuolization Membrane integrity DNA fragmentation DNA condensation Annexin V Caspase-8, -3 PARP cleavage Vacuolization Cell viability LC3-II induction Beclin induction Cell viability LC3-II induction Cell viability Membrane integrity BNIP-3 induction Cell viability MTT LC3-II induction GAIP phosphorylation Autophagic LDH sequestration Proteolysis LDH sequestration 3h MDC incorporation Human osteosarcoma cell line HOS and U2OS Human colon adenocarcinoma HCT-15 42 h 4h FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS [83] [84] [85] [87] [88] [66] ERK and cell death S Cagnol and J.-C Chambard Table Continued Stimuli inducing cell death Duration of ERK activation promoting cell death Lindane 24 h Dihydrocapsaicin 4h Human OVCA-420 PEA-15 48 h Primary human fibroblast IMR90 RasV12 Constitutive In vivo ⁄ cellular model Mouse 42GPA9 Sertoli cell line Primary human lung fibroblasts WI38 (DAPK) expression [87], could induce apoptosis without any other stimulus Moreover, in rare cases, activation of the Raf ⁄ ERK pathway alone mediated by Raf1 [88,89], RasV12S35 [89], insulin-like growth factor type I (IGF-I) receptor [90] expression or by DRaf1:ER induction [91] was sufficient to promote cell death Mechanisms of Ras ⁄ Raf ⁄ ERK-mediated apoptosis ERK activity has been associated with classical markers of apoptosis execution, such as effector caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, annexin-V staining, DNA condensation or DNA fragmentation (see Table for details) Depending on the cell type and the nature of the injury, activation of the Ras ⁄ Raf ⁄ ERK pathway is associated with the intrinsic apoptotic pathway characterized by the release of cytochrome c from mitochondria [22,38,57,77,79] and activation of initiator caspase-9 [20,27,29,57,59,82] or with the extrinsic apoptotic pathway, which relies on the activation of an initiator caspase-8 [27,28,40,42,68,82] ERK promotes caspase-8 signaling and activation The Ras ⁄ Raf ⁄ ERK pathway potentiates activation of death receptors by increasing the level of death ligands such as TNFa [28] or FasL [51] or death receptors such as Fas [39,89,91], DR4 [44,46] or DR5 [42,44,46] ERK activity also promotes the induction of FADD, an adaptator of caspase-8 to the death receptors [39,91] Because ERK-mediated caspase-8 activation requires de novo protein synthesis [68,91], it may reflect the activation of transcription factors regulated by ERK, such as c-Fos, which has been associated with Characteristics of cell death LC3 relocalization Vacuolization LC3-II induction Caspase-3, -7 LC3 relocalization Membrane integrity Acidic vacuoles LC3-II induction Evidence implicating MEK-ERK in cell death PD98059 U0126 PD98059 ERK siRNA ERK siRNA Reference [117] [118] [119] [120] the upregulation of DR4 and DR5 [44] However, we have shown that ERK-induced caspase-8 activation could be independent of Fas and FADD upregulation, suggesting death receptor-independent modes of caspase-8 activation by ERK [91] FADD bears a death effector domain that mediates caspase-8 activation A very similar structure is found to bind ERK in vanishin [92] and PEA-15 [93], proteins that regulate both ERK and FADD activity [94] These observations suggest that differential interactions between death effector domain-containing proteins that bind either ERK or caspase-8 could mediate death receptorindependent activation of the extrinsic pathway of apoptosis The control of cytochrome c release by Bcl-2 family proteins As shown above, ERK activity is associated with DNA-damaging agents and antitumor compoundinduced apoptosis, which are often described as inducing the intrinsic pathway of apoptosis Therefore, several studies have suggested that the Ras ⁄ Raf ⁄ ERK pathway is involved in this pathway Indeed, ERK activity has been shown to directly affect mitochondrial function by decreasing mitochondrial respiration [25,58] and mitochondrial membrane potential [58,63,79], which could lead to mitochondria membrane disruption and cytochrome c release [22,38,57,77,79] Interestingly, active ERK has been found to be localized to mitochondrial membranes [25,58,63] ERK activity could also promote cytochrome c release by modulating Bcl-2 family protein expression MEK ⁄ ERK activity has been associated with the upregulation of proapoptotic members of the Bcl-2 family, such as Bax [20,30,40,62,67,77], p53 upregulat- FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS S Cagnol and J.-C Chambard ed modulator of apoptosis (PUMA) [20,86] and Bak [75], as well as the downregulation of antiapoptotic members, such as Bcl-2 [13,18,20,23,41,45,89] and BclXl [23,45] In addition, ERK-activated caspase-8 induces the release of cytochrome c through proteolytic activation of the proapoptotic member Bid [45] ERK promotes p53 stability and activity The regulation of Bcl-2 family proteins has been tightly associated with transcriptional activity of the tumor suppressor gene p53 Apoptosis induced by DNA-damaging agents correlates with p53 upregulation and modulation of Bcl-2 family proteins in an MEK-dependent manner [13,18,20,23,24,33,40,41,48, 77,95] ERK-mediated p53 upregulation is associated with p53 phosphorylation on serine 15 [20,24,33,70, 86,95,96], which stabilizes p53 protein and promotes its accumulation by inhibiting an association with Mdm2 [96] This is supported by the ability of ERK to bind p53 [18,95] and to phosphorylate p53 on serine 15 in vitro [95,96] Moreover, Mdm2 phosphorylation on serine 166, which is associated with its ubiquitin ligase activity toward p53, is inhibited upon sustained ERK activation [97] ERK activity is implicated in p53 phosphorylation on threonine 55, promoting DNAbinding activity and Bcl-2 downregulation [18] c-Myc, which is stabilized by ERK through phosphorylation on serine 62, increases the proapoptotic functions of p53 [98] Interestingly, when combined with c-Myc overexpression, constitutive activation of ERK is sufficient to induce apoptosis in Rat-1 cells [85] and to potentiate TRAIL-induced apoptosis in primary fibroblasts [43] The use of p53-deficient cells [35,83], p53 siRNA [70], p53 antisense [33,77], p53 inhibitor pifithrin-a [20,33,41], temperature-sensitive allele of p53 [86] or inducible p53 [24] showed that ERK-mediated p53 expression is required for apoptosis However, in other studies, the Ras ⁄ Raf ⁄ ERK pathway is able to induce apoptosis independently of p53 [24,35,36,41] Together, these data suggest that upregulation of the tumor suppressor p53 may be an important mechanism of Ras ⁄ Raf ⁄ ERK-induced apoptosis Other mediators of Ras ⁄ Raf ⁄ ERK-induced apoptosis Cytosolic phospholipase A2 (cPLA2) is a potential mediator of Ras ⁄ Raf ⁄ ERK pathway-induced apoptosis through intrinsic as well as extrinsic pathways The Fas receptor in Sertoli cells [51], B cell receptor (BCR) in B lymphoma [99] or leptin in adipocytes [79], all ERK and cell death promote MEK-dependent cPLA2 induction and activation ERK can directly activate cPLA2 by phosphorylation at serine 505 [100,101] The use of cPLA2 inhibitor AACOCF3 suggests that cPLA2 was necessary for ERK-induced apoptosis by a mechanism that promotes FasL induction [51] or cytochrome c release [79] Like death receptors and FADD, DAPK contains a death domain ERK was shown to bind to DAPK and increase its catalytic activity by phosphorylation on serine 735 [87] DAPK activation results in apoptosis due to cell detachment [87] or increase in TNF receptor function [47] In MCF-7 cells and primary osteoblasts, activation of Raf ⁄ ERK pathway-induced apoptosis was the consequence of cellular detachment from the matrix, which was in this case due to a decrease in integrin b1 expression [88,89] DNA-damaging agents have been shown to mediate sustained ERK activation through the protein kinase ataxia telangiectasia mutated [13,102,103] Implication of Ras ⁄ Raf ⁄ ERK pathway during apoptosis in vivo Following tissue injury ERK activity has been clearly implicated in neurodegenerative diseases and brain injury following ischemia ⁄ reperfusion in rodents (for a review see [6,104,105]) The Ras ⁄ Raf ⁄ ERK pathway also plays a key role in mouse models of acute renal failure induced by cisplatin [28] or lung injury induced by hyperoxia [57], as treatment with MEK inhibitors prevents apoptosis and tissue destruction in these models During development Proapoptotic ERK activity has also been reported in developmental models During germinal cell development, PEA-15, a cytoplasmic death domain-containing protein that binds and sequesters ERK, is highly expressed in the cytoplasm of Sertoli cells, spermatogonia and spermatocytes, inducing a cytoplasmic ERK localization Interestingly, testis isolated from PEA-15deficient mice display an abnormal nuclear accumulation of ERK in germinal cells, which correlates with increased apoptosis [106] In Caenorhabditis elegans, loss-of-function alleles of lin-45 (RAF homolog), mek-2 (MEK homolog) and mpk-1 (ERK homolog), have presented genetic evidence for a direct role of the Ras ⁄ Raf ⁄ ERK pathway in germinal cell apoptosis [72] In unfertilized eggs of starfishes Asterina pectinifera FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS ERK and cell death S Cagnol and J.-C Chambard and Marthasterias glaciali, v-mos Moloney murine sarcoma viral oncogene homolog (MOS)-dependent sustained ERK activity led to protein synthesis-dependent synchronous apoptosis [107–109] Moreover, maintaining ERK activity in fertilized eggs by MOS injection is sufficient to induce apoptosis [107] During metamorphosis in ascidian Ciona intestinalis, sustained nuclear activity of ERK homolog (Ci-ERK) in the tail is required for the induction of apoptosis (caspase-3like activity) and necessary for tail regression [110] Finally, during limb development in chick embryos, ERK activity is inhibited in the mesenchyme by FGF8-induced DUSP6 activity When activated by the expression of constitutively active MEK1, downregulation of DUSP6 or by the expression of a phosphataseinactive mutant of DUSP6 (C294S), sustained ERK activity induces massive apoptosis and prevents limb development [111] These results strongly indicate that Ras ⁄ Raf ⁄ ERK pathway-mediated apoptosis is not only associated with in vitro manipulation of cell lines, but also plays a key role in vivo during development and following tissue injury The role of the Raf ⁄ ERK pathway in the induction of cell death should not be restricted to apoptosis, i.e caspase-dependent cell death In some cases, the methods used to assess cell viability, based on cell metabolism or membrane permeabilization measurements (see Table 1), cannot distinguish between apoptosis and other forms of cell death, such as necrosis or autophagy Cytoplasmic vacuolization: lysosomal cell death and autophagy Autophagy is a genetically regulated program, initially identified as a cell survival mechanism to protect from nutrient deprivation However, in certain conditions, autophagy results in a form of cell death now described as type II programmed cell death [112] We and others have shown that constitutive activation of ERK by active Raf [88,91], cadmium [68] or IGF-I receptor [90] induced a form of cell death that correlated with extensive cell rounding and the formation of cytoplasmic macrovacuoles, which pushed the nucleus and the cytoplasm to the side of the dying cell Although cell death was associated with caspase-8 activation [68,91], this massive vacuolization is unrelated to the classical features of apoptosis This morphology could be a sign of autophagic programmed cell death, but also of paraptosis, a form of caspase-independent cell death associated with cytoplasmic vacuolization [90] Interestingly, other studies using cadmium [69,113] or TNFa treatment [49] have clearly associated ERK activation with autophagic programmed cell 10 death rather than with apoptosis This is supported by several studies that have associated ERK activity with neuron autophagic cell death in the course of a neurodegenerative disease [6,104,105] In addition, ERK activity has also been associated with autophagy and autophagic cell death in many non-neuronal cellular models (see Table 1) in response to different stresses, such as amino acid depletion [114] and aurintricarboxylic acid (ATA) [115] in human colorectal cancer cell line HT29, soyasaponins [116] in human colon adenocarcinoma HCT-15, lindane [117] in the mouse Sertoli cell line, dihydrocapsaicin [118] in WI38 lung fibroblasts, cadmium in mesengial MES-13 cells [69,113] and TNFa treatment in MCF-7 [49] and L929 cells [48] Interestingly, in human ovarian cancer cells, cytoplasmic sequestration of ERK by PEA-15 has been shown to promote autophagy [119] Moreover, direct ERK activation by overexpression of constitutively active MEK can promote autophagy without any other stimulus [117] ERK-dependent autophagic activity is associated with classical markers of autophagy, such as induction of LC3 and conversion of LC3-I to LC3-II [48,49,118], induction of beclin [48], induction of BNIP-3 [66] and activation of G-interacting protein (GAIP) by phosphorylation on serine 151 [114] p53 is also associated with the autophagic process, as ERK-mediated phosphorylation of p53 on serine 392 [48] was involved in TNFa-induced autophagy The lysosomal compartment plays an important role in autophagy by fusing with autolysosome vacuoles In NIH3T3 and in human colon carcinoma HCT-116 cells, oncogenic forms of Ras, respectively, v-H-Ras and K-Ras, lead to increased sensitivity to the lysosomal cell death pathway induced by cisplatin or etoposide in an MEK-dependent manner In these models, constitutive ERK activation leads to a decrease in the levels of lysosome-associated membrane protein-1 and -2 due to the induction and activation of cysteine–cathepsin B [15] In humans, ERK activity is potentially associated with limb sporadic inclusion body myositis, a disease characterized by cytoplasm vacuolization of muscle fibers Interestingly, immunostaining of muscle samples from patients revealed a strong ERK accumulation in cytoplasmic vacuoles [120] Together, these results suggest that the Ras ⁄ Raf ⁄ ERK pathway can mediate autophagic type II programmed cell death ERK-induced cytoplasm vacuolization associated with autophagy has some similarity with senescenceassociated vacuoles Interestingly, autophagy has recently been reported to be required for the efficient FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS S Cagnol and J.-C Chambard ERK and cell death establishment of senescence induced by a constitutively active form of Ras or MEK [121] process is often prevented by use of MEK inhibitors (See Table 2) The Ras ⁄ Raf ⁄ ERK pathway and senescence Mechanisms of ERK-induced senescence Cellular senescence is an irreversible form of cell cycle arrest that prevents proliferation of damaged cells or cells that have surpassed their capacity to proliferate In response to oncogenic hyperproliferative signals, primary cells undergo cell cycle arrest leading to premature oncogene-induced senescence [122] Although aberrant activation of the Ras ⁄ Raf ⁄ ERK pathway promotes oncogenic transformation of immortalized cells, it is also tightly associated with senescence of primary cells In human and rodent primary fibroblastic and melanocytic cells, senescence is triggered by constitutively active forms of Ras [123–126], PAK4 [125,127], Raf [97,126,128–133] or MEK [123,124,132,134] This Ras ⁄ Raf ⁄ ERK pathway-induced senescence correlates with increased b-galactosidase activity and induction of classical senescence-associated genes, such as p16 ⁄ INK4A, p53, p21 and p14-p19 ⁄ ARF (Table 2), senescence-associated heterochromatic foci [127,132] and DNA damage foci [132] In human primary fibroblasts IMR90, when senescence is provoked by inducible activation of Raf1:ER, it correlates with inhibition of AKT and dephosphorylation of Mdm2, which lead to p53 accumulation and growth arrest [97] In the case of human BJ foreskin primary fibroblasts, senescence induced by ectopic expression of RasV12 or constitutively activated forms of MEK (MEKCA) requires ERK-induced p38 activation [124] Interestingly, a Table Models of ERK-mediated senescence In vivo ⁄ cellular model Primary human fibroblastic IMR90 Induction of ERK activity Mouse immortalized NIH 3T3 fibroblast Primary human fibroblast IMR90 Ras V12 MEKCAQ56P RasV12 MEKCA Ras V12 PAK4 DRaf1:ER DRaf1:ER Human prostate cancer LNCaP cells DRaf1:ER Mouse embryonic fibroblast Primary human fibroblast IMR90 DRaf1:ER Raf-CAAX DRaf1:ER B-Raf V600E Primary human fibroblastic BJ cells Primary mouse fibroblastic cells Primary human melanocytes Primary human fibroblast BJ In vivo ⁄ human naevi Primary human melanocytes In vivo ⁄ human Naevi B-Raf V600E In vivo ⁄ mouse lung tumor model B-Raf V600E Primary human melanocytes B-Raf V600E MEKCAQ56P Primary human melanocytes Primary human intestinal epithelial cells B-Raf V600E NRasQ61R MEKCA SS218 ⁄ 222DD Primary human fibroblast WI38 DUSP4 (MKP-2) FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS Markers of senescence MEK activity Reference b-galactosidase activity p21 p53 and p16 ⁄ INK4A b-galactosidase activity p16 ⁄ INK4A b-galactosidase activity p21, p19 ⁄ ARF and p16 ⁄ INK4A p21 b-galactosidase activity p21 and p16 ⁄ INK4A b-galactosidase activity p21 p21, p53 and p19 ⁄ Arf b-galactosidase activity PD98059 [123] U0126 [124] PD98059 [125] PD98059 [128] [129] b-galactosidase activity p21, p53 and p16 ⁄ INK4A Heterochromatic foci b-galactosidase activity Heterochromatic foci p16 ⁄ INK4A p19 ⁄ ARF Dec1 Heterochromatic foci b-galactosidase activity p16 ⁄ INK4A, cH2AX Heterochromatic foci b-galactosidase activity p53 and p16 ⁄ INK4A b-galactosidase activity p21 p53 and p16 ⁄ INK4A b-galactosidase activity [130] [131] [97] [127] [135] [133] [132] [126] [134] [136] 11 ERK and cell death S Cagnol and J.-C Chambard phenotypic comparison between RasV12-, RasR61- and B-RafE600-induced senescence in human melanocytes suggests that the senescence programs are different: Ras-induced senescence was faster and was associated with massive cytoplasmic vacuolization, whereas B-Rafexpressing cells exhibited a more rounded morphology [126,132] However, human and rodent cells induce different senescence programs The use of mouse embryonic fibroblasts derived from knock-out mouse models suggested that Ras ⁄ Raf ⁄ ERK pathway-induced senescence relies on the induction of cell cycle regulators, such as p16 ⁄ INK4A [123,125], p21 [126], p53 [123], p19 ⁄ ARF [125] In human primary fibroblasts, however, knock-down or inhibition of either p16 [127] or p53 [126] was not sufficient to reverse senescence, suggesting that these genes may have a redundant function controlling human senescence Oncogene-induced senescence prevents transformation of human primary cells unless overridden by the presence of a cooperating oncogene, such as Myc Indeed, overexpression of c-Myc in normal human melanocytes suppressed B-Raf- or N-Ras-induced senescence [126] Myc expression is then continuously required for transformation, as downregulation of c-Myc in tumor-derived melanoma cells was shown to induce senescence [126] Senescence and subcellular ERK localization ERK-induced senescence has been associated with an aberrant control of its spatial activity Kim et al [135] found that reactive oxygen species (ROS) produced during senescence of human primary fibroblasts inactivate the cytosolic ERK phosphatase DUSP6, resulting in cytoplasmic sequestration of active ERK However, other studies have suggested that senescence could also be the result of inhibition of nuclear ERK activity due to an increase in nuclear DUSP4 activity [136,137] Thus, coordinate gene expression induced by nuclear ERK might be required to prevent the completion of a senescence program induced by increased cytoplasmic ERK activity Implication of the Ras ⁄ Raf ⁄ ERK pathway in senescence in vivo Senescence induced by ectopic expression of an oncogene might reflect an artificially high expression level, as discussed in the study by Tuveson et al [138] of the oncogene KRasD12 However, several results based on the expression of B-RafE600 at physiological level under the control of its own promoter, support the idea that Ras ⁄ Raf ⁄ ERK pathway-induced senescence is a physiological cellular response For instance, in 12 humans, naevi (moles) can be considered as an in vivo example of B-Raf-driven senescence Naevi are melanocyte-derived benign tumors restrained from malignant progression by engagement of senescence Naevi frequently harbor the oncogenic B-RafE600 or NRasR61 mutation [139] (which promote senescence of primary melanocytes [126,127,132]) and display markers of senescence such as b-galactosidase activity and high p16 expression [127,140] Recently, a mouse model of inducible tumorigenesis in lung epithelium driven by the B-RafE600 oncogene revealed that expression of BRafE600 alone was not sufficient to promote a severe tumoral phenotype, leading instead to benign hyperplastic lesions undergoing senescence-associated growth arrest [133] In this model, p53 invalidation was necessary to promote B-RafE600-mediated transformation and malignant tumor formation [133] The hallmarks of ERK-mediated cell death: sustained and sequestered activity ROS as mediators of ERK-induced cell death In the majority of the studies related to cell death induced by the Ras ⁄ Raf ⁄ ERK pathway, ERK activation is unusually prolonged, i.e ERK is maintained phosphorylated for between and 72 h (see Table 1) Moreover, delayed treatments with U0126, a MEK inhibitor, have revealed that ERK activity is continuously required to induce cell death [91] Despite constitutive activation of the pathway by oncogenes, levels of ERK phosphorylation in tumor cells are very variable [141], presumably due to phosphatase-driven feedback mechanisms Because ERK-specific phosphatases are sensitive to ROS, we speculate that the main cause of sustained ERK activation is the presence of ROS, perhaps reflecting the levels of ROS scavengers in each particular model The use of different ROS inhibitors demonstrated that ERK activation requires ROS production to induce cell death [14,54,56,57,60,61,65,76,78] Indeed, chemical oxidants, such as H2O2, peroxynitrite ONOO) or NO (see Table 1), induce ERK, whereas many stimuli implicating ERK in cell death promote the production of ROS [14,35,54,55,71,76,82] Moreover, DNA-damaging agents, such as doxorubicin, cisplatin or etoposide, catalyze the formation of ROS [142] In addition, ERK activity could be directly responsible for ROS production by upregulating inducible NO synthase [80] Thus, ROS-mediated prolonged ERK activation might be the crucial mechanism implicating the functions of the Ras ⁄ Raf ⁄ ERK pathway in cell death FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS S Cagnol and J.-C Chambard ROS promote sustained ERK activation Mechanisms of ROS-mediated ERK activation upstream of ERK ROS can stimulate the Ras ⁄ Raf ⁄ ERK pathway by promoting the activation of tyrosine kinase receptors, such as platelet-defined growth factor receptor or EGF receptor [26,60,61], and adaptor proteins, such as Shc [143] ROS can also increase signaling by direct oxidation of residue C118 on Ras, a reaction that potentiates recruitment and activation of Raf at the plasma membrane [144,145] Other proteins implicated in Raf activation, such as Src [61], protein kinase C (PKC)-d [25,29,61] or the cGMP pathway [146], could also be activated by ROS Moreover, direct oxidation of cysteine residues in the cystein-rich domain of Raf promotes its autoactivation [147] Downstream of Raf, peroxynitrite ONOO) can also cause nitration and autophosphorylation of MEK [61] ROS and inhibition of ERK phosphatases Finally, ERK activity could be prolonged through the inhibition of tyrosine phosphatases and DUSP by ROS Indeed, enzymatic activity of DUSP and tyrosine phosphatases requires a catalytic cysteine residue sensitive to oxidation [3,4] ROS have been shown to inhibit ERK-directed phosphatases, DUSP1 and DUSP6, by oxidation of their catalytic cysteine residues, C258 and C293, respectively [148,149], as well as ERK tyrosine phosphatases PP1 ⁄ 2A by oxidation of their conserved catalytic residue C62 [135] Thus, the control of phosphatases that downregulate ERK activity plays a crucial role in the outcome of Ras ⁄ Raf ⁄ ERK pathway signaling Together, these data indicate that ROS can initiate and sustain ERK activation by different mechanisms Interestingly, cell death has been associated with a biphasic activation of ERK, which could reflect this dual control of ERK activity by ROS [62,68] The importance of subcellular localization of ERK activity In most cases, prolonged ERK activation alone, such as in models expressing constitutively active forms of upstream kinases, is not sufficient to promote cell death [15,63,64,66,82,85–87] In normal cells, subcellular localization of ERK is tightly regulated by scaffold proteins and docking phosphatases that allow nuclear accumulation of dephosphorylated ERK to terminate signaling [2] Thus, in addition to a sustained ERK activity, the outcome of ERK-mediated ERK and cell death cell death might also rely on an aberrant subcellular localization Indeed, apoptosis induced by estradiol [73], tamoxifen [74], zinc [54,55] cephaloridine [76], doxorubicin [20], revestratol [33] or dominant negative mutant of Rac or Cdc42 [84] correlated with sustained nuclear ERK activity As mentioned previously, nuclear activation of ERK is also associated with apoptosis during Ciona intestinalis development [106] and in mouse testis deficient for PEA-15 [106] Interestingly, some of the compounds that induce nuclear ERK activity are associated with the production of ROS [54,55,76], which could promote nuclear accumulation of active ERK due to inhibition of DUSP In MDA-MB-231 human breast cancer cells, taxolinduced apoptosis was abrogated by induction of nuclear DUSP1 In this study, DUSP1 induction clearly inhibited both ERK and JNK activity [150] Because nuclear DUSPs (especially DUSP1 and -4) also control JNK and p38 phosphorylation [3,4], any modification of DUSP activity or expression could also increase cell death by activation of the stress pathways Cytoplasmic sequestration of ERK has also been associated with different forms of cell death Cytoplasmic sequestration of ERK by binding to PEA-15 promotes autophagy [119], whereas sustained cytoplasmic ERK activity induces senescence in human primary fibroblasts [135–137] Together, these data suggest that sustained activation of ERK in different subcellular compartments is not tolerated and results in different forms of cell death (see Fig 1) The limits of ERK1/2-mediated cell death studies, the specificity of MEK inhibitors In many studies, implication of the Ras ⁄ Raf ⁄ ERK pathway in the induction of cell death is based uniquely on the sole use of MEK1 ⁄ inhibitors PD05059 or U0126 (see Table 1) The weakness of these inhibitors is that they inhibit both MEK1 ⁄ and MEK5 [151] Interestingly, it has recently been shown that constitutive activation of the MEK5 ⁄ ERK5 pathway could promote apoptosis of meduloblastoma cells [152] or thymocytes [153,154], through Nur77-dependent mechanisms [153,154] As a consequence, in those types of cell, some of the effect attributed to ERK1 ⁄ might also be caused by the MEK5 ⁄ ERK5 pathway The use of PD184352, a more recent MEK1 ⁄ inhibitor that does not target MEK5 [155], could help to distinguish between the effects of MEK1 ⁄ and MEK5 in those cells FEBS Journal 277 (2010) 2–21 ª 2009 The Authors Journal compilation ª 2009 FEBS 13 ERK and cell death S Cagnol and J.-C Chambard Fig The hallmarks of ERK-mediated cell death: sustained and sequestered activity ERK activity induces the expression of many genes, including its own regulators, the DUSP (ERK-specific phosphatases) Thus, ERK activity rapidly reaches a steady state and its death-promoting activity remains at low levels (gray line) Any agent that provokes a sustained activation of ERK, such as ROS that inhibit DUSP, induces the progressive accumulation of death-promoting factors up to a level that induces cell death The activation of ERK (arrow) might also transiently increase death-promoting activities of other death stimuli, such as chemotherapeutic agents In addition to DUSP inhibition by ROS, ERK-mediated cell death is characterized by a deregulation of subcellular active ERK localization Sustained cytoplasmic ERK activity might promote senescence or autophagy, whereas sustained nuclear sequestration of ERK activity might trigger apoptosis In both conditions, sequestration of ERK depends on subcellular anchors, such as PEA-15, in the cytoplasm Conclusions Together, these data clearly demonstrate that the Ras ⁄ Raf ⁄ ERK pathway plays a critical role in promoting several forms of cell death in response to numerous stress stimuli both in vitro, with various cellular models, and in vivo A common hallmark of this response is the sustained activation of ERK, which contrasts with the transient nature of ERK stimulation found in situations where ERK regulates other cell fates As depicted in Fig 1, ERK activates its own phosphatases, inducing a feedback loop that, within hours, restores a basal level of ERK activity At least in the cellular models depicted in Table 1, ERK stimulation induces the expression of gene products with death-promoting activity We can 14 speculate that the feedback loop decreasing subcellular ERK activity over time prevents these death-promoting factors reaching a threshold concentration that triggers cell death Consequently, any agent affecting the kinetics of ERK activity in a given cellular compartment (such as the ROS that inhibit DUSP in Fig 1) has a potential to induce cell death Given the importance of the spatiotemporal regulation of ERK activity for the control of cell division [2], the induction of cell death could be seen as negative feedback mechanism preventing uncontrolled cell proliferation The Ras ⁄ Raf ⁄ ERK pathway is among the most commonly deregulated pathways identified in tumors, as indicated by frequently observed activating mutations in Ras or B-Raf oncogenes Thus, this pathway is currently the target of new antitumor strategies, based on the inhibition of upstream ERK regulators However, because ERK activation is implicated in DNA-damaging agent-induced cell death (see Table 1), inhibiting ERK activity in combination therapy with classical antitumor compounds, such as cisplatin or doxorubicin, might affect the efficiency of such compounds Because prolonged ERK activation has been shown to promote the death of human cancer cell lines from different origins (see Table 1), this property of the Ras ⁄ Raf ⁄ ERK pathway to induce cell death could be used to target cancer cells However, although tumor cells escape Ras ⁄ Raf ⁄ ERK pathway-induced senescence by inactivating effectors of senescence, such as p53 or p16 ⁄ INK4A, mechanisms involved in ERKinduced cell death might also be silenced in tumor cells Tumor cells with high ERK activity might also have re-modeled the ERK signaling to escape ERKmediated cell death Thus, the crucial biochemical events underlying sensitivity or resistance to ERKmediated cell death remain to be fully understood We propose the hypothesis that in tumor cells harboring strong ERK activity, the alteration of compensating pathways (PI3K ⁄ AKT, Wnt, etc…) would unleash the cell killing 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