Role of sirtuin 6 in melanoma development and progression

Wm S. Middleton Memorial Veterans Hosp, Madison WI

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Abstract

Malignant melanoma is one of the deadliest forms of cancer among Veterans, and the existing therapeutic options have not been fully effective in melanoma management, primarily owing to acquired drug resistance. Therefore, novel target-based approaches are needed for the management of this neoplasm. The mammalian sirtuins belong to a family of seven members (SIRT1 – SIRT7) with NAD+-dependent protein deacetylase and/or ADP-ribosyltransferase activities, which play critical roles in important cellular processes, and are involved in a variety of diseases, including cancer. We have an ongoing program to determine the roles and functional significance of sirtuins in melanoma. Our published and preliminary data has shown a pro- proliferative role of SIRT6 in melanoma in vitro and in vivo. Aberrant expression of SIRT6 has been shown to enhance melanoma growth through an autophagy-dependent manner. Further, our ongoing active VA Merit funding has provided compelling data suggesting that SIRT6 has a pro-proliferative role in melanoma and can serve as a target for melanoma management via its small molecule inhibition. However, an understanding of the mechanisms of the biological actions of SIRT6 in melanoma is far from complete. This may lead to the identification of additional strategies for melanoma management. Further, NOTCH1 is a promising therapeutic target, which is considered as a primary oncogenic factor in melanoma and linked with its metastasis. Thus, development of efficacious novel inhibitors of SIRT6 and NOTCH1 is needed for potential future use against melanoma (and other cancers). Interestingly, SIRT6 and NOTCH1 were found to be the target gene of the micro-RNA (miR)-34a. Importantly, miR-34a is shown to be significantly downregulated in melanoma tissues. However, the association of miR-34a with SIRT6 and NOTCH1 in melanoma is not known. Thus, based on our published and preliminary data and available literature, we propose to test a hypothesis that miR-34a- SIRT6-NOTCH1 axis plays a critical role in melanoma development and progression. A corollary to this hypothesis is that novel SIRT6 and NOTCH1 inhibitors (identified via virtual screening using library of pharmacologically active compounds) may be useful for melanoma management. To test the hypothesis, we propose two thematically connected specific aims to 1) to determine the involvement of miR-34a-SIRT6- NOTCH1 axis in melanoma development and progression, and 2) identify and develop novel small molecule inhibitor(s) of SIRT6 and NOTCH1 via virtual screening and to test them in vitro and in vivo against melanoma. Under specific aim 1, we will determine the expression profile of miRNA (in serum and melanoma tissue vs normal individuals) and SIRT6 and NOTCH1 mRNA and proteins (in melanoma tissues vs normal skin) followed by a correlation analysis between circulating miRNA-34a levels and expression of these proteins. This will be followed by in vitro experiments to determine if miRNA-34a functions as an upstream effector of SIRT6 and NOTCH1, employing miR-34a mimics; antagomiR-34a, overexpression and knockdown of SIRT6 to determine their effects in melanoma cells. We will do rescue experiments by NOTCH1 manipulation to analyze effects on miR-34a and SIRT6 mediated cellular effects. Under specific aim 2, we will first identify novel small molecule inhibitor(s) of SIRT6 and NOTCH1 via a virtual screening for focused testing on the SIRT6 and NOTCH1 targets. The top 10 identified inhibitors of SIRT6 and NOTCH1 will be used to determine in vitro therapeutic index (TI) using multiple melanoma cells. Finally, the top 2 compounds based on the highest in vitro TI and their combinations will be used to assess the effects on melanoma development and growth, in vivo using BRAFV600E/PTENNull mouse model. The outcome of our study will i) define the role of miR-34a- SIRT6-NOTCH1 axis in melanoma, and ii) lead to identification of novel diagnostic/prognostic markers as well as drug candidates for an efficient management of melanoma in Veterans as well as general population.