Abstract 1424: Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism

Abstract

Abstract Proliferating cancer cells require an active Krebs cycle for generating anabolic precursors, in addition to energy production. Diversion of the Krebs cycle intermediates to meet anabolic demands cannot be sustained without anaplerosis. The two major anaplerotic pathways are the deamidation of glutamine to form glutamate catalyzed by glutaminase (GLS) or glutaminolysis, and the carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (PC). We determined the expression of PC and GLS proteins in fresh tumor tissue and paired adjacent benign tissue from 86 human NSCLC patients. PC was elevated (median 8-10 fold) in 94% of the tumor tissues, whereas GLS expression did not differ significantly between tumor and paired non tumorous lung tissue. Using 13C6 glucose as tracer and stable isotope-resolved metabolomics (SIRM), we also observed elevated PC activity in vivo in human patients with early stage NSCLC. To examine the importance of PC in the growth and survival of NSCLC, 5 NSCLC cell lines were transduced with a lentiviral vector containing an shRNA against PC. PC knockdown slowed proliferation, induced multinucleation, and reduced colony formation in soft agar. To validate attenuated PC activity and to profile the metabolic effect of PC knockdown, A549 cells were grown in 13C6 glucose or 13C5 glutamine and the incorporation of 13C into various metabolic pathways was measured by NMR and MS by SIRM. We found reduced entry of both glucose and glutamine carbon into the Krebs cycle metabolites, fatty acyl chains of lipids, and nucleotides, suggesting that both energy production and anabolic pathways were hindered and blocking the PC pathway was not compensated by GLS activity. In addition, glutathione biosynthesis and homeostasis were perturbed by PC suppression, leading to compromised anti-oxidation capacity. We further found that PC knockdown in A549 cells reduced their growth rate in mouse xenografts, and recapitulate the metabolic perturbations seen both in cell culture and in human patients. Together, these results suggest that PC is indispensible for the growth and anabolism of NSCLC. This work was funded by 5P20RR018733, 1R01CA118434-01A2, 1P01CA163223-01A1, 1R01ES022191-01, and 3R01CA118434-02S1; and the KLCRP, CTSPGP, and the KY Challenge for Excellence. Citation Format: Katherine E. Sellers, Matthew P. Fox, Michael Bousamra, Jun Yan, Mariia Yuneva, Richard M. Higashi, Andrew N. Lane, Teresa WM Fan. Enhanced pyruvate carboxylation is crucial to non-small cell lung cancer proliferation and anabolism. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1424. doi:10.1158/1538-7445.AM2014-1424