Abstract 1005: Mitochondrial inhibition decreases the malignant phenotype of esophageal adenocarcinoma cells through the induction of autophagy

Abstract OBJECTIVE Esophageal adenocarcinoma (EAC) is a lethal disease for which novel therapies are needed. Malignant cell metabolism often shifts from mitochondrial respiration to aerobic glycolysis (Warburg effect). However, glucose alone does not meet the metabolic requirements of cancer cells; therefore, glutamine becomes an essential nutrient. A critical step in glutamine metabolism is the conversion of glutamine to glutamate by the enzyme glutaminase-1 (Gls-1), a potential therapeutic target. The present study was undertaken to identify the effect of targeting glutamine utilization by the mitochondrion in EAC. METHODS Assays were performed in 3 EAC and 1 Barrett's cell lines: Flo-1, NCI-SB-ESC2 (Esc2), OE33, and CP-C. qRT-PCR and immunoblot were used for Gls-1 expression. Cyquant® and Millipore Invasion Kit measured proliferation and invasion. Flow cytometry, SA-β-galactosidase assay, and immunoblot assessed cell cycle, apoptosis, senescence, and autophagy. Seahorse Extracellular Flux Analyzer was used to quantitate mitochondrial respiration and glycolytic capacity. Cells were treated with bis-2 [5-phenylacetamido-1,2,4-thiadiazol-2-yl] ethylsulfide (BPTES), an inhibitor of Gls-1, and/or metformin, a mitochondrial electron transport chain inhibitor. Glycolysis was inhibited with 2-deoxyglucose (2-DG). Flo-1 and Esc2 were transduced with shRNA targeting Gls-1. RESULTS Glutamine withdrawal decreased proliferation of Flo-1, OE33, and CP-C more than Esc2. Similarly, BPTES caused a more significant decrease in proliferation in Flo-1, OE33, and CP-C than in Esc2. BPTES growth inhibition was reversed by α-ketoglutarate, a metabolite of glutamate. Unlike BPTES, metformin decreased proliferation in all cell lines which was augmented with 2-DG inhibition of glycolysis. Metabolic and knockdown experiments were performed in Flo-1 and Esc2. Glucose partially blocked mitochondrial spare capacity in Flo-1, but completely abrogated the spare capacity in Esc2 suggesting a higher glycolytic dependency in Esc2. Also, knockdown of Gls-1 blocked mitochondrial spare capacity and increased glycolysis in both cell lines. Knockdown of gls-1 decreased proliferation and invasion. Glutamine withdrawal induced autophagy as evidenced by increased LC3 and pAMPK and decreased p70 S6 Kinase in both cell lines. Apoptosis and senescence were not observed. To test glutamine's effect on glycolysis, glutamine repressed the negative regulator of glycolysis, thioredoxin interacting protein (TXNIP), suggesting that glutamine can increase glycolysis while simultaneously fueling the mitochrondria. CONCLUSIONS Mitochondrial respiration is mediated by both glucose and glutamine in EAC cells. Glutamine exerts an effect on glycolysis through repression of TXNIP. Whereas EAC cells have unique metabolic profiles, targeting common metabolic steps such as Gls-1 may be novel strategies to treat EAC. Citation Format: Deborah R. Depew, Paul L. Feingold, Kate Brown, Yuan Xu, Mahadev Rao, Michael Moses, Leonard M. Neckers, David S. Schrump, R Taylor Ripley. Mitochondrial inhibition decreases the malignant phenotype of esophageal adenocarcinoma cells through the induction of autophagy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1005.