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A Novel C-Met Inhibitor, MK8033, Synergizes with Carboplatin Plus Paclitaxel to Inhibit Ovarian Cancer Cell Growth

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A Novel C-Met Inhibitor, MK8033, Synergizes with Carboplatin Plus Paclitaxel to Inhibit Ovarian Cancer Cell Growth ONCOLOGY REPORTS 29: 2011-2018, 2013 A novel c-Met inhibitor, MK8033, synergizes with carboplatin plus paclitaxel to inhibit ovarian cancer cell growth DOUGLAS C. MARCHION1,2, ELONA BICAKU1,2, YIN XIONG1,2, NADIM BOU ZGHEIB1, ENTIDHAR AL SAWAH1,2, XIAOMANG BA STICKLES1, PATRICIA L. JUDSON1,2,4, ALEX S. LOPEZ3,4, CHRISTOPHER L. CUBITT5, JESUS GONZALEZ-BOSQUET1,4, ROBERT M. WENHAM1,2,4, SACHIN M. APTE1,4, ANDERS BERGLUND6 and JOHNATHAN M. LANCASTER1,2,4 1Department of Women's Oncology, 2Experimental Therapeutics Program, 3Department of Anatomic Pathology, 4Department of Oncologic Sciences, 5Translational Research Core, 6Cancer Informatics Core, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA Received December 10, 2012; Accepted January 29, 2013 DOI: 10.3892/or.2013.2329 Abstract. Elevated serum levels of hepatocyte growth factor 47-gene signature to be associated with MK8033-carboplatin (HGF) and high tumor expression of c-Met are both indica- plus paclitaxel response. PCA modeling indicated an associa- tors of poor overall survival from ovarian cancer (OVCA). tion of this 47-gene response signature with overall survival In the present study, we evaluated the role of the HGF from OVCA (P=0.013). These data indicate that HGF/c-Met signaling pathway in OVCA cell line chemoresistance and pathway signaling may influence OVCA chemosensitivity and OVCA patient overall survival as well as the influence of overall patient survival. Furthermore, HGF/c-Met inhibition HGF/c-Met signaling inhibition on the sensitivity of OVCA by MK8033 represents a promising new therapeutic avenue to cells to combinational carboplatin plus paclitaxel therapy. The increase OVCA sensitivity to carboplatin plus paclitaxel. prevalence of the HGF receptor, c-Met, was determined by immunohistochemistry in primary OVCA samples (n=79) and Introduction OVCA cell lines (n=41). The influence of the c-Met-specific inhibitor MK8033 on OVCA cell sensitivity to combinations Ovarian cancer (OVCA) is the fifth leading cause of cancer- of carboplatin plus paclitaxel was examined in a subset of related mortality among women in the United States and OVCA cells (n=8) by CellTiter-Blue cell viability assays. Western Europe, with the highest mortality rate of all Correlation tests were used to identify genes associated with gynecologic malignancies. Approximately 75% of patients response to MK8033 and carboplatin plus paclitaxel. Identified with OVCA are diagnosed at an advanced stage (III/IV) genes were evaluated for influence on overall survival with disseminated intraperitoneal metastases (1). Although from OVCA using principal component analysis (PCA) the majority of patients initially experience a complete modeling in an independent clinical OVCA dataset (n=218). clinical response to primary surgery followed by platinum/ Immunohistochemistry analysis indicated that 83% of OVCA taxane-based chemotherapy, most patients eventually develop cells and 92% of primary OVCA expressed the HGF receptor, platinum/taxane-resistant persistent or recurrent disease. c-Met. MK8033 exhibited significant anti-proliferative effects These patients have a poor prognosis. Despite cytoreductive against a panel of human OVCA cell lines. Combination index surgery and aggressive chemotherapy, the majority of patients values determined by the Chou-Talalay isobologram equation with OVCA succumb to the disease within 5 years (2). These indicated synergistic activity in combinations of MK8033 and dismal statistics highlight the need for research into the carboplatin plus paclitaxel. Pearson's correlation identified a cellular basis of platinum response, as well as the design of targeted therapeutic strategies. The hepatocyte growth factor (HGF) signaling pathway may be a viable target for the development of directed thera- peutic regimens for the treatment of OVCA. HGF is the only Correspondence to: Dr Johnathan M. Lancaster, Department of known ligand of the HGF receptor (c-Met), which is expressed Women's Oncology, H. Lee Moffitt Cancer Center and Research in approximately 70% of OVCAs (3,4). Overexpression of HGF Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA and/or c-Met has been associated with poor clinical outcome E-mail: [email protected] in OVCA (5-7), whereas c-Met overexpression has further Key words: c-Met expression, combination index, ovarian cancer, been associated with a chemo-resistant subset of ovarian clear- carboplatin, immunohistochemistry cell adenocarcinomas (8,9). The targeted inhibition of c-Met has been shown to reduce c-Met expression, block OVCA cell proliferation, and reduce tumor burden in pre-clinical mouse models (10-12). 2012 MARCHION et al: c-MET EXPRESSION IN OVCA In this study, we analyzed the expression of the HGF tions (Qiagen, Valencia, CA, USA). Quality of the RNA was receptor, c-Met, in primary OVCA and OVCA cell lines, measured using an Agilent 2100 Bioanalyzer. The targets and tested the activity of the HGF/c-Met signaling inhibitor, for Affymetrix DNA microarray analysis were prepared MK8033, alone and in combination with standard of care according to the manufacturer's instructions. For OVCA cell therapy (carboplatin plus paclitaxel) in a panel of OVCA lines (n=41), targets were hybridized to customized Human cells. We then identified the genes associated with sensitivity Affymetrix HuRSTA gene chips (HuRSTA-2a520709) [GEO to MK8033 plus carboplatin-paclitaxel and used the prin- accession number GSE34615 (14)]. cipal component analysis (PCA) to define a gene expression signature of OVCA cell response. Finally, this PCA response Statistical analyses. Expression data were subjected to signature was tested in an independent survival dataset of background correction and normalization using the Robust primary OVCA for which response to chemotherapy and Multichip Average algorithm in the Affymetrix Expression overall survival were known. Console (http://www.affymetrix.com/products/software/ specific/expression_console_software.affx). Pearson's corre- Materials and methods lation test was performed on individual gene expression and IC50 values. Probe sets with P<0.001 were considered to have Cell culture. OVCA cell lines were obtained from the significant correlations with IC50 values. American Type Culture Collection (Manassas, VA, USA) (CAOV3, OV90, OVCAR3 and SKOV3); from the European Cell viability assays. For cells cultured in the presence Collection of Cell Cultures (Salisbury, England) (A2780CP of cisplatin, resistance was quantified using MTT prolif- and A2780S); from Kyoto University (Japan) (CHI, CHIcisR, eration assay, in accordance with manufacturer's instructions M41, M41CSR, Tyknu and TyknuCisR); or were kind gifts (Promega, Madison, WI, USA). Cells (5,000/well) were seeded from Dr Patricia Kruk, Department of Pathology, College in 96-well plates, allowed to adhere, and incubated with of Medicine, University of South Florida, Tampa, FL, and increasing doses of cisplatin for 48 h at 37˚C. Following drug Professor Susan Murphy, Department of OBGYN/Division of incubation, plates were read at 570 nm using a SpectraMax 190 Gynecologic Oncology, Duke University, Durham, NC, USA microplate reader (Molecular Devices, USA). For drug combi- (A2008, C13, CAOV2, HeyA8, IGR-OV1, IMCC3, IMCC5, nation experiments, CellTiter-Blue cell viability assays were MCAS, OV2008, OVCA420, OVCA429, OVCA432, OVCA433, performed using 384-well plates and an automated pipetting FUOV1, PEO1, PEO4, SK-OV-6, T8, TOV-112D, TOV-21-G, station. Briefly, for these assays, 1.2x103 cells (24-µl volume) Dov13, BG1, Ovary1847, OVCAR10, OVCAR8, OVCAR5, were plated in each well and allowed to adhere overnight at OVCAR4, OVCAR2 and SK-OV-4). Cell lines were maintained 37˚C and 5% CO2. Drug dilutions were then added using the in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) supplemented same pipetting station, and the plate was incubated for 72 h. with 10% fetal bovine serum (Fisher Scientific, Pittsburg, Following drug incubation, fluorescence was measured using PA, USA), 1% sodium pyruvate, 1% penicillin/streptomycin a Synergy 4 microplate reader (Bio-Tek Instruments, Inc.). The (Cellgro, Manassas, VA, USA), and 1% nonessential amino fluorescence data were transferred to a spreadsheet program acids (HyClone, Hudson, NH, USA). Mycoplasma testing was to calculate the percent viability relative to untreated cells. performed every 6 months in accordance with the manufac- Experiments were repeated a minimum of three times. turer's protocol (Lonza, Rockland, ME, USA). Synergism analyses. Drug combination experiments were Staining and scoring of c-Met expression in OVCA. Slides were analyzed for synergistic, additive, or antagonistic effects using stained using a Ventana Discovery XT automated system as per the combination index method developed by Chou and Talalay. the manufacturer's protocol with proprietary reagents (Ventana For the application of this method, the drug concentration Medical Systems, Tucson, AZ, USA). Briefly, slides were dilutions of the drugs were used at fixed dose ratios based on deparaffinized on the automated system with EZ Prep solu- the IC50 values of each drug obtained from preliminary experi- tion (Ventana). The heat-induced antigen retrieval method was ments (e.g., 50:1, 2:5, 1:250). Briefly, the dose-effect curve for used in Cell Conditioning 1 (Ventana). The mouse monoclonal each drug alone is determined based on experimental obser- antibody that reacts to c-Met (#18-7366; Invitrogen) was used vations using the median-effect
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