Abstract 501: Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis
Authors: Shimonosono Masataka , Kijima Takashi , Maekawa Hisatsugu , Takada Satoshi , Tanaka Koji , Sahu Varun , Guha Manti , Klein-Szanto Andres J. , Diehl J. Alan , Bass Adam J. , Rustgi Anil K. , Philipone Elizabeth , Yoon Angela J. , Nakagawa Hiroshi 
Published: September 2020

Abstract
Introduction: Heavy alcohol (EtOH) drinking and tobacco smoking increase the risk for upper digestive (e.g. oral and esophageal) squamous cell carcinomas (SCC) in individuals with polymorphic mutations in aldehyde dehydrogenase 2 (Aldh2). Mutant Aldh2E487K protein delays mitochondrial clearance of acetaldehyde, the chief metabolite of EtOH and constituents of tobacco smoke, aka a major human carcinogen. The role of Aldh2 in tumor biology remains elusive.
Methods: Single cell-derived three-dimensional (3D) organoids were generated from murine oral and esophageal preneoplastic and SCC lesions carrying wild-type Aldh2WT or mutant Aldh2E487K as well as human SCC cell lines with Aldh2E487K, the latter subjected to xenograft transplantation in immunodeficient mice. 3D organoids and xenograft tumors were analyzed by morphology and flow cytometry to determine CD44, autophagy (cyto-ID), proliferation (EdU), and reactive oxygen species (ROS) following EtOH exposure ex vivo (0.1-2% EtOH) or in vivo (10% EtOH in drinking water) along with or without inhibitors of alcohol metabolism (4-methylpyrazole; 4MP, siRNA against Adh1B and Cyp2E1), and autophagy flux (chloroquine; CQ or siRNA against Atg7). Flow cytometry was performed to determine cancer stem-like cells with high CD44 expression (CD44H cells).
Results: EtOH stimulated xenograft tumor growth and increased highly proliferative intratumoral CD44H cells with increased autophagy. EtOH-stimulated tumor growth and induction of CD44H cells were sharply suppressed by concurrent 4MP treatment, indicating that EtOH is directly metabolized in SCC cells to promote tumor growth via CD44H cells. These EtOH-induced effects were recapitulated in 3D organoids where Aldh2E487K augmented EtOH-mediated induction of CD44H cells and organoid formation with increased cellular atypia. EtOH induced mitochondrial superoxide and ROS-dependent autophagy activation in CD44H cells. Inhibition of autophagy prevented EtOH from enriching CD44H cells in 3D organoids, suggesting that autophagy may limit alcohol-induced oxidative stress as a cytoprotective mechanism.
Conclusions: These studies provide not only mechanistic insights into the role of Aldh2 mutation in oral and esophageal SCC pathogenesis but also establish the utility of the 3D organoid system to test the functional interplay between genetic and environmental factors which may subsequently be targeted for pharmacological intervention in the setting of personalized medicine.
Citation Format: Masataka Shimonosono, Takashi Kijima, Hisatsugu Maekawa, Satoshi Takada, Koji Tanaka, Varun Sahu, Manti Guha, Andres J. Klein-Szanto, J. Alan Diehl, Adam J. Bass, Anil K. Rustgi, Elizabeth Philipone, Angela J. Yoon, Hiroshi Nakagawa. Aldh2 mutation promotes oral and esophageal cancer stem cells via autophagy-mediated redox homeostasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 501.