Abstract
Abstract Introduction: Few proteins have been found to be master-regulators of pancreatic ductal adenocarcinoma (PDA) cell metabolism (e.g., LKB1, KRAS). We recently demonstrated that the regulatory RNA binding protein, HuR, binds to numerous metabolic mRNA transcripts and regulates their expression. Additionally, HuR silencing with small interfering RNAs(siRNAs) inhibited growth of PDA cells in vitro under glucose deprivation. Herein, we manipulated HuR expression levels in PDA cells and performed isotope tracer fate association studies to better understand HuR's role in metabolic reprogramming. Methods: We used 13C-labeled nutrients to map carbon flux in BxPC3 pancreatic cancer cells that were either transiently transfected with siRNA oligos against HuR (siHuR) or scrambled control (siCTRL). Cells were incubated with standard DMEM or mild glucose deprivation (5 mM glucose) for 48 hours, and media were supplemented with [1,2-13C2]-D-glucose or [U-13C5]-L-glutamine tracers for the final 24 hours. A total of 24 samples were snap frozen and prepared for GC-MS isotopomer analysis (3 replicates X 2 different siRNA oligos X 2 isotope tracers X 2 glucose concentrations). Transfection experiments were validated for reduced HuR expression (>70% reduction) and reduced protein expression of at least one metabolic and established HuR target (e.g., IDH1) by immunoblot. Results: HuR silencing directly impaired fatty acid (Table, line 1-3), ribose (line 5) and glycogen synthesis (line 6). Reductive carboxylation of glutamine-derived isocitrate was impaired and futile carbon exchange fluxes were prevalent (not shown). Conclusions: HuR enhances metabolic efficiency in PDA cells by directly regulating multiple metabolic pathways. Ongoing microarray studies will highlight which metabolic transcripts are post-transcriptionally regulated by HuR, resulting in the observed phenotype. Altered metabolites due to HuR silencing in PDA cells ([1,2-13C2]-D-glucose tracer)PathwayMetabolitesiHuR 25mM glucosesiHuR 5 mM glucoseCorrelation1Myristate (C:14) Intracell13C enrichment70.271.01.02Myristate (C:14) intracellFNS (direct)56.060.41.03Oleate (C:18-1) IntracellIndirect synthes-m187.689.51.04Glutam extracell [C2-C5]13C-m1 (m/z198)97.497.91.05RNA-ribose Intracell [C1-C4]13C-m3(m/z242)99.399.10.9846Glucose intracell [C3-C6]Peak area, Glycog-gluc74.180.10.983 Citation Format: Fernando F. Blanco, Mahsa Zarei, Jonathan R. Brody, Laszlo G. Boros, Jordan M. Winter. The RNA binding protein, HuR, regulates pancreatic cancer cell metabolism. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1191. doi:10.1158/1538-7445.AM2015-1191
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