Abstract 4619: Sirt2 protein is required for activation of mTOR signaling in colorectal cancer cells
Authors: Wang Qingding , Zhou Yuning , Weiss Heidi L. , Evers B. Mark 
Published: July 2016

The human sirtuin proteins (Sirt1-7) possess protein deacetylase activities and are involved in the regulation of metabolism, differentiation and cell survival. Sirt2 is upregulated in some cancers; however, some studies have suggested its role as that of a tumor suppressor. mTOR regulates many processes that control growth, including protein synthesis, oxidative metabolism, glucose homeostasis, cell growth, and lipogenesis. Deptor protein binds mTOR through the PDZ domain and inhibits mTOR activity. Our previous studies have shown that mTOR is a central regulator of colorectal cancer (CRC) cell proliferation and survival. The purpose of this study was to investigate: i) the regulation of Deptor expression by Sirt2, and ii) the role of Sirt2 protein in mTOR activation in CRC and normal intestinal cells. METHODS. Human CRC cell lines, HT29 and Caco-2, the rat normal intestinal cell line, IEC-6, and the human embryonic kidney cell line, HEK293, were transfected with siRNA targeting Sirt2 or control nontargeting siRNA. Cells were also infected with lentiviral vectors containing the control shRNA or shRNA to human Sirt2; stably expressing cells were selected with puromycin. Sirt2 overexpression was achieved by infecting cells with an adenovirus encoding Sirt2. Cell proliferation was assessed by counting the number of viable cells. The expression of Sirt2, Deptor, p-Akt (S473), Akt, p-S6 (S235/236), S6, LC3B, Cyclin D1, c-Myc, and survivin was analyzed by western blot. Deptor mRNA levels were determined by real time RT-PCR. RESULTS. Knockdown of Sirt2 inhibited mTOR signaling, decreased expression of cyclin D1, c-Myc, and survivin, and inhibited CRC cell growth. In agreement with the inhibition of mTOR activation, knockdown of Sirt2 significantly increased Deptor mRNA and protein expression in CRC cells. Conversely, overexpression of Sirt2 decreased Deptor expression and activated mTOR signaling. Knockdown of Sirt2 increased, while knockdown of Deptor decreased, the protein expression of LC3B, an autophagy marker, suggesting Sirt2 inhibition increases autophagy through induction of Deptor. Knockdown of Sirt2 inhibited HT29 cell proliferation; this inhibition was enhanced by treatment with 3MA, an autophagy inhibitor. In contrast, knockdown or overexpression of Sirt2 did not affect either the expression of Deptor or the activation of mTOR in HEK293 and IEC6 cells, suggesting a differential regulation of Deptor/mTOR signaling by Sirt2 in CRC and normal intestinal cells. CONCLUSIONS. We provide evidence that the Sirt2 protein increases mTOR activity and contributes to CRC cell proliferation. Importantly, our results indicate a novel role of Sirt2 in the activation of mTOR signaling in CRC cells but not in normal intestinal cells and suggests that targeted inhibition of Sirt2 could represent a novel and safe strategy for the treatment of CRC.

Citation Format: Qingding Wang, Yuning Zhou, Heidi L. Weiss, B. Mark Evers. Sirt2 protein is required for activation of mTOR signaling in colorectal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4619.