Abstract 4505: miR-122 regulates hepatic glutamine metabolism by directly targeting mitochondrial glutaminase

Abstract

Abstract It is well established that the liver specific miR-122, a bona fide tumor suppressor, plays critical role in lipid homeostasis [1]. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine is an important energy source, we monitored glutamine metabolism in the wild type (WT) and miR-122 knockout (KO) mice by SIRM (Stable Isotope Resolved Metabolomics) studies. To this end, 6-8 weeks old mice were injected with [U-13C,15N]-L-glutamine (Gln) (7 mg/mouse, 2x at 15 min interval) and sacrificed 15 min after the last injection. Analysis of polar metabolites from snap-frozen liver tissues by NMR and IC-MS showed elevated 13C-glutamine, -glutamate, -α-ketoglutarate, -isocitrate and -citrate levels without significant changes in succinate, malate or fumarate levels in KO livers suggest enhanced glutaminolysis and a break in the latter half of the Krebs cycle in miR-122 depleted livers. Reduced conversion of [13C,15N-glutamine] to 13C3- and or/ 13C6-glucose-6-phosphate and -fructose-6-phosphate implicates reduced gluconeogenesis from glutamine in KO livers while that to 13C-glutathiones suggests decreased synthesis and/or increased utilization. To elucidate the underlying mechanism, we focused on HITS-CLIP and RNA-seq data of the WT and KO mice, which identified functional miR-122 targets in the liver transcriptome [2]. The results showed that Gls is a direct miR-122 target, which was validated by luciferase reporter assay. Importantly, Gls expression was suppressed in glutamine dependent hepatoma cells by ectopic expression of miR-122 mimic. These results explain that the increased conversion of [U-13C,15N]-L-glutamine to glutamate and other Krebs cycle metabolites is likely due to the upregulation of Gls in miR-122 depleted livers. TCGA-LIHC database analysis showed that GLS expression in tumors inversely correlated with miR-122 expression. Upregulation of GLS protein level in primary HCCs relative to the matched benign liver tissues suggests enhanced use of glutamine as energy source by the tumor cells. Collectively, these results show that miR-122 modulates glutamine metabolism both in vitro and in vivo. These results implicate therapeutic potential of glutaminase inhibitors in patients bearing HCCs that bear low levels of miR-122. (supported in part, by R01CA193244 grant from NIH and Pelotonia Idea grant) 1. Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest. 2012 Aug;122(8):2871-83. 2. Argonaute CLIP Defines a Deregulated miR-122-Bound Transcriptome that Correlates with Patient Survival in Human Liver Cancer. Mol Cell. 2017 Aug 3;67(3):400-410.e7. Citation Format: Dipanwita Sengupta, Teresa Cassel, Kun-yu Teng, Peng Hu, Teresa Fan, Kalpana Ghoshal. miR-122 regulates hepatic glutamine metabolism by directly targeting mitochondrial glutaminase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4505.