Novel downstream molecular targets of SIRT1 in melanoma: A quantitative proteomics approach

Abstract

Melanoma is one of the most lethal forms of skin cancer and its incidence is continuing to rise in the United States. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. SIRT1, a NAD(+)-dependent class III histone deacetylase, has been implicated in a variety of physiological processes and pathological conditions. We recently demonstrated that SIRT1 is upregulated in melanoma and its inhibition by a small-molecule, tenovin-1, inhibits cell proliferation and clonogenic survival of melanoma cells, possibly via activating p53. Here, we employed a gel free quantitative proteomics approach to identify the downstream effectors and targets of SIRT1 in melanoma. The human malignant melanoma, G361 cells were treated with tenovin-1 followed by protein extraction, in liquid trypsin digestion, and peptide analyses using nanoLC-MS/MS. A total of 1091 proteins were identified, of which 20 proteins showed significant differential expression with 95% confidence interval. These proteins were subjected to gene ontology and Ingenuity Pathway Analysis (IPA) to obtain the information regarding their biological and molecular functions. Real-Time qRT-PCR validation showed that five of these (PSAP, MYO1B, MOCOS, HIS1H4A and BUB3) were differentially expressed at mRNA levels. Based on their important role in cell cycle regulation, we selected to focus on BUB family proteins (BUB3, as well as BUB1 and BUBR1) for subsequent validation. The qRT-PCR and immunoblot analyses showed that tenovin-1 inhibition of SIRT1 resulted in a downregulation of BUB3, BUB1 and BUBR1 in multiple melanoma cell lines. Since tenovin-1 is an inhibitor of both SIRT1 and SIRT2, we employed lentivirus mediated silencing of SIRT1 and SIRT2 in G361 cells to determine if the observed effects on BUB family proteins are due to SIRT1- or SIRT2- inhibition. We found that only SIRT1 inhibition resulted in a decrease in BUB3, BUB1 and BUBR1. Our study identified the mitotic checkpoint regulator BUB family proteins as novel downstream targets of SIRT1. However, further validation is needed in appropriate models to confirm our findings and expand on our observations.

How myosin organization of the actin cytoskeleton contributes to the cancer phenotype

“…Of the class 1 myosins, MYO1B is of particular interest as it is most strongly implicated in metastasis. MYO1B (Myr1/MM1 ) [28] is expressed at high levels in metastatic prostate cancer tissue [5] and the metastatic prostate cancer cell line (PC-3), head and neck cancers [29] and melanomas [30]. High levels of MYO1B expression are likely to affect the organization of the actin cytoskeleton.…”

AC-93253 triggers the downregulation of melanoma progression markers and the inhibition of melanoma cell proliferation

“…Unfortunately, little is known regarding the possible roles of sirtuins in melanomas and their development. Thus far, almost all investigations have focused on the most extensively studied SIRT1, which was found to be overexpressed in non-melanoma skin cancers [52] and in melanoma cells [53,54]. The usage of pharmacological inhibitors of SIRT1, i.e., tenovin-1, sirtinol, and EX-527, resulted in melanoma cell proliferation arrest [54,55].…”

Sirtuin deacetylases: A new target for melanoma management

“…5,7,8 In addition to the finding that SIRT1 inhibition has effects on p53 protein levels in wild-type p53 melanoma cells, another recent paper by our lab found that several other pathways may be affected by SIRT1 inhibition, including a possible role in cell cycle regulation. 9 In our recent paper, we showed that SIRT1 is overexpressed in melanoma cell lines and human tissue samples. In melanoma cells, chemical inhibition of SIRT1 with the small molecule inhibitor Tenovin-1 led to a decrease in cellular proliferation and viability which was accompanied Keywords: cellular localization, melanoma, PI3K, SIRT1, SIRT1 inhibitors by increased protein levels of p53 and p21.…”