Abstract B41: Post-transcriptional regulation of IDH1 by the RNA-binding protein HuR is important for pancreatic cancer cell survival under nutrient deprivation

Abstract

Abstract Introduction: Isocitrate dehydrogenase 1(IDH1) has been prioritized in the recent cancer biology literature because of mutations occurring in some tumor types. Additionally the wild type isoenzyme is important for metabolic reprogramming under hypoxic stress. Importantly, the role of IDH1 under other forms of metabolic stress, such as nutrient deprivation (hallmark of the PDA microenvironment) has not been explored, and the regulatory mechanism of IDH1 expression remains unknown. We recently showed that the regulatory RNA binding protein, HuR, binds and directly regulates IDH1 expression in multiple PDA cell lines. The regulatory protein becomes biologically engaged under nutrient deprivation by translocating with the IDH1 transcript form the nucleus to the cytoplasm under glucose deprivation, and HuR silencing sensitized cells under these conditions. Here, we hone in on the regulatory HuR binding site to the IDH1 transcript, explore the importance of HuR in the context of additional metabolic stressors and utilize stable isotope metabolomic profiling to gain mechanistic insight into how HuR is protective under nutrient deprivation. Methods: IDH1 and HuR expression were knocked down by siRNA, and cell viability was determined by PicoGreen and Trypan blue exclusion assays. Immunofluorescence was used to image HuR subcellular localization under glutamine deprivation. Based on computational predictions of 5 HuR binding sites in the IDH1 mRNA 3'UTR, we subcloned this entire region into a luciferase reporter construct to further study this regulatory interaction. In order to determine the impact of HuR expression in cellular metabolism targeted tracer fate association studies were performed using GC-MS of pellets from cells cultured with 13C-labeled glucose and glutamine in BxPC3 pancreatic cancer cells that differed only in HuR expression. We calculated Pearson correlations of measured metabolites after HuR silencing. Results: Cell viability was impaired by depletion of both IDH1 and HuR (vs. controls) upon glutamine and glucose withdrawal. Moreover, HuR silencing resulted in potent suppression of IDH1 at the mRNA and protein levels. HuR silencing substantially decremented luciferase activity in the IDH1 3'UTR construct compared to the control (> 2-fold decrease). HuR translocated to the cytoplasm under glutamine deprivation, as previously published observed with glucose deprivation by our group. Metabolic tracer fate association studies revealed that HuR silencing impaired carbon flux from glutamine into fatty acid end products (myristate and palmitate, > 0.9-Pearson's Correlation) under low glucose conditions, implicating HuR's regulatory role in the IDH1-mediated reductive carboxylation step of this metabolic pathway. Additionally, HuR silencing impaired ribose and glycogen synthesis from glucose, and futile carbon exchange fluxes were prevalent. Conclusions: HuR is important for pancreatic cancer cell survival under glutamine deprivation, as previously observed for glucose deprivation. Carbon flux from glutamine to fatty acid end products suggests a role for HuR in reductive carboxylation of glutamine-derived α-ketoglutarate by IDH1, as a way to maintain adequate lipid synthesis under glucose deprivation. Our Studies provide a rationale to pursue pharmacologic strategies that target HuR or its regulation of IDH1 as a novel treatment of PDA. Citation Format: Mahsa Zarei, Fernando F. Blanco, Laszlo G. Boros, Charles J. Yeo, Jonathan R. Brody, Jordan M. Winter. Post-transcriptional regulation of IDH1 by the RNA-binding protein HuR is important for pancreatic cancer cell survival under nutrient deprivation. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B41.