Abstract
Abstract We have conducted a comprehensive proteomic analysis of an ovarian cancer cell line (A2780) and its cisplatin resistant daughter line (A2780-CP20) to identify key regulators and signaling pathways that confer cisplatin resistance. Quantitative proteomic profiling was conducted of defined biological compartments including that from global lysates, the nuclear fraction, the phosphoproteome and the secretome. Total cell lysates and nuclear fractions were harvested from A2780 and A2780-CP20 cultured in the presence or absence of cisplatin (3 µM) for 72 h. The secretome fractions were collected from A2780 and A2780-CP20 cultured in serum-free, phenol red-free media in the presence or absence of cisplatin (3 µM) for 24 h. Phosphopeptides were enriched from peptide digests of total lysates of A2780 and A2780-CP20 cultured in the presence or absence of cisplatin (3 µM) for 15 min using TiO2. Sample digests were analyzed by high-resolution LC-MS/MS that resulted in the identification of 2703, 2286, 1815, and 1362 proteins from the global, nuclear, secretome, and phosphoproteome fractions, respectively. Spectral counting was utilized to quantify the relative abundance of proteins identified. Nuclear proteins identified at elevated abundance in the cisplatin resistant A2780-CP20 including an array of chromatin remodeling proteins, such as SATB1, SATB2, coilin, AT-rich interactive domain-containing protein 3A (ARID3A), ARID3B, and ARID3Cwere validated by Western blot and qPCR in A2780/A2780-CP20 as well as other gynecologic cancer cell lines. Modulation of the abundance level of these candidates and the phenotypic impact on cisplatin resistance utilizing shRNA knockdown will be described. Differential proteins selected from the secretome analysis for western blot validation include Fras1-related extracellular matrix protein 2 (FREM2), stanniocalcin-1 (STC1), angio-associated migratory cell protein (AAMP), plasminogen activator inhibitor 1 (SERPINE1), high mobility group protein B1 (HMGB1), and chloride intracellular channel protein 2 (CLIC4), stromelysin-2 (MMP10), interstitial collagenase (MMP2), and CD166 antigen. Validated candidate protein targets from the secretome analysis will be tested by IHC on formalin fixed tissue sections as well as ELISA and multiple reaction monitoring (MRM) by mass spectrometry in serum from recurrent and non-recurrent ovarian cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 799. doi:1538-7445.AM2012-799
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