Abstract SY31-02: Autophagy, cancer cell survival, and mitochondrial homeostasis
Authors: Debnath Jayanta 
Published: May 2019

Abstract
Autophagy is a tightly regulated lysosomal self-digestion process that serves as an important adaptive mechanism during stress or starvation. My laboratory recently discovered that autophagy is strongly induced in epithelial cells deprived of extracellular (ECM) contact, which protects them from apoptosis. In further work, we have uncovered that autophagy is robustly induced in epithelial cells transformed by mutationally active oncogenes, raising the hypothesis that autophagy functions as a survival mechanism in cancerous cells deprived of proper ECM contact. We are currently dissecting whether detachment-induced autophagy contributes to oncogenic cell survival and transformation in vitro, and promotes the dissemination and metastasis of tumor cells in vivo.
In addition, we are uncovering novel cellular functions for ATGs, the proteins that regulate autophagy. ATG12 is an ubiquitin-like modifier required for autophagy, which to date, has been proposed to possess a single conjugation target, another autophagy regulator called ATG5. Now, we identify ATG3 as a novel substrate for ATG12 conjugation. ATG3 is the E2-like enzyme necessary for ATG8/LC3 lipidation during autophagy. ATG12-3 complex formation requires ATG7 as the E1 enzyme and ATG3 autocatalytic activity as the E2, resulting in the covalent linkage of ATG12 onto a single lysine on ATG3. Mutation of this single lysine abolishes ATG12-ATG3 complex formation.
To uncover the biological functions of ATG12-ATG3, we reconstituted ATG3-deficient cells with wild type ATG3 (ATG3WT) or a mutant version unable to conjugate to ATG12 (ATG3KR). Remarkably, disrupting ATG12-3 complex formation has no observable effect on autophagy. In fact, during nutrient starvation and rapamycin- mediated mTORC1 inhibition, ATG3WT and ATG3KR-expressing cells exhibit equal rates of both LC3 lipidation (LC3-II) and turnover in the lysosome, as well as the robust proteolytic degradation of autophagy substrates like p62/SQSTM.
Instead, the most obvious consequence of disrupting ATG12-3 is an expansion in mitochondrial mass due to autophagy- and mitophagy-independent mechanisms. Lack of ATG12 conjugation to ATG3 also produces an increase in mitochondrial fragmentation, arising from a defect in mitochondrial fusion. Furthermore, cells lacking ATG12-3 exhibit resistance to the intrinsic apoptotic pathway. Most importantly, these unique functions of the ATG12-3 complex can be completely separated from the established roles of either individual ATG on autophagosome formation.
Overall, these results reveal unexpected novel roles for ATG12-3 in mitochondrial homeostasis and cell death, and for the first time, implicate the ATG12 conjugation system in new cellular functions distinct from the early steps of autophagosome formation.
Citation Format: Jayanta Debnath. Autophagy, cancer cell survival, and mitochondrial homeostasis [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SY31-02