Abstract A45: Beclin1-independent autophagy promotes the survival of metastatic ovarian cancer cells
Authors: Correa Rohann , Ramos-Valdes Yudith , Peart Teresa , Fazio Elena , Bertrand Monique , McGee Jacob , Préfontaine Michel , Sugimoto Akira , Shepherd Trevor G. , DiMattia Gabriel E. 
Published: August 2014

Epithelial ovarian cancer (EOC) metastasis proceeds by an intra-peritoneal route, involving detachment of cells from the primary tumor and dissemination throughout the peritoneal cavity as multicellular aggregates, or spheroids. Herein, we demonstrate that ascites-derived cells upregulate the process of macroautophagy (autophagy), a highly-conserved self-digestion mechanism that functions to mitigate cellular stresses. Both non-adherent culture of these cells to form in vitro spheroids as well as exposure to an allosteric AKT inhibitor stimulated this process, as evidenced by Western blot and immunofluorescence staining for the autophagy marker MAP1LC3B (LC3). Likewise, expression of an eGFP-labeled LC3 protein revealed a shift from diffuse to ‘punctate’ cytoplasmic localization, demonstrating the formation of intracellular autophagic vesicles (autophagosomes). Additionally, we performed transmission electron microscopy and autophagy flux assays, confirming that autophagic degradation proceeded to completion. To determine the role of autophagy upregulation, we attempted to block this process using siRNAs targeting critical autophagy-related (ATG) genes. Interestingly, we found that depletion of Beclin1/ATG6 had no effect on autophagy, despite its role as a canonical inducer of the process. Conversely, depletion of the autophagy regulators ATG5 & ATG7 efficiently blocked LC3 processing and autophagosome formation. Likewise, autophagy was also blocked pharmacologically using the classical inhibitor Chloroquine and the novel agent Spautin-1 (the first targeted autophagy inhibitor to be published). Autophagy blockade by any of these mechanisms reduced EOC cell viability in both adherent and spheroid cultures, suggesting that the upregulation of this process serves to promote cell survival. Moreover, when autophagy blockade was combined with AKT inhibition, a synergistic reduction in cell survival was observed based on combination index analysis. Taken together, this work describes spheroid formation as a stimulus for a non-canonical, Beclin1-independent autophagy program, but also reveals autophagy to be a promoter of tumor cell survival during ovarian cancer metastasis, particularly in cells subjected to AKT/mTOR inhibition. In light of these findings, we propose that clinical autophagy inhibition could enhance the effectiveness of current or investigational ovarian cancer therapies.

Citation Format: Rohann Correa, Yudith Ramos-Valdes, Teresa Peart, Elena Fazio, Monique Bertrand, Jacob McGee, Michel Préfontaine, Akira Sugimoto, Trevor G. Shepherd, Gabriel E. DiMattia. Beclin1-independent autophagy promotes the survival of metastatic ovarian cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A45.