Abstract 2177: A novel role for sirtuins in modulating ER stress: implications for neuroblastoma therapy.

Sirtuins (SIRTs) are NAD+ dependent deacetylases, which play an important role in cancer cell survival and resistance to chemotherapy. SIRT1, the most studied sirtuin, is overexpressed in many drug resistant cancers including neuroblastoma. SIRTs mediate cellular processes by deacetylating histone and non-histone proteins including those involved in the oxidative stress response, DNA repair and apoptosis. Here we define a novel role for Sirts in modulating the unfolded protein response (UPR) and cell survival following endoplasmic reticulum (ER) stress. Cellular stresses such as hypoxia, nutrient deprivation and chemotherapy can reduce the protein folding capacity of the ER leading to the accumulation of unfolded proteins which triggers activation of the UPR. The UPR is an adaptive mechanism that functions to restore ER and cellular homeostasis and as such plays a key role in cancer cell survival and tumor growth. To induce ER stress we exposed neuroblastoma cell lines NB1691, SK-N-BE2 and SH-SY5Y to the glucose analog, 2-deoxy-D-glucose (2-DG). In addition to inhibiting glycolysis, 2-DG has been shown to activate the UPR by interfering with N-linked glycosylation and proper protein folding. 2-DG (2mM) induced the UPR markers grp78, grp94 and CHOP as determined by western blot analysis. Treatment of neuroblastoma cell lines with the SIRT inhibitor, sirtinol (50μM), blocked the induction of grp78 by 2-DG, attenuated grp94 and increased CHOP. Recently it has been shown that activation of SIRT1 and AMP-activated protein kinase (AMPK) exerts similar effects and may regulate the activity of each other. AMPK can increase the transcription of the NAD+ biosynthetic enzyme Nampt, which in turn increases the NAD+/NADH ratio We found that 2-DG induced rapid activation of AMPK, as indicated by robust induction of phospho-ACC (serine 79) and inhibition of AMPK with compound C prevented grp78 induction by 2-DG. To determine if this effect of SIRT inhibition on grp78 induction was unique to 2-DG, we also treated neuroblastoma cells with the ER stressors velcade (bortezomib) and the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Inhibiting SIRTs significantly decreased the induction of grp78 and enhanced CHOP levels. Furthermore, sirtinol significantly increased 2-DG, velcade, and thapsigargin induced cell death as determined by MTS assay. The UPR promotes cancer cell survival in response to cellular stressors including chemotherapy. Therapeutic targeting of the UPR is a novel anti-cancer approach. Inhibiting the activity of SIRTs modulates the UPR in neuroblastoma and represents an innovative way to enhance the effect of chemotherapy in a cancer with minimal survival. Citation Format: Regina M. Graham, Jayanti Singh, Keith A. Webster, Steven Vanni. A novel role for sirtuins in modulating ER stress: implications for neuroblastoma therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2177. doi:10.1158/1538-7445.AM2013-2177