Abstract 2276: Coordinate autophagy and PI3K/Akt/mTOR pathway inhibition enhances cell death in melanoma

Abstract

Abstract The PI3K/Akt/mTOR pathway is a critical survival pathway in numerous malignancies, including melanoma. Inhibitors of this pathway in cancer cells, in particular mTOR, are commonly cytostatic rather that cytotoxic, which may potentially limit efficacy of these agents. mTOR inhibition also induces the survival pathway of autophagy, a catabolic process whereby cells collect and recycle cellular components to sustain energy homeostasis. Thus we tested the hypothesis that autophagy induction by mTOR inhibitors directly limited their efficacy for the treatment of melanoma. Here we found that melanoma exhibits high basal levels of autophagy, as evidenced by a) detection of autophagosomes using LC3 fluorescence and autophagic flux in multiple melanoma cell lines and b) increased LC3 fluorescence in human primary and metastatic melanomas compared to benign nevi. Knockdown of Atg7, a key element of autophagy machinery, resulted in cell death, indicating that survival of melanoma cells is autophagy dependent. We also showed that the autophagy inhibitor hydroxychloroquine (HCQ) shows synergy when combined with the mTOR inhibitor CCI-779 (temsirolimus), inducing cell death via increased apoptosis in melanoma cell lines and in 3-D spheroid cell cultures. Since autophagy eliminates mitochondria that are a source of toxic reactive oxygen species (ROS) and mediators of apoptosis, we tested the possibility that this contributed to melanoma survival. To test this potential mechanism, we examined the role of the transcription factor Nrf2, a master regulator of antioxidant defense. Treatment with CCI-779 inactivated Nrf2 by blocking its nuclear translocation and creating the requirement for autophagy to eliminate ROS-producing mitochondria. Combination treatment with CCI-779 and HCQ then prevented mitochondrial elimination by autophagy, resulting in increased ROS production compared to either agent alone and increased cell death. Conversely, knockdown of the Nrf2 binding protein and inhibitor Keap1 increased Nrf2 activity, resulting in increased cell culture survival and resistance to treatment with CCI-779 and HCQ. These data suggest that coordinate inhibition of the PI3K/Akt/mTOR and autophagy pathways promotes ROS-mediated apoptosis and is a new therapeutic paradigm for the treatment of melanoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2276. doi:1538-7445.AM2012-2276