Abstract 5394: HuR affects pancreatic cancer metabolism through post-transcriptional control of core metabolic enzymes.

Abstract

Abstract Background: HuR is an RNA-binding protein that translocates from the nucleus to the cytoplasm with its mRNA cargo in response to stress, and regulates mRNA stability and translation of these transcripts. HuR has been implicated as an important regulator of pro-survival pathways in cancer, and increased cytoplasmic expression of HuR has been observed in numerous cancer types. The regulatory mechanisms of metabolic pathways under metabolic stress are poorly understood. We investigated HuR's role in regulating pancreatic cancer metabolism. Methods: We measured HuR cytoplasmic expression in pancreatic cancer cells in response to glucose deprivation. Ribonucleoprotein precipitation (RIP) of HuR was performed to survey candidate metabolic mRNA targets. HuR levels were depleted with siRNA oligos under metabolic stress, and effects on cell survival, biochemicals in the media, and metabolic enzyme targets were determined. Experiments were performed in MiaPaca2, BXPC3, Panc1 cell lines. Results: Cytoplasmic protein expression of HuR was increased by immunoblot and immunofluorescence after 6 hours of glucose deprivation in all 3 cell lines (vs. 25 mM glucose). In addition, HuR depletion with siRNA impaired survival in of all 3 cell lines in low glucose media (<10 mM) at 7 days by PicoGreen. Overexpression of HuR had a protective effect under glucose deprivation in Panc1 cells. Surprisingly, HuR depletion resulted in increased glucose uptake in MiaPaca2 and BxPC3 cells. RIP-qPCR and immunoblot experiments ruled out GLUT1 as a mediator of this effect. In all 3 cell lines, lactate production decreased with HuR depletion. RIP assays of enzymes involved in carbohydrate metabolism identified 11 novel metabolic targets of HuR in MiaPaca2 cells: glycogen metabolism (GBE1, GSK3β); glycolysis (GPI, PGK1, HK2); Krebs cycle (IDH1, IDH3A, CS); and pentose phosphate pathway (PRPS2, RPIA, and RPE). Validation RIP-qPCR was performed for 3 enzymes (GPI, IDH1, PRPS2) in BXPC3 and Panc1 cells. In addition, HuR depletion resulted in decreased mRNA expression of these 3 genes in MiaPaca2 cells. Decreased protein expression of these 3 targets was observed in each of the cells lines. Incubation of MiaPaca2 cells in 1 mM glucose resulted in increased cytoplasmic HuR protein expression at 12 hours, and increased protein levels of all 3 metabolic targets at 48 hours. Conclusions: HuR cytoplasmic expression is critical for pancreatic cancer cell survival under glucose deprivation. HuR likely supports a ‘Warburg’ phenotype characterized by increased lactate production. Surprisingly, HuR activation led to decreased glucose uptake. Novel metabolic targets of HuR were identified, and coordination of these targets may be responsible for the observed findings. Improved understanding of how HuR acutely regulates metabolism in response to stress should uncover novel therapeutic targets against pancreatic cancer. Citation Format: Jordan M. Winter, Richard A. Burkhart, Danielle M. Pineda, Saswati Chand, Joseph Cozzitorto, Charles J. Yeo, Jonathan R. Brody. HuR affects pancreatic cancer metabolism through post-transcriptional control of core metabolic enzymes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5394. doi:10.1158/1538-7445.AM2013-5394