Abstract 5466: Mitochondrial superoxide inhibits autophagy and induces apoptosis through SQSTM1-mediated mechanism

Abstract

Abstract Metastatic melanoma has a dismal survival rate for patients, despite recent advances in targeted and immune-therapies. Recent studies show that elevated autophagy and redox imbalance in melanoma cells contributes to chemotherapeutic resistance, making these biological processes ideal therapeutic targets. However, gaps still exist regarding the mechanism by which pro-oxidant therapeutics affect autophagic signaling and the ultimate impact on melanoma cell fate. We previously demonstrated that pro-oxidant compound Nexrutine (NX) selectively inhibits melanoma cell survival and autophagy through autophagy scaffolding protein SQSTM1/p62. However, the mechanism by which NX-induced oxidative stress inhibits autophagy through p62 remained unknown. Thus, the primary objective of this study was to determine how p62 regulates cell fate during oxidative damage. We used a panel of primary melanocyte and melanoma cells to evaluate i) proteasomal degradation of p62 during oxidative stress-induced autophagy blockade, ii) the requirement for p62 during NX-induced apoptosis, iii) effect of NX-induced superoxide on autophagy and cell survival, iv) association of p62 with caspase-8 during oxidative damage, and v) subcellular localization of NX. Proteasomal degradation was evaluated using MG132 and western blotting for p62 following NX. We used genetic approaches (RNAi and overexpression using HA-p62) to determine the role of p62 in cell fate as determined by survival, apoptosis, and autophagy measurements. Fluorescence microscopy was used to determine the subcellular localization of NX. Generation of superoxide was determined using mitoSOX and mitoTEMPO pre-treatment was used to determine specificity. We found that i) NX-induced ROS inhibits p62 protein by stimulating proteasomal degradation, ii) p62 mediates apoptosis in response to high levels of oxidative damage through caspase-8 iii) NX generates superoxide which inhibits autophagy and melanoma cell survival, and iv) NX accumulates selectively in mitochondria. Overall, we show that oxidative stress induced by NX occurs upstream of its inhibitory effect on p62, autophagy and cell survival. Further, we demonstrate that NX induces superoxide, which leads to autophagy inhibition through increased p62 degradation, that ultimately inhibits melanoma cell survival, while not affecting primary melanocytes. We also show that p62 mediates apoptosis through increased association with caspase-8 in melanoma cells during superoxide generated by NX. We also demonstrate for the first time that NX localizes to mitochondria of melanoma cells and results in membrane potential loss due to its generation of high amounts of superoxide. This study demonstrates that pro-oxidants like NX can successfully target two pro-survival melanoma features simultaneously and may be useful melanoma therapeutic agents. Supported by R21 CA125719 & ACRCF (RG); NIDCR T32 DE14318/COSTAR (HGH) Citation Format: Heather G. Hambright, Addanki P. Kumar, Rita Ghosh. Mitochondrial superoxide inhibits autophagy and induces apoptosis through SQSTM1-mediated mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5466. doi:10.1158/1538-7445.AM2017-5466