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Wip1 Promotes RUNX2-Dependent Apoptosis in P53-Negative Tumors and Protects Normal Tissues During Treatment with Anticancer Agents

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Wip1 Promotes RUNX2-Dependent Apoptosis in P53-Negative Tumors and Protects Normal Tissues During Treatment with Anticancer Agents Wip1 promotes RUNX2-dependent apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents Anastasia R. Goloudinaa, Kan Tanoueb, Arlette Hammanna, Eric Fourmauxa, Xavier Le Guezennecc, Dmitry V. Bulavinc, Sharlyn J. Mazurb, Ettore Appellab, Carmen Garridoa,d,e, and Oleg N. Demidova,d,1 aInstitut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche 866, University of Burgundy, 21078 Dijon, France; bLaboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; cInstitute of Molecular and Cell Biology, Cell Cycle Control and Tumorigenesis Group, Singapore 138673; dFaculty of Medicine and Pharmacy, University of Burgundy, 21078 Dijon, France; and eCentre Hospitalier Universitaire Dijon, 21000 Dijon, France Edited by Carol Prives, Columbia University, New York, NY, and approved October 17, 2011 (received for review May 11, 2011) The inactivation of the p53 tumor suppressor pathway in many have clinical implications given the potential therapeutic uses of cancers often increases their resistance to anticancer therapy. Here compounds that interfere with Wip1 suppression of p53 activity. we show that a previously proposed strategy directed to Wip1 Indeed, expression of a Wip1 antisense transcript in the breast inhibition could be ineffective in tumors lacking p53. On the carcinoma cell line MCF-7 resulted in increased p53-dependent contrary, Wip1 overexpression sensitized these tumors to chemo- apoptosis (17). Several studies in mice have shown that the tumor- therapeutic agents. This effect was mediated through interaction suppressive phenotype of Wip1 deletion_is p53-dependent and between Wip1 and RUNX2 that resulted, in response to anticancer that p53 loss completely reverses the effect in these systems. Thus, treatment, in RUNX2-dependent transcriptional induction of the Wip1 inhibition is probably only effective in tumors with preserved proapoptotic Bax protein. The potentiating effects of Wip1 over- WT p53. expression on chemotherapeutic agents were directed only to tu- Currently, the majority of anticancer therapies use the p53 mor cells lacking p53. The overexpression of Wip1 in normal tissues pathway to induce tumor cells death (18). Following activation by provided protection from cisplatin-induced apoptosis through de- anticancer drugs, p53 induces the expression of numerous genes, creased strength of upstream signaling to p53. Thus, Wip1 phos- including proapoptotic genes such as Bax (19). Elevated expres- phatase promotes apoptosis in p53-negative tumors and protects sion of proapoptotic genes leads to initiation of the apoptotic normal tissues during treatment with anticancer agents. program and eventually to cell death. In contrast, tumors bearing mutant p53 often exhibit resistance to anticancer drugs (20, 21). caspases | Bcl-2 family | dephosphorylation | intestine Effective therapy of tumors with inactive p53 continues to present a challenge for modern oncology. To overcome this issue, several he tumor suppressor p53 is a transcriptional factor that is ac- strategies have been proposed. For example, inactivation of Chk1 Ttivated by various stresses and initiates cell cycle arrest, DNA kinase in p53-negative tumors compromises G2 arrest in response repair, and apoptosis (1). The importance of p53 in suppressing to anticancer therapy and induces mitotic catastrophe, eliminating tumor initiation and growth is indicated by the fact that more than tumor cells (22). Unfortunately, Chk1 inhibition could be highly half of all human cancers lose p53 function through mutation or toxic to normal tissues and may induce severe side effects (23). depletion of the p53 gene (2). The consequences of p53 loss are Here we report an alternative approach, based on activation of multiple; not only does it influence growth, genomic stability, and Wip1 phosphatase, toward sensitizing tumors containing inactive other tumor characteristics, but it also affects the efficacy of an- p53 to anticancer drugs, while at the same time protecting normal ticancer treatment. tissues. In response to anticancer drugs in tumors with inactive p53, Alternatively, tumor cells could use negative regulators of p53 to Wip1 overexpression led to the induction of Bax through de- suppress its activity. For example, a significant number of human phosphorylation-dependent activation of the transcription factor RUNX2. In normal tissues, Wip1 suppressed p53 hyperactivation cancers exhibit Mdm2 gene amplification and/or overexpression (3). in response to anticancer therapy, thereby decreasing normal tissue Mdm2 gene encodes an E3 ubiquitin ligase that interacts with p53 damage. Thus, in tumors lacking functional p53, Wip1 acts as and mediates p53 proteasomal degradation. P53 activates Mdm2 a sensitization factor to anticancer drugs while protecting normal expression, forming a negative feedback regulatory loop (4, 5). tissues bearing WT p53 from side effects of anticancer therapies. Another feedback mechanism in the p53 pathway is connected fi to its posttranslational modi cation. After stress, p53 undergoes Results various posttranslational modifications that increase p53 stability Wip1 Overexpression Increases Sensitivity of Tumor Cells with and/or potentiate its transcriptional activity (6). Among the genes Inactive p53 to Anticancer Drugs. Wip1 inhibition was proposed as whose expression is regulated by p53 is the PP2C serine-threonine a novel anticancer strategy directed to nongenotoxic activation of phosphatase Wip1 (gene name PPM1D). Similarly to Mdm2,the Wip1 gene (PPM1D)isamplified in many tumor types (7, 8). Wip1 overexpression leads to dephosphorylation of crucial activating Author contributions: A.R.G. and O.N.D. designed research; A.R.G., K.T., A.H., E.F., and phosphoserines or phosphothreonines found in the DNA damage O.N.D. performed research; X.L.G., D.V.B., and E.A. contributed new reagents/analytic response protein kinases ATM, Chk2, and Chk1, among others. tools; A.R.G., K.T., D.V.B., S.J.M., E.A., C.G., and O.N.D. analyzed data; and A.R.G., S.J.M., DNA damage response kinases are important transducers of sig- E.A., C.G., and O.N.D. wrote the paper. nals from damaged DNA to p53. The inhibition of the ATM/Chk2 The authors declare no conflict of interest. kinase cascade by Wip1 prevents p53 activation (9–11). Addition- This article is a PNAS Direct Submission. ally, it was shown that phospho-Ser15 in p53, which is important for Freely available online through the PNAS open access option. p53 activity, could be directly dephosphorylated by Wip1 (12). 1To whom correspondence should be addressed. E-mail: [email protected]. Wip1-KO mice exhibit a tumor-suppressive phenotype in several See Author Summary on page 361. cancer models such as lymphomagenesis and mammary gland or This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. intestinal tumorigenesis (13–16). Therefore, Wip1 inhibition may 1073/pnas.1107017108/-/DCSupplemental. E68–E75 | PNAS | January 10, 2012 | vol. 109 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.1107017108 Downloaded by guest on October 2, 2021 p53 (24). At the same time, in several mouse models of tumori- To examine the effect of Wip1 overexpression on the sensitivity PNAS PLUS genesis, the tumor-resistant phenotype of Wip1-null mice was of tumor cells to anticancer drugs, we generated two human cell abrogated by p53 deletion, supporting the interpretation that lines, U2OS-Wip1-on (WT p53) and Saos-2-Wip1-on (p53 de- effects of Wip1 deletion are mediated through the p53 pathway letion), in which Wip1 expression was regulated by a tetracycline (15). The p53 tumor suppressor mutated or lost in approximately inducible promoter (Fig. S1B). Induction of Wip1 in the cells with 50% of human tumors. Therefore, the status of p53 may affect the WT p53 mildly protected the cells from anticancer treatment with response of tumor cells to Wip1 inhibition. First, we analyzed the cisplatin. In sharp contrast, in the highly cisplatin-resistant, p53- effect of Wip1 depletion on the sensitivity to an anticancer agent of negative Saos-2 cells, previous Wip1 induction strongly increased several tumor cell lines with or without p53. As expected, Wip1 Saos-2-Wip1-on cell death in response to cisplatin (Fig. 1B and depletion (Fig. S1A) increased sensitivity to cisplatin (i.e., CDDP) Fig. S1C). Interestingly, Wip1 overexpression in Saos-2-Wip1-on in tumor cell lines expressing WT p53, the osteosarcoma cell line cells increased cell death only after cisplatin treatment. Wip1 in- U2OS and the colon cancer cell line HCT116, but it had no effect duction had no effect on survival of nontreated Saos-2-Wip1-on − − on the sensitivity in HCT116 p53 / or the p53-negative osteo- cells. Thus, Wip1 overexpression sensitized Saos-2 cells to cis- sarcoma cell line Saos-2 (Fig. 1A). platin-induced cell death, but did not induce cell death by itself. Fig. 1. Wip1 sensitizes tumor cells to chemotherapeutic agents. (A) Down-regulation of Wip1 sensitized cancer cell lines to cisplatin (CDDP) only in the presence of p53. WT p53-expressing cells (human osteosarcoma U2OS cells and colorectal cancer HCT116 cells) and p53-negative cells (human osteosarcoma Saos-2 and colorectal cancer HCT116 p53−/− cells) were transfected with control scrambled siRNA or Wip1 siRNA. Twenty-four hours after transfection, cells were nontreated (NT) or treated with 25 μM CDDP for 48 h, harvested, and subjected to Guava ViaCount cell death assay. (B) Sensitivity of Saos-2 and U2OS cells to cisplatin (CDDP) with or without doxycycline-induced Wip1 expression. Wip1 was induced by doxycycline for 24 h in several clones of established U2OS- Wip1-on and Saos-2-Wip1-on cell lines (Fig. S1B). Cells with or without Wip1 induction were treated with 25 μM cisplatin for 48 h, harvested, and subjected to Guava ViaCount cell death assay (*P < 0.05). (C) Overexpression of phosphatase inactive Wip1 does not sensitize Saos-2 cells to cisplatin. Saos-2 cells were MEDICAL SCIENCES transfected with mutant Wip1 D314A expressing vector DNA; Saos-2 Wip-on cells were cultivated with or without doxycycline.
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