Cancer Therapy: Preclinical Mitogen-Activated Protein Kinase Phosphatase-1 in Human Breast Cancer Independently Predicts Prognosis and Is Repressed by Doxorubicin

Purpose: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dephosphorylates mitogen-activated protein kinase [extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38], mediates breast cancer chemoresistance, and is repressible by doxorubicin in breast cancer cells. We aimed to characterize doxorubicin effects on MKP-1 and phospho-MAPKs in human breast cancers and to further study the clinical relevance of MKP-1 expression in this disease. Experimental Design: Doxorubicin effects on MKP-1, phospho-ERK1/2 (p-ERK1/2), phospho-JNK (p-JNK), and phospho-p38 were assayed in a panel of human breast cancer cells by Western blot and in human breast cancer were assayed ex vivo by immunohistochemistry (n = 50). MKP-1 expression was also assayed in a range of normal to malignant breast lesions (n = 30) and in a series of patients (n = 96) with breast cancer and clinical follow-up. Results: MKP-1 was expressed at low levels in normal breast and in usual ductal hyperplasia and at high levels in in situ carcinoma. MKP-1 was overexpressed in ∼50% of infiltrating breast carcinomas. Similar to what was observed in breast cancer cell lines, ex vivo exposure of breast tumors to doxorubicin down-regulated MKP-1, and upregulated p-ERK1/2 and p-JNK, in the majority of cases. However, in a proportion of tumors overexpressing MKP-1, doxorubicin did not significantly affect MKP-1 or phospho-MAPKs. With regard to patient outcome, MKP-1 overexpression was an adverse prognostic factor for relapse both by univariate (P < 0.001) and multivariate analysis (P = 0.002). Conclusions: MKP-1 is overexpressed during the malignant transformation of the breast and independently predicts poor prognosis. Furthermore, MKP-1 is repressed by doxorubicin in many human breast cancers. There is a need to find novel targets to improve the therapeutic options for breast cancer patients (1, 2). A recently proposed target is the mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1; refs. 3, 4). MAPKs, the substrates of MKPs, play important roles in proliferation, stress responses, apoptosis, and immune response (3–7). There are three well-known MAPK subfamilies: extracellular signal-regulated kinases (ERK), c-Jun NH2-terminal kinases (JNK), and p38 MAPK isoforms. MAPKs are activated through a cascade of sequential phosphorylation events. The phosphorylation of MAPKs on threonine and tyrosine residues by specific upstream MAPK kinases (MEKs or MKKs) leads to their activated state. Authors' Affiliations: Cancer Research Program, IMIM-Hospital del Mar; Pathology Service, Hospital del Mar; Breast Disease Pathology Unit, Hospital del Mar; Medical Oncology Service, Hospital del Mar-IMAS, Barcelona, Spain; Pathology Service and Department of Immunology, Fundación Jiménez Díaz; Instituto de Investigaciones Biomédicas CSIC/ UAM, Translational Oncology Unit, Madrid, Spain; and Departament de Medicina, Universitat Autonoma de Barcelona, Bellaterra, Spain Received 8/1/08; revised 2/16/09; accepted 2/18/09; published OnlineFirst 5/5/09. Grant support: Spanish Health Ministry “Fondo de Investigación Sanitaria” grants PI051305 (R. Perona), PI052019 (B. Bellosillo, A. Rovira, C. Campas, J.M. Corominas, I. Tusquets, andC.Suárez), andPI061513 (J.Albanell, A. Rovira, andS.Serrano) and RedTemáticade InvestigaciónCooperativa enCáncer grant 06/0020/19 (I.GonzálezNavarrete, A. Dalmases, S. Menéndez, J.M. Corominas, I. Tusquets, A. Rovira, and J. Albanell). B. Bellosillo and J. Albanell have been receptors of “Ayuda a la intensificación de investigación” ISCiii. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Joan Albanell, Medical Oncology Service, Hospital del Mar, Passeig Maritim 25-29, 08003 Barcelona, Spain. Phone: 34932483137; Fax: 34-932483366; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2070 3530 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Research. on April 14, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from Conversely, MKPs, also known as dual-specificity phosphatases, dephosphorylate MAPKs on tyrosine and threonine residues (3, 5, 8). The prototypic member of the family, MKP-1 is an inducible nuclear phosphatase able to dephosphorylate ERK, JNK, and p38. MKP-1 is induced by many of the same stimuli that activate MAPKs, including growth factors and stress (5). Furthermore, MAPKs can increase MKP-1 protein activity in two distinct ways: firstly, MKP binding to its MAPK target causes a subtype-specific enhancement of its catalytic activity (9), and secondly, by phosphorylation by ERK, which inhibits MKP-1 degradation through the ubiquitin pathway (10). This mechanism is viewed as a feedback control to attenuate MAPK signaling (4, 5, 11, 12). MKP-1 seems to play an important role in tumorigenesis (4, 13, 14) and counterbalances the cytotoxicity of various anticancer drugs (4, 14–21). In this regard, anthracyclines, alkylating agents, taxanes, cisplatin, or proteasome inhibitors induce apoptosis in part by activation of the JNK pathway (15, 16). Notably, high levels of MKP-1 may dephosphorylate JNK and therefore limit the cytotoxicity of these agents (14, 16, 17, 19–24). Conversely, down-modulation of MKP-1 might be proapoptotic by facilitating a persistent JNK phosphorylation (4, 17). Several reports suggest MKP-1 as a potential target in breast cancer. In breast cancer cells, MKP-1 was a significant mediator of chemoresistance to anthracyclines, alkylating agents, and taxanes (15, 18, 19). Proteasome inhibitors induce MKP-1 and this induction played an antiapoptotic role (16, 20–23). Dexamethasone also induced MKP-1 and limited paclitaxel cytotoxicity (18, 19). In contrast, anthracyclines decrease expression of MKP-1 (15, 24). This repression is involved in the potentiation of the cytotoxicity of alkylating agents by anthracyclines (15). Aplidin also down-modulates MKP-1 (25). Complementing preclinical data, MKP-1 is overexpressed in human breast cancers (26, 27). These observations justify an interest in MKP-1 inhibitors for cancer therapy (3, 4, 19, 28, 29). Here, we aimed to further characterize MKP-1 and phospho-MAPK regulation by doxorubicin in breast cancer cells and in an ex vivo model (30, 31). We also analyzed the expression of MKP-1 in breast malignant transformation and in breast cancer patients with clinical follow-up. Materials and Methods Reagents, antibodies, and cells. Doxorubicin and Ro-31-8220 (both from Calbiochem; ref. 32) were purchased for use. Doxorubicin was freshly dissolved in water and Ro-31-8220 was dissolved in DMSO at stock concentrations of 10 mmol/L. Elite avidin-biotin complex method kit was from Vector Laboratories. Enhanced chemiluminescence detection kit was from Amersham Pharmacia Biotech. All tissue culture materials were from Life Technologies. The following antibodies were used: anti–phospho-ERK1/2 (p-ERK1/2; Thr/Tyr), anti-ERK1/2, anti-JNK, anti-p38 MAPK, and anti–phospho-p38 (p-p38) MAPK (all from Cell Signaling Technology). Anti–phospho-JNK (p-JNK; Thr/ Tyr) from Promega and Cell Signaling Technology was used for immunohistochemical and Western blot analysis, respectively. To detect MKP-1, two antibodies were used: one for Western blot and one for immunohistochemical assays. Antibodies to detect expression of estrogen receptor, progesterone receptor, and HER2 (HercepTest) were purchased from Dako. HER2 amplification was assayed by HER2 fluorescence in situ hybridization pharmDx (Dako). The human breast cancer cell lines BT-474, SK-BR3, MDA-MB-468, MDA-MB-453, MDAMB-231, and MCF-7 (from the American Type Culture Collection) were maintained as previously reported (9). Real-time quantitative reverse transcription-PCR. Specific PCR primer combinations and fluorogenic probes (5′-FAM, 3′-TAMRA) for the target MKP-1 mRNA and housekeeping RNA (endogenous control, 18S, β-actin, and RPLPO) were purchased from Applied Biosystems. Total RNAwas isolated using RNeasy kit (Qiagen) and reverse transcribed to cDNA using SuperScript reverse transcriptase (Invitrogen). cDNAs were combined with primers and probes specific for each gene of interest along with predeveloped Taqman Gene Expression Master Mix (Applied Biosystems) for the following genes: MKP-1 (Hs-00610256-g1), human β-actin (Hs99999903_m1), human 18S (Hs99999901_s1), and human RPLPO (large ribosomal protein PO; Hs99999902_m1). The PCR protocol was 50°C for 2 min and 95°C for 10 min followed by 50 cycles at 95°C for 15 s and at 60°C for 1 min. Negative controls were included and yielded no products. Real-time PCR analysis was carried out on an ABI7500HT. Ct values were determined using SDS v2.2 software (Applied Biosystems) and compared using the Ct method. Western blot analysis. Western blot analysis in cultured cells was done following as previously reported (20). Briefly, cells were washed in PBS and scraped, and whole-cell lysates were prepared. Frozen breast tumor samples were analyzed by Western blot, also as described previ-