Abstract 2509: Mechanism of anti-proliferative effects of SIRT1 inhibition in melanoma cells: a proteomics approach.

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Abstract An analysis of the SEER (Surveillance, Epidemiology, and End Results) program data has suggested that the age-adjusted annual incidence of melanoma in both young men and women has increased over the past 20 years. The current therapeutic options have shown little success in melanoma management. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. The mammalian SIRT1 or sirtuin (silent mating type information regulation 2 homolog) 1 is a NAD(+)-dependent class III histone deacetylase (HDAC) that has been shown to play important roles in a variety of biological processes and diseases including cancer. Previous studies from our laboratory have demonstrated that 1) SIRT1 is overexpressed in human melanoma cells and tissues; 2) SIRT1 inhibition by Tenovin-1 resulted in decreased growth, viability, and clonogenic survival of melanoma cells; 3) Tenovin-1 treatment resulted in decreased level of SIRT1 protein and increased level of the tumor suppressor p53 (In: AACR Annual Meeting: Proceedings; 2012 March 31-April 4, Chicago, IL; Abstract # 4722). In this study we employed a proteomics approach using gel free Liquid Chromatography-Mass Spectrometry (LC-MS) to identify the downstream molecular targets of SIRT1 in melanoma. The human melanoma G361 cells were treated with Tenovin-1 (25 μM; 48 h) followed by protein extraction. Extracted proteins were precipitated (using 10% TCA and 0.05% DTT), pelleted and washed with acetone and air dried. Samples were re-suspended, labelled, trypsin digested and subjected to LC-MS/MS on a linear trap quadrupole (LTQ) Orbitrap XL. The resultant peptide sequence data were matched against the uniprot_human_FR_CP_090710 database using Mascot (Matrix Sciences, London, UK). Scaffold (version Scaffold_3.6.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Protein identifications were accepted if they could be established at greater than 95% probability and contained at least two identified peptides. The differentially expressed protein data were collected from three different biological replicates. We found 14 proteins (from a total of 1092) which showed +2 fold differential expressions with statistical significance (T-test, p value <0.05). Among these proteins, three proteins were found to be newly expressed whereas one was found to completely disappear following SIRT1 inhibition, under the experimental conditions employed. The differentially expressed proteins identified in our study are associated with RNA processing-, mitochondrial checkpoint- and apoptosis- pathways. Currently, we are in process of validating and ascertaining the functional relevance of these downstream targets. Our provides an improved understanding of SIRT1 downstream targets, which may lead to better strategies for melanoma management. Citation Format: Chandra K. Singh, Jasmine George, Raj Kumar, Nihal Ahmad. Mechanism of anti-proliferative effects of SIRT1 inhibition in melanoma cells: a proteomics approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2509. doi:10.1158/1538-7445.AM2013-2509