Chk1 inhibition activates p53 through p38 MAPK in tetraploid cancer ...

[Cell Cycle 7:13, 1956-1961; 1 July 2008]; ©2008 Landes Bioscience

Brief Report

Chk1 inhibition activates p53 through p38 MAPK in tetraploid cancer cells Ilio Vitale,1-3 Laura Senovilla,1-3 Lorenzo Galluzzi,1-3 Alfredo Criollo,1-3 Sonia Vivet,1-3 Maria Castedo1-3,† and Guido Kroemer1-3,†,* 1INSERM, †These

U848; Unit “Apoptosis, Cancer and Immunity”; Villejuif, France; 2Institut Gustave Roussy; Villejuif, France; 3Université Paris Sud-XI; Villejuif, France

authors share senior co-authorship.

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Abbreviations: ATM, ataxia teleangiectasia mutated; ATR, ATM related kinase; BSA, bovine serum albumine; Bub1, budding uninhibited by benzimidazole 1; BubR1, Bub1-related protein; Chk1, checkpoint kinase 1; Chk2, checkpoint kinase 2; ΔΨm; mitochondrial transmembrane potential; DiOC6(3); 3,3'-dihexyloxacarbocyanine iodide; DYRK2, dual-specificity tyrosine-phosphorylation-regulated kinase 2; EndoG, endonuclease G; Env, HIV-1 envelope glycoprotein complex; ES, embryotic stem; Mad2, mitotic arrest deficiency 2; mTOR, mammalian target of rapamycin; p53S15P, p53 phophorylated on serine 15; p53S46P, p53 phophorylated on serine 46; p38T180/Y182P, p38 phophorylated on threonine 180 and tyrosine 182; p38 MAPK, p38 mitogen-activated protein kinase; PI, propidium iodide; SAC, spindle assembly checkpoint; siRNA, small interfering RNA, UCN-01, 7-hydroxystaurosporine

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Key words: apoptosis, genomic instability, mitosis, polyploidy, spindle assembly checkpoint

proteins has been shown to promote tetraploidization or to allow for the survival of tetraploid cells. This applies to APC6 or Lats2,7 whose suppression induces tetraploidization, as well as to p53,8 and its downstream effectors p21,9 and Bax,10 whose downregulation allows for the survival of recently generated tetraploid cells (which would be eliminated in normal circumstances). Conversely, the activation of oncogenes such as Aurora kinase A11 and papillomavirus E6,12 induces tetraploidization. Tetraploid cells can undergo asymmetric, often multipolar cell divisions and/or lose chromosomes, which leads to their aneuploidization.2 Experimental tetraploidization of p53-/- mammary epithelial cells coupled to chemical mutagenesis leads to the formation of tumors.13 Similarly, retroviruses are able to induce the transformation of human cells in vitro as a result of cell fusion,14 demonstrating that tetraploidization way constitute an oncogenic event. Pre-malignant and malignant tetraploid cells have been detected in precancerous and cancerous lesions including Barrett’s esophagus,15 laryngeal dysplasia,16 chronic ulcerative colitis dysplasia17 and pre-invasive lesions of the uterine cervix.12,18 Tetraploid cells are particularly resistant against radiotherapy and an array of DNA-damaging chemotherapeutic agents (e.g., cisplatin, oxaliplatin, etoposide, camptothecin).19 In response to chemotherapy or hypoxia,20 polyploid cells accumulate in tumors, suggesting that polyploidization may have a role in therapeutic failure. In yeast, the survival of tetraploid cells depends on enhanced DNA repair activity (in particular by homologous recombination), chromosomal cohesion and proficiency of the mitotic spindle checkpoint.21,22 Depletion of endonuclease G (EndoG) is lethal for tetraploid cells, both in yeast and in mammals.23 Moreover, mammalian tetraploid cells die upon knockdown of the checkpoint kinase 1 (Chk1), and its pharmacological inhibiton with 7-hydroxystaurosporine (UCN-01) is sufficient to kill tetraploid cells, both in vitro

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We have previously shown that tetraploid cancer cells succumb through a p53-dependent apoptotic pathway when checkpoint kinase 1 (Chk1) is depleted by small interfering RNAs (siRNAs) or inhibited with 7-hydroxystaurosporine (UCN-01). Here, we demonstrate that Chk1 inhibition results in the activating phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK). Depletion of p38 MAPK by transfection with a siRNA targeting the α isoform of p38 MAPK (p38α MAPK) abolishes the phosphorylation of p53 on serines 15 and 46 that is induced by Chk1 knockdown. The siRNA-mediated downregulation and pharmacological inhibition of p38α MAPK (with SB 203580) also reduces cell death induced by Chk1 knockdown or UCN-01. These results underscore the role of p38 MAPK as a pro-apoptotic kinase in the p53-dependant pathway for the therapeutic elimination of polyploidy cells.

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During oncogenesis and tumor progression, cancer cells and their precursors accumulate genetic and epigenetic changes, while genomic instability determines an ever more malignant phenotype. One of the multiple mechanisms to genomic instability involves a metastable phase of polyploidization (mostly tetraploidization), which may result from endoreplication (DNA replication without mitosis),1,2 endomitosis (karyokinesis without cytokinesis),2,3 or aberrant cell fusion between diploid cells.4,5 The loss of several tumor suppressor *Correspondence to: Guido Kroemer; INSERM; U848; Institut Gustave Roussy, PR1; 39, rue Camille Desmoulins; Villejuif F-94805 France; Tel.: 33.1.42.11.60.46; Fax: 33.1.42.11.60.47; Email: [email protected] Submitted: 04/07/08; Accepted: 04/08/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/6073 1956

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p38 MAPK in tetraploid tumor cell death

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Materials and Methods

Cell lines and culture conditions. Tetraploid HCT 116 colon carcinoma cells were generated from diploid HCT 116, as previously described.10,19 Cells were routinely maintained in McCoy’s 5A medium supplemented with 10% FCS (at 37°C in a 5% CO2 atmosphere) and seeded onto the appropriate support for cell culture (6-, 12-, 24- or 96-well microtiter plates, 35 or 100 mm Ø Petri dishes) 24 h before the beginning of the experiment. Media and supplements for cell culture were purchased from Gibco-Invitrogen (Carlsbad, USA), whereas plasticware was obtained from Corning B.V. Life Sciences (Schiphol-Rijk, The Netherlands). Chemicals. The p38 MAPKα inhibitor SB 203580 and the inactive chemically related compound SB 202474 were purchased from Merck Chemicals (Nottingham, UK). The Chk1 inhibitor UCN-01 and the ATM inhibitor KU-55933 were obtained from the National Cancer Institute (Bethseda, MD) and from KuDOS (Cambridge UK), respectively. Caffeine, an inhibitor of ATM and ATR, and the mTOR inhibitor rapamycin were obtained from Sigma-Aldrich (St. Louis, MO) and Tocris Bioscience (Ellisville, USA), respectively. Stock solutions were prepared following the manufacturer’s recommendations. Transfection and RNA interference. The knockdown of the proteins indicated in Supplementary Table 1 was performed with previously validated, specific siRNAs purchased from Sigma-Proligo (The Woodlands, TX) and from Invitrogen. The siRNAs for the downregulation of p38α MAPK (siGENOME Smart-pool, M-003512) were purchased from Dharmacon Inc., (Chicago, Il). As a control, a siRNA with an unrelated, scrambled sequence was employed (SCR). Tetraploid HCT 116 cells were cultured in 12-well plates and transfected at 30–40% confluence by means of the HiPerFect transfection reagent (Qiagen, Hilden, Germany) as previously described.26 72 h later, the transfection efficiency was determined by immunoblot. Immunofluorescence. Cells were fixed with 4% (w/v) paraformaldehyde in PBS and then immunostained with a rabbit antibody specific for p53 phophorylated on serine 46 (p53S46P) or with a mouse antibody specific for p53 phophorylated on serine 15

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Chk1 knockdown causes p38 MAPK-dependent p53 phosphorylaton in tetraploid cancer cells. When Chk1 is knocked down with a specific small interfering RNA (siRNA) (Suppl. Table 1), tetraploid HCT 116 cells manifest signs of apoptosis including the dissipation of the mitochondrial transmembrane potential (ΔΨm, detectable with the ΔΨm-sensitive fluorochrome DiOC6(3)) and the subsequent loss of viability (determined with the vital dye propidium iodide, PI) (Fig. 1A). This lethal effect is specific for tetraploid cells (and hence affects less than 20% of diploid cells),24 and could not be further enhanced by abolition of the spindle assembly checkpoint with siRNAs targeting budding uninhibited by benzimidazole 1 (Bub1), Bub1-related protein (BubR1) or mitotic arrest deficiency 2 (Mad2) (Fig. 1B). This agrees with the hypothesis that Chk1 inhibition induces tetraploid cell death through the inhibition of cell cycle checkpoints.24 We have previously shown that the death of tetraploid cells induced by Chk1 depletion is accompanied by the activating phosphorylation of p53 on serine 15 (which increases the stability of p53 protein),29,30 and on serine 46 (which stimulates the transactivation of pro-apoptotic p53 target genes).31,24 When searching for potential p53 kinases that might be activated after Chk1 inhibition, we found that p38 MAPK exhibited an activating phosphorylation on threonine 180 and tyrosine 182, as detectable by immunoblots with a phospho-epitope-specific antibody (Fig. 1C). Depletion of the dominant p38 MAPK α isoform with specific siRNAs (Fig. 1C) inhibited the phosphorylation of p53 on serine 46, as detected by immunochemistry (Fig. 1D) or immunofluorescence (Fig. 1E). Depletion of Chk1 (but not of the checkpoint kinase 2, Chk2) enhanced the frequency of tetraploid cells displaying phosphorylated p53 in their nuclei, and p53 phosphorylation (both on serines 15 and 46) was inhibited by depleting p38α MAPK, but was not affected by downregulating other kinases known to phosphorylate p53 on serine 46 (i.e., ataxia teleangiectasia mutated, ATM; ATM related kinase, ATR; dual-specificity tyrosine-phosphorylationregulated kinase 2, DYRK2) or by pharmacologically inhibiting mammalian target of rapamycin (mTOR),32 which has been previously described to phosphorylate p53 on serine 15 in polyploid cells33 (Fig. 1E and F). Altogether, these data indicate that p38 MAPK, a kinase that can directly phosphorylate p53 on serines 15 and 46,34-36 is activated in Chk1-depleted tetraploid cells and that p38MAPK is required for p53 activation in these circumstances. Interference with p38 MAPK reduces apoptosis induction by Chk1 inhibition. Since chemical inhibition, knockdown or knockout of p53 suppresses the death of tetraploid cells depleted for Chk1,24 we postulated that blocking the phosphorylation of p53 should have a similar outcome. In line with this hypothesis, we observed that depletion of p38 MAPKα (but not that of ATM, ATR or

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Results and Discussion

DYRK2) reduced tetraploid cell death induced by Chk1 knockdown (Fig. 2A). Similar results were obtained when tetraploid cells were killed by the addition of UCN-01, a pharmacological agent that inhibits Chk1,37 but that may also interfere with other kinases38 (including p38α MAPK, though at higher concentrations).39 Knockdown of p38α MAPK (but not that of ATM, ATR or DYRK2) reduced killing by UCN-01 (Fig. 2B), yet did not modify the apoptotic response to the general tyrosine kinase inhibitor staurosporine (data not shown). Finally, chemical inhibition of p38 MAPK with SB 203580 (which reportedly acts in a highly selective fashion on the p38 MAPK isoforms α and β)40,41 diminished the apoptosis of tetraploid cells induced by Chk1 depletion, while a chemically related compound, (SB 202474, which does not inhibit p38α MAPK)42 as well as blockers of ATM (KU-55933,43 caffeine), ATR (caffeine) and mTOR (rapamycin) had no cytoprotective effects (Fig. 2C). In conclusion, p38α MAPK is required for the optimal induction of tetraploid cell death triggered by depletion/inhibition of Chk1.

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and in vivo.24 This therapeutic effect of Chk1 depletion/inhibition is accompanied by the inactivation of the spindle assembly checkpoint, by mitotic aberrations, and by the activaton of a p53-dependent apoptotic program.24,25 Here, we demonstrate that p38 MAPK is involved in the activating phosphorylation of p53 on serines 15 and 46, thus constituting an obligatory link between Chk1 inhibition and p53-dependent apoptosis of tetraploid cells.

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Figure 1. For figure legend, see page 1959.

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Figure 1. p38 MAPK-dependent phosphorylation/activation of p53 in tetraploid HCT 116 cells upon Chk1 depletion. (A and B) Tetraploid human colon carcinoma HCT 116 cells were subjected to siRNA-mediated downregulation of Chk1 or transfected with a control siRNA (SCR), alone or in combination with siRNAs directed against spindle assembly checkpoint (SAC) proteins (Bub1, BubR1 or Mad2) for 72 h, followed by cytofluorometric quantification of viability (with propidium iodide, PI) and mitochondrial transmembrane potential (ΔΨm, with DiOC6(3)). Representative FACS pictograms are shown in (A) and data are quantified in (B). White and black columns report the percentage of cells (mean ± SEM, n = 3) exhibiting ΔΨm loss alone (ΔΨmlow) and in combination with plasma membrane rupture (PI+), respectively. Asterisks (*) depict statistical significant results (p < 0.05). (C and D) HCT 116 cells were transfected for 72 h with a control or a Chk1-specific siRNA alone or together with a p38α MAPK (p38α)-depleting siRNA. Thereafter, proteins were purified from cellular extracts, and subjected to immunoblotting with antibodies that recognize p38 MAPK and phospho-p38 MAPK (Thr 180/Tyr 182, p38T180/Y182P) (C) or with antibodies specific for p53 and phospho-p53 (Ser 46, p53S46P) (D). GAPDH was detected to ensure control equal loading. Extracts of cells treated for 18 h with 15 μM cisplatin (CDDP) were used as a positive control of p53 activated phosphorylation. (E and F) Tetraploid HCT 116 Chk1-transfected cells were left untreated, co-transfected for 72 h with siRNAs specific for kinases known to phosphorylate p53 (ATM, ATR, p38, Chk2, DYRK2) or treated with 10 μM rapamycin (rap, an mTOR inhibitor). Thereafter, cells were immunostained with antibodies specific for the phosphorylated forms of p53 on serine 15 (p53S15P, red fluorescence) and on serine 46 (p53S46P, green fluorescence, insert of E). Hoechst 33342 (emitting in blue) was employed as nuclear counterstain. Representative images are shown in (E) (scale bars = 20 μm). White and black columns in (F) report the percentage (mean ± SEM, n = 3) of cells characterized by the phosphorylation of p53 on serine 15 and 46, respectively. Asterisks (*) depict statistical significant differences (Student’s t-test, p < 0.05) in the percentage of cells displying p53 phosphorylation.

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Concluding Remarks

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Technology Inc.,), p53 (Santa Cruz Biotechnology, San Jose, USA) and GAPDH (Millipore), which was monitored as loading control.

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Since polyploid cells constitute potential intermediates of carcinogenesis, and represent a chemoresistant population of tumor cells, their removal might have a prophylactic and/or therapeutic impact. Here, we show that Chk1 depletion/inhibition causes the death of tetraploid cancer cells, through a pathway that involves the activation of p38α MAPK, which in turn participate in the pro-apoptotic phosphorylation of p53 on serines 15 and 46. These results, which have been obtained in tetraploid cancer cells, are incompatible with multiple reports suggesting that p38 MAPK, once activated, may act as a positive effector of cell cycle arrest in diploid cells. In normal and transformed diploid cells, p38α MAPK plays an important role as a positive effector in the spindle assembly checkpoint44 and is able to mediate a G2/M arrest in response to physical or chemical DNA damage.45,46 This effect reportedly contributes to cell survival, perhaps due to the avoidance of catastrophic mitoses,47 yet may be independent of p38 MAPK kinase activity.48 In normal unstressed cells, p38 MAPK may control mitotic entry by phosphorylating Cdc25B,49 but may also act as an essential “back up system” in DNA-damaged p53-deficient cells to avoid mitotic catastrophe.39 According to one report,50 p38 MAPK and Chk1 may even collaborate in non-transformed cells to prevent mitotic entry upon hydroxyurea treatment. This is again at odds with our results, which suggest that Chk1 inhibition (not activation) is linked to p38 MAPK activation. There is only one report suggesting that Chk1 inhibition can lead to the activation of p38 MAPK, thereby mediating an S phase arrest in mouse embryonic stem (ES) cells51 (which are notoriously deficient in normal G1/S checkpoint activation after DNA damage).52,53 Thus, tetraploid and ES cells may share some peculiarities in the regulation of cell cycle checkpoints that remain to be investigated at the mechanistic level. p38 MAPK is activated after fusion of non-synchronized cells with polyethylene glycol or after formation of syncytia beetween cells expressing the HIV-1 envelope glycoprotein complex (Env) and cells bearing at their surface the Env receptor CD4 and suitable co-receptors of the chemokine receptor family.36,54 In this system, p38 MAPK physically interacts with p53, thereby causing its phosphorylation on serines 15 and 46.36 Both p38 MAPK and p53 phosphorylation can be detected in syncytia, in tissues from HIV-1 carriers,36 suggesting

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(p53S15P) (both from Cell Signaling Technology Inc., Danvers, USA).10 Thereafter, slides were rinsed in 1% (w/v) bovine serum albumin (BSA) in PBS and incubated for 1 h at 37°C with secondary goat anti-rabbit or anti-mouse IgG conjugated to Alexa Fluor® 568 or to Alexa Fluor® 488 fluorochromes (Molecular Probes-Invitrogen, Eugene, USA) in 2% (w/v) BSA in PBS. Nuclei were counterstained with 10 μM Hoechst 33342 (Molecular Probes-Invitrogen). Fluorescence images were captured on an IRE2 microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a DC300F camera and image analysis was performed with the open source software Image J (freely available from the National Institute of Health, Bethesda, USA at the address http://rsb.info.nih.gov/ij/). In each of three independent experiments, the percentage of cells that exhibited p53 phosphorylation on serine 15 and 46 was estimated among a population of 200 cells (mean ± SEM, n = 3). In this context, statistical significance of was assessed by independent, two-tail Student’s t-test (p values < 0.05 are depicted by asterisks in figures, when relevant for the aim of this study). Cytofluorometric studies. For the simultaneous quantification of plasma membrane integrity and mitochondrial transmembrane potential (ΔΨm), live cells were collected and stained with 1 μg/mL propidium iodide (PI, which only incorporates into dead cells, from Sigma-Aldrich) and 40 nM of the ΔΨm-sensitive dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3), from Molecular Probes-Invitrogen) for 30 min at 37°C.27 Cytofluorometric acquisition of red (PI) and green (DiOC6(3)) fluorescence was carried out by means of a FACSCalibur or a FACScan (Becton Dickinson, San Jose, CA) equipped with a 70 μm nozzle. Data were statistically evaluated using CellQuest™ software (Becton Dickinson). Only the events characterized by normal forward scatter and side scatter parameters were included in subsequent analysis. Statistical significance was determined by independent, two-tail Student’s t-test (asterisks indicate p values < 0.05, when important for the scope of the article). Western blotting. HCT 116 cells were lysed in a buffer containing 1% NP40, 20 mM HEPES (pH 7.9), 10 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM orthovanadate, 1 mM PMSF, 1 mM dithiothreitol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 10 μg/ml pepstatin, as previously described.28 Thereafter, protein extracts (80 μg/lane) were subjected to standard separation on SDS-PAGE, followed by immunoblotting with antibodies specific for p38 MAPK (Millipore, Billerica, USA), phospho-p38 MAPK (Thr 180/Tyr 182, p38T180/ Y182P), phospho-p53 (Ser 46, p53S46P) (both from Cell Signaling www.landesbioscience.com

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Figure 2. Apoptosis induced by Chk1 depletion/ inhibition in tetraploid cells is controlled by p38 MAPK. (A and B) Tetraploid colon carcinoma HCT 116 cells were transfected for 72 h with a Chk-1 specific or a control siRNA (SCR) (A) or incubated with 500 nM UCN-01 (a pharmacological inhibitor of Chk1) (B), alone or in combination with transfection of the indicated siRNAs, and then processed for the cytofluorometric quantification of mitochondrial transmembrane potential (ΔΨm) and viability (with propidium iodide, PI). (C) Tetraploid HCT 116 cells transfected with a a control (SCR) or a siRNA targeting Chk1 were left untreated or treated with the p38a MAPK inhibitor SB 203580, the chemically related compound SB 202474 (which does not inhibit p38α MAPK), or with inhibitors of ATM (KU-55933, caffeine), ATR (caffeine) and mTOR (rapamycin, rap) at the indicated concentration, followed by cytofluorometric assessment of apoptosis-associated parameters as in (A). White and black columns depict the percentage of cells (mean ± SEM, n = 3) that have dissipated their ΔΨm (ΔΨmlow) and lost plasma membrane integrity (PI+), respectively. Asterisks (*) indicate statistically significant difference (Student’s t-test, p < 0.05) as compared to the indicated control.

that this event is relevant to HIV-1 pathogenesis. Of note, in HIV-1-elicited syncytia, checkpoint kinases reportedly act as negative regulators of cell death, meaning that transfection of cells with a construct encoding for dominant-negative checkpoint kinase 2 or treatment with debromohymenialdesine (a chemical inhibitor of checkpoint kinases) enhanced the demise of syncytia, through the induction of mitotic catastrophe.55,56 Here, we report that Chk1 inhibition activates p38 MAPK in yet another model of polyploidy, namely tetraploid cancer cells. This suggests that, in conditions of polyploidy, p38 MAPK is under the control of checkpoint kinases, through a mechanism that remains to be elucidated. Regardless of these details, our results delineate a novel pathway to induce the therapeutic death of tetraploid cells. In this cascade of molecular events, Chk1 inhibition results in the activation of p38 MAPK, thereby causing p53-dependent apoptosis. Acknowledgements

UCN-01 was a generous gift of the National Institute for Cancer (NCI, Bethesda, USA), KU-55933 of KuDOS (Cambridge, UK). GK is supported by the Ligue Nationale contre le Cancer (Equipe labellisée), European Commission (Active p53, Apo-Sys, RIGHT, TransDeath, ChemoRes, DeathTrain), Cancéropôle Ile-de-France, and 1960

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Supplementary materials can be found at: www.landesbioscience.com/supplement/VitaleCC7-13-Sup.pdf

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