Melanoma is the most notorious and fatal of all skin cancers and the existing treatment options have not been proven to effectively manage this neoplasm, especially the metastatic disease. Sirtuin (SIRT) proteins have been shown to be differentially expressed in melanoma. We have shown that SIRTs 1 and 2 were overexpressed in melanoma and inhibition of SIRT1 imparts anti-proliferative responses in human melanoma cells. To elucidate the impact of SIRT 1 and/or 2 in melanoma, we created stable knockdowns of SIRTs 1, 2, and their combination using shRNA mediated RNA interference in A375 human melanoma cells. We found that SIRT1 and SIRT1&2 combination knockdown caused a decreased cellular proliferation in melanoma cells. Further, the knockdown of SIRT 1 and/or 2 resulted in a decreased colony formation in melanoma cells. To explore the downstream targets of SIRTs 1 and/or 2, we employed a label-free quantitative nano-LC-MS/MS proteomics analysis using the stable lines. We found aberrant levels of proteins involved in many vital cellular processes, including cytoskeletal organization, ribosomal activity, oxidative stress response, and angiogenesis. These findings provide clear evidence of cellular systems undergoing alterations in response to sirtuin inhibition, and have unveiled several excellent candidates for future study. Significance Melanoma is the deadliest form of skin cancer, due to its aggressive nature, metastatic potential, and a lack of sufficient treatment options for advanced disease. Therefore, detailed investigations into the molecular mechanisms of melanoma growth and progression are needed. In the search for candidate genes to serve as therapeutic targets, the sirtuins show promise as they have been found to be upregulated in melanoma and they regulate a large number of proteins involved in cellular processes known to affect tumor growth, such as DNA damage repair, cell cycle arrest, and apoptosis. In this study, we used a large-scale label-free comparative proteomics system to identify novel protein targets that are affected following knockdown of SIRT1 and/or 2 in A375 metastatic melanoma cell line. Our study offers important insight into the potential downstream targets of SIRTs 1 and/or 2. This may unravel new potential areas of exploration in melanoma research.
|Number of pages||11|
|Journal||Journal of Proteomics|
|State||Published - Jan 6 2018|
Bibliographical noteFunding Information:
This work was supported by funding from the NIH ( R01AR059130 and R01CA176748 to NA, and R01CA157429 , R01CA192894 , R01CA196835 , R01CA196634 to XL), and the Department of Veterans Affairs (VA Merit Review Awards I01BX001008 and I01CX001441 ; and a Research Career Scientist Award IK6BX003780 to NA). We also acknowledge support from the NIH in form of the High-End, Shared Instrumentation grant ( 1S10RR029531 ) to the Analytical Instrumentation Center of the School of Pharmacy, for mass spectrometric analyses. In addition, we acknowledge the core facilities supported by the Skin Diseases Research Center (SDRC) Core Grant P30AR066524 from NIH/NIAMS.
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