Abstract 548: The effects of Sirtuin 6 inhibition on autophagy-related pathways in melanoma

Melanoma is an aggressive skin cancer that can rapidly metastasize to become fatal, if not diagnosed early. Despite therapeutic advances, clinical management of melanoma remains challenging. The existing chemotherapeutic drugs either fail to achieve greater than 25% response or ultimately develop resistance to therapy. Therefore, novel molecular targets and treatments are required for an effective management of melanoma. In order to design novel and more effective approaches for lasting cure of melanoma, a deeper understanding of the mechanisms involved in melanomagenesis are needed. In our laboratory, we are assessing the role and functional and therapeutic significance of sirtuin proteins in melanoma. We have previously demonstrated that Sirtuin 6 (SIRT6) is significantly overexpressed in human melanoma cells and clinical tissues, and its genetic knockdown resulted in a marked anti-proliferative response in human melanoma cells (Genes & Cancer; In Press). A limited number of studies have implicated SIRT6 in autophagy regulation. Autophagy forms part of a basic cellular process that copes with cellular stress and is considered to be an important metabolic mechanistic essential for degradation and recycling of unnecessary intracellular components. In melanoma autophagy is believed to be a tumor suppressing process in early stages of cancer, but tumor-promoting in established tumors. This study was designed to determine the connection between SIRT6 and autophagy in melanoma. Employing lentiviral short hairpin RNA-mediated knockdown of SIRT6 in A375 melanoma cells, we determined the modulation in autophagy related pathways. We employed a PCR array containing 84 genes that are involved in the regulation of autophagy. Our data demonstrated that 17 of the 84 genes were significantly modulated (two-fold or more) upon SIRT6 knockdown. Of these 17 genes, 2 were upregulated (ATG10 and GAA) and 15 were downregulated (AKT1, ATG12, ATG3, ATG7, BAK1, BCL2L1, CLN3, CTSB, CTSS, DRAM2, HSP90AA1, IRGM, NPC1, SQSTM1, and TNF). Modulated genes were analyzed using Ingenuity Pathway Analysis (IPA) for predicted gene-gene interactions and functional networks. Many of the network genes were found to have definitive links to cancer. These genes were found to be associated with cell transformation and tumor invasion. Further validation at mRNA and protein levels, confirmed the significant modulations in autophagy markers BECN1, SQSTM1, ATG3, ATG7, ATG10 and GAA, upon SIRT6 knockdown. Moreover, SIRT6 knockdown was found to cause a marked decrease in the LC3 II protein level, which is a central protein in the autophagy pathway. Taken together, our data suggest that SIRT6 knockdown significantly alters pathways related to autophagy in melanoma cells. Future studies are required to carefully study the role of SIRT6 and autophagy in early versus late melanomas. Citation Format: Liz M. Garcia-Peterson, Mary A. Ndiaye, Chandra K. Singh, Gagan Chhabra, Nihal Ahmad. The effects of Sirtuin 6 inhibition on autophagy-related pathways in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 548.