Abstract 5061: ALDH2 restricts esophageal stem/progenitor cell expansion via autophagy under alcohol-induced mitochondrial stress

Abstract

Abstract Introduction: Esophageal cancer is linked to heavy alcohol (EtOH) drinking and polymorphic aldehyde dehydrogenase 2 (ALDH2) mutation. Mutant ALDH2E487K protein delays mitochondrial clearance of acetaldehyde, the chief metabolite of EtOH and a major human carcinogen. Autophagy maintains mitochondrial redox homeostasis and may act as a tumor suppressor by decreasing oxidative stress. The role of ALDH2 and autophagy in esophageal epithelial homeostasis remains elusive. Methods: We analyzed EtOH-exposed esophageal epithelial cells and EtOH-fed Aldh2E487 or Aldh2WT mice by immunoblotting, immunofluorescence, flow cytometry and respirometry to determine mTORC1 signaling, autophagy, mitochondrial dysfunction and superoxide production. Mitochondrial superoxide was documented via mitochondria-targeted antioxidant mito-CP. Autophagy flux was blocked pharmacologically by chloroquine (CQ) or RNA interference (RNAi) against Beclin, a mediator of autophagy. Mitochondria-targeted autophagy (mitophagy) was further evaluated by RNAi against Parkin. To evaluate esophageal stem/progenitor cell function, we assessed the formation of single cell-derived three-dimensional (3D) organoids from mice with Aldh2E487K or Atg7loxP/loxP (autophagy mediator). We determined organoid formation following EtOH exposure, autophagy inhibition by CQ or adenoviral Cre-mediated Atg7 deletion. Rapamycin was used to inhibit mTORC1 and activate autophagy. Results: EtOH induced mitochondria damage in esophageal epithelial cells in vitro and in vivo with enhanced mitochondrial dysfunction by Aldh2E487K. EtOH induced mitochondrial dysfunction and superoxide production was antagonized by mito-CP. EtOH robustly suppressed mTORC1 signaling with concurrent activation of autophagy flux. Pharmacological or genetic inhibition of autophagy or mitophagy aggravated EtOH-induced mitochondrial dysfunction, superoxide production and cell death. Recapitulating esophageal epithelial proliferation-differentiation gradient ex vivo, Aldh2E487K murine esophageal cells displayed increased 3D organoid formation capability where resulting organoids contained more basaloid (undifferentiated) cells with mitochondrial dysfunction as compared to Aldh2WT cells. Moreover, EtOH stimulated 3D organoid formation by Aldh2E487K cells in a dose-dependent manner. Aldh2 dysfunction was phenocopied by CQ or Atg7 deletion, which increased 3D organoid formation. Interestingly, rapamycin suppressed organoid formation. Conclusions: Our innovative approach reveals that autophagy and ALDH2 protect against EtOH-induced mitochondrial dysfunction to maintain redox homeostasis and prevent abnormal propagation of esophageal stem/progenitor cells, providing novel mechanistic insights into the role of dysfunctional alcohol metabolism and autophagy in esophageal carcinogenesis. Citation Format: Prasanna Modayur Chandramouleeswaran, Kelly A. Whelan, Noah Engel, Jason Correnti, Koji Tanaka, Satish Srinivasan, Manti Guha, Rotonya Carr, Andres J. Klein-Szanto, Balaraman Kalyanaraman, Che-hong Chen, Daria Mochly-Rosen, Narayan G. Avadhani, Hiroshi Nakagawa. ALDH2 restricts esophageal stem/progenitor cell expansion via autophagy under alcohol-induced mitochondrial stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5061.