Comparative Proteomic Analysis of Paclitaxel Sensitive A2780

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Comparative Proteomic Analysis of Paclitaxel Sensitive A2780 Epithelial Ovarian Cancer Cell Line and Its Resistant Counterpart A2780TC1 by 2D-DIGE: The Role of ERp57 Lucia Cicchillitti,†,# Michela Di Michele,*,‡,# Andrea Urbani,§,⊥ Cristiano Ferlini,† Maria Benedetta Donati,| Giovanni Scambia,† and Domenico Rotilio‡ Department of Oncology, “RE ARTU” Laboratory of Analytical Techniques and Proteomics, and “RE ARTU” Research Laboratories, “John Paul II” Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy, and Department of Internal Medicine, University of Rome Tor Vergata, and Laboratory of Proteomics and Metabonomics, EBRI/S, Lucia Foundation, Rome, Italy Received October 13, 2008

Epithelial ovarian cancer is the leading cause of gynecological cancer mortality. Despite good response to surgery and initial chemotherapy, chemoresistance occurrence represents a major obstacle to a successful therapy. To better understand biological mechanisms at the basis of paclitaxel resistance, a comparative proteomic approach based on DIGE coupled with mass spectrometry (MALDI-TOF and LC-MS/MS) was applied to the human epithelial ovarian cancer cell lines A2780 and its paclitaxel resistant counterpart A2780TC1. Most of the differentially expressed proteins between the two cell lines belong to the class of stress response (29%), metabolism (21%), and cell cycle and apoptosis (17%). We focused on proteins which were most strongly modulated by paclitaxel resistance and in particular on the disulphide isomerase ERp57, which may represent a chemoresistance biomarker. ERp57 was found to interact with class III β-tubulin (TUBB3), involved in paclitaxel resistance in ovarian and other cancers. Moreover, we demonstrated a novel localization of this protein in cytoskeleton and described that ERp57/TUBB3 interaction occurs also in the nuclear compartment and in association with a multimeric complex formed by nucleolin, nucleophosmin, hnRNPK, and mortalin. Our data suggest that ERp57 plays an important role in chemoresistance mechanisms in ovarian cancer by modulating the attachment of microtubules to chromosomes following paclitaxel treatment through its interaction with TUBB3. Keywords: biomarker • 2D-DIGE • chemoresistance • ERp57 • mass spectrometry • ovarian cancer • paclitaxel • proteomics

Introduction Epithelial ovarian cancer is the sixth most common cause of death from cancer and the first from gynecological cancer in the Western countries.1 It is characterized by few and nonspecific early symptoms and a typical presentation only at an advanced stage, thus, explaining the poor survival statistics.2 Currently, debulking surgery followed by taxanes-based chemotherapy is an effective strategy in the treatment of advanced ovarian carcinoma. Among taxanes, paclitaxel is a potent cytotoxic diterpene effective against ovarian carcinoma * Corresponding author: Michela Di Michele, “RE ARTU” Laboratory of Analytical Techniques and Proteomics, “John Paul II” Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Largo A. Gemelli, 1, 86100 Campobasso, Italy. E-mail, michela.dimichele@ moli-sani.org; tel, 0039 0874 312285; fax, 0039 0874 312710. † Department of Oncology, Catholic University. ‡ “RE ARTU” Laboratory of Analytical Techniques and Proteomics, Catholic University. # These authors contributed equally to this work. § Department of Internal Medicine, University of Rome Tor Vergata. ⊥ Laboratory of Proteomics and Metabonomics, EBRI/S, Lucia Foundation. | “RE ARTU” Research Laboratories, Catholic University.

1902 Journal of Proteome Research 2009, 8, 1902–1912 Published on Web 02/09/2009

and a wide range of solid tumors, as single agent or in combination with other chemotherapeutic drugs.3 It binds to β-tubulin subunits and inhibits microtubule dynamics, thereby blocking cell cycle progression during mitosis and activating cell apoptosis.4,5 Despite the relevant contribution of paclitaxel in ameliorating the quality of life and overall survival of cancer patients, the occurrence of intrinsic or acquired tumor chemoresistance remains the major determinant of chemotherapy failure and unfavorable clinical outcome.6 At present, resistance can only be determined retrospectively after patients have experienced the burden and toxicity of ineffective therapy. Outcomes for women with ovarian cancer could be improved by a better understanding of mechanisms at the basis of chemoresistance and the identification of biomarkers capable of identifying resistant tumors and patients. So far, a variety of mechanisms have been proposed to explain paclitaxel chemoresistance, such as the overexpression of P-glycoprotein or the selective expression of class III β-tubulin isotypes (TUBB3),7,8 but studies on the overall protein expression have never been reported. 10.1021/pr800856b CCC: $40.75

 2009 American Chemical Society

Comparative Proteomic Analysis of Paclitaxel Sensitive A2780 Recently, ERp57 gene was found to be associated with drug resistance in a study based on an array comparative genomic hybridization (aCGH) and microarray expression profiling in epithelial ovarian cancer tissues.9 ERp57 belongs to the family of protein disulfide isomerases which act as chaperones and are involved in the proper folding and in the formation and reshuffling of the disulfide bridges of newly synthesized proteins in the endoplasmic reticulum (ER).10 Though mainly localized in the ER, ERp57 was also found in the cytosol11 and in the nucleus.12 In the cytosol, it is involved in signal transduction and transcription regulation by activation of STAT313 and is associated with a sodium chloride co-transporter,14 while in the nucleus, it plays an important role in the cytotoxic response to DNA modified by incorporation of anticancer nucleoside analogues.15 ERp57 expression is induced by a variety of stress conditions, such as glucose deprivation or hypoxia, and in particular physiological situations, such as neoplastic transformation. Moreover, in our previous work,5 several chaperones and proteins that catalyze protein folding were found to interact with TUBB3, which has an important role as a clinical marker of drug resistance and poor prognosis in ovarian, lung, gastric, and breast cancer.7 Proteomics represents a powerful tool to perform highthroughput studies at protein level, allowing the detection of the appearance of new proteins resulting from gene expression changes, differences in the amount of expressed proteins, and changes in PTM. Among proteomic techniques, differential ingel electrophoresis (DIGE) circumvents many of the issues associated with traditional 2DE, such as reproducibility and limited dynamic range, and allows more accurate and sensitive quantitative proteomics studies.16,17 This innovative technology18 relies on pre-electrophoretic labeling of samples with one of three CyDye fluors (Cy2, Cy3, Cy5), each with a unique fluorescent wavelength, allowing for two experimental samples and an internal standard to be simultaneously separated on the same gel. The internal standard is a pool of an equal amount of all the experimental samples and facilitates accurate data normalization among gels, increasing statistical confidence in quantitative comparative analysis. In this study, a human epithelial ovarian cancer cell line, A2780, and its paclitaxel resistant counterpart, A2780TC1, were analyzed by DIGE coupled with MS to screen differentially expressed proteins. Proteins modulated in paclitaxelresistant and sensitive ovarian cancer cells are likely to be involved in pathways that regulate the sensitivity of ovarian cancer to paclitaxel and are plausible candidates for treatment response biomarkers and therapeutic targets. In particular, we hypothesized a major role of ERp57 in the mechanisms of paclitaxel resistance in ovarian cancer, opening new possibilities for future steps to design more personalized chemotherapeutic treatment strategies and to develop novel drugs to avoid resistance.

Materials and Methods Chemicals and Reagents. General chemicals were obtained from Sigma-Aldrich (Poole, U.K.), while reagents for 2D-DIGE experiments were purchased from Amersham Biosciences (Uppsala, Sweden), unless otherwise indicated. Bradford assay kit was from Bio-Rad (Hercules, CA). Anti-TUBB3 (PRB-435P) polyclonal antibody was purchased from Covance (Berkeley, CA); anti-tubulin (H-235) and anti-ERp57 (sc-23886) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); anti-

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R-tubulin (SP06) was purchased from Calbiochem (San Diego, CA). Chemicals and biochemicals used were of analytical grade. Cell Lines and Paclitaxel Citotoxicity. The human ovarian cancer cell line A2780 was purchased from the European Collection of Cell Cultures (Salisbury, United Kingdom). A2780TC1 was previously produced by our group19 and is a cell clone derived from A2780 cells chronically exposed to paclitaxel (100 nmol/L). Growth conditions and paclitaxel toxicity testing were the same as previously described.19 Immunoblotting of the Subcellular Fractions. Cells, harvested in cold phosphate-buffered saline, were extracted for 30 min at 4 °C in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 1% NP-40) containing 1 mM DTT, protease inhibitor cocktail (Roche), and phosphatase inhibitors (50 mM NaF, 0.2 mM Na3VO4) or treated with 25 mM N-ethylmaleimide (NEM) for 5 min before harvesting to preserve mixed disulphides. After centrifugation, supernatants (300 µg from total lysates) were immunoprecipitated at 4 °C overnight in lysis buffer by adding protein A/G plus-agarose beads (Santa Cruz Biotechnology) after 2 h of incubation with 2 µg of the antiERp57 monoclonal antibody. Immunoblots were developed using Supersignal West Femto chemiluminescent reagents (Pierce Biotechnology, Rockford, IL). For subcellular fractionation, cells were scraped, rinsed with phosphate-buffered saline, and lysed with cytoplasm lysis buffer (10 mM HEPES, pH 8.0, 40 mM KCl, 3 mM MgCl2, 5% glycerol, 2 mM DTT, and 0.5% NP-40) supplemented with a protease inhibitor cocktail (Roche), on ice for 10 min. The cytosolic fraction was prepared by performing high-speed centrifugation (6000 rpm for 5 min at 4 °C); then, the supernatant was stored at -80 °C. The nuclei fraction was obtained by suspending the pellet with nuclei lysis buffer (10 mM HEPES, pH 8.0, 420 mM NaCl, 1.5 mM MgCl2, 25% glycerol, and 0.5 mM DTT) on ice for 10 min. After centrifugation (6000 rpm for 5 min at 4 °C), the supernatant was collected as nuclear fraction and stored at -80 °C. Microtubules proteins were isolated was performed as previously described.5 For two-dimensional nonreduced/reduced SDS-polyacrylamide gel, proteins were mixed in nonreducing SDS-PAGE sample buffer (0.25 mM Tris-HCl, pH 6.8, 2% (w/v) SDS, 20% (v/v) glycerol, 0.004% (w/v) bromophenol blue) and separated by SDS-PAGE on a 10% acrylamide gel. Gels were silver stained and gel lanes were excised and incubated in buffer containing 50 mM DTT for 10 min and further 10 min in 100 mM iodocaetamide before separation through a second SDS-PAGE gel (10%). 2D-DIGE. Four independent extractions were carried out from A2780 and A2780TC1 cell lines. The pellet of 107 cells was solubilized in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris, pH 8.5) containing antiprotease cocktail (Sigma) and incubated for 1 h on ice. The cells were lysed by sonication (3 × 5 s pulses) and centrifuged at 20 000g for 15 min. The supernatant was saved as the cell lysate and brought to pH of 8.5 to optimize fluorescent tagging. The protein concentration was determined in triplicate by the Bio-Rad Dc protein assay as described by the manufacturers (Bio-Rad), with bovine serum albumin as the standard. Half of the samples from each cell line were labeled with Cy3 and half were labeled with Cy5 to minimize potential dye artifacts. Additionally, all gels contained a third sample which was a pool of all the samples in the experiment to be used as internal standard for quantitative comparisons, made up of Journal of Proteome Research • Vol. 8, No. 4, 2009 1903

research articles equal amounts of proteins from each of the individual sample and labeled with Cy2 fluorescent dye. A total of 50 µg of proteins from internal standard and paclitaxel sensitive or resistant cells was labeled as indicated by the manufacturers. The first dimension was carried out on an IPGphor system (Amersham) using pH 3-10 gel strips of 13 cm. Strips were actively rehydrated at 30 V for 16 h in 260 µL of sample. The IEF was performed at 20 °C under the following conditions: 2 h at 300 V, 3 h at 1000 V, 2 h at 8000 V in gradient, 3 h at 8000 V. IPG strips were incubated for 15 min in equilibration buffer I (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 6.5 mM DTT) and in equilibration buffer II for an additional 15 min (Buffer II was identical to buffer I with 2% iodoacetamide instead of DTT). The second-dimensional separations were carried out at 2 mA/gel for 1 h and at 10 mA/gel until the bromophenol blue dye front reached the end of the gels. Gel Image Analysis. Labeled proteins were visualized using the Typhoon Trio imager (Amersham Biosciences). The Cy2, Cy3, and Cy5 components of each gel were individually imaged using mutually exclusive excitation/emission wavelengths of 488/520 nm for Cy2, 532/580 nm for Cy3, and 633/670 nm for Cy5. Gel analysis was performed using Decyder 2-D Differential Analysis Software v6.5 (GE Healthcare, Amersham Bio-sciences) to co-detect and quantify the protein spots in the images. A protein abundance ratio >1 corresponds to an increase in escape compared with control samples, whereas a ratio