Abstract B53: Effects of metformin and ATP-competitive inhibitor of mTOR on targeted-metabolomic profile in HPAF-II pancreatic cancer cell lines

Abstract

Abstract Introduction: Pancreatic cancer (PC) is a devastating disease attributable to late diagnosis and is resistant to conventional treatment. Thus, there is an urgent need to develop new strategies for intervention of this lethal disease. Pancreatic ductal adenocarcinoma (PDAC) is the most common and most aggressive type of PC. Recent reports indicate the 50% of PDAC patients are diabetic at the time of diagnosis. To that end, tumor-related diabetes is now considered Type 3cDM. The mammalian Target of Rapamycin mTOR (also known as mechanistic TOR) is a conserved serine/threonine kinase and master regulator of metabolism, cell growth and proliferation. mTOR is dysregulated in a variety of tumors including pancreatic cancer. Furthermore, the anti-diabetic drug, metformin, which indirectly inhibits mTOR by activation of AMPK, has emerged as a potential therapeutic target in the treatment of PC, particularly when associated with type 3c Diabetes Mellitus. Mounting evidence links glycolytic metabolic disturbances to the adverse pancreatic cancer prognosis. The objective of this study is to determine the targeted-metabolomic profile in human PDAC cell line (HPAF-II) and the possible synergism between pan mTOR inhibition by ATP competitors (which inhibits both mTORC1 and mTORC2) and metformin administration as potential targets for therapeutic intervention in pancreatic cancer. Methods: Well differentiated pancreatic cancer cell line HPAF-II (CRL# 1997) were cultured in DMEM media supplemented with 10% fetal bovine serum in the presence of either Torin 2 (ATP-competitive mTOR inhibitor), metformin, both, or vehicle control for 1 hour and 24 hours in five replicate samples per group. We utilized targeted LC/MS/MS to characterize the alterations in the glycolytic and tricarboxylic acid (TCA) cycle metabolomics, and employed Western Blot analysis for cell signaling activation by phosphorylation. Comparisons between groups were analyzed using one-way Analysis of Variance (ANOVA) for detection of the overall statistical significance followed by secondary post-hoc analysis to determine differences between groups. The significance level was assessed at p < 0.05. Results: After 1 hour incubation with Torin 2, AMP concentration was reduced compared to the control and other treatment groups (p < 0.03). After 24 hours incubation, Torin-2 significantly decreased the glycolysis intermediates (fructose 1, 6 bisphosphate (FBP), and 2 phosphoglycerate/3 phosphoglycerate (2PG/3PG), TCA intermediate metabolites (acetyl CoA, citrate/isocitrate, and malate), as well as the electron acceptors (NAD+, and FAD). Metformin treatment alone reduced FBP and citrate/ citrate/isocitrate ratio. However, when HPAF-II cells were incubated with both Torin-2 and metformin, there was significant reduction in nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD), suggesting decreased levels of energy equivalents available to the electron transport chain. To confirm that Torin 2 blocked both mTORC1 and mTORC2, whole cell lysates from all treatment and control groups where subjected to Western Blot analysis. Torin 2 treatment decreased the autophosphoyration of mTOR (S2481), and phosphorylation of its downstream target phospho S6 (pS6) confirming the inhibition of mTORC1, and decreased Akt phosphorylation at S473 (phospho-Akt-S473) indicating the inhibition of mTORC2, while rapamycin had no effect on Akt phosphorylation. Conclusion: The targeted metabolomics data indicate that mTORC1 and mTORC2 inhibition by Torin 2 reduced glycolytic intermediates and TCA metabolites in HPAF- II and may synergize with metformin to decrease the electron donors NAD+ and FAD by the TCA cycle which may lead to reduced energy production. The work on this abstract utilized Metabolomics Core Services supported by grant U24 DK097153 of NIH to the University of Michigan. Citation Format: Ghada A. Soliman, Asserewou Etekpo. Effects of metformin and ATP-competitive inhibitor of mTOR on targeted-metabolomic profile in HPAF-II pancreatic cancer cell lines. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B53.