Abstract 5144: RNA-binding protein HuR supports post-transcriptional regulation of pancreatic cancer cell metabolism
Authors: Richard Burkhart, Danielle Pineda, Joseph Cozzitorto, Charles Yeo, Jonathan Brody, Jordan Winter
Journal: Cancer Research (2012)

Abstract Metabolic transformation towards a glycolytic phenotype is well described in pancreatic ductal adenocarcinoma (PDAs) and other cancers, yet the underlying molecular mechanisms remain largely unknown. To date, literature has focused principally on genetic changes in cancers such as somatic mutations. However, it is more likely that post-transcriptional regulatory elements play a significant role in metabolic adaptive responses given the rapid and dynamic nature of conditions in vivo. One element, the RNA-binding protein HuR, has been shown to direct the dynamic cellular response to various cancer-associated stressors by translocating from the nucleus to the cytoplasm and augmenting target expression. Recently discovered HuR-metabolic targets include HIF-1α and the glucose transporter GLUT-1. We hypothesize therefore that HuR is an important post-transcriptional regulator of cellular metabolism in pancreatic cancer. We began by exposing pancreatic cancer cell lines (MiaPaca-2, PL5, PL45, BxPC-3, ASPC-1, and Panc1) to the known glycolytic inhibitors citrate and hypoglycemia. When citrate was added to culture media at doses between 0.1 to 10 mM (for 2-12 hrs), we identified robust HuR cytoplasmic translocation by both western blot and immunofluorescence. When these cells are exposed to a known α-ketoglutarate agonist and hypoxia mimetic, Dimethyloxalylglycine, HuR is also activated to the cytoplasm at doses between 0.1 and 1 mM. Similarly, reducing the glucose in standard media (25mM) to physiologic levels (5mM) and below (down to 0.5 mM) induces HuR cytoplasmic translocation. Interestingly, with continued metabolic stress cytoplasmic HuR protein returned to pre-stress levels after 24 hours of treatment. We then studied the affects of these metabolic stressors (4–6 hours) on validated HuR mRNA targets (dCK, HIF-1α, and SUMO1), and observed upregulation using quantitative PCR (qPCR) analysis of total RNA. Finally, we performed ribonucleoprotein-immunoprecipitation (RNP-IP) of HuR bound mRNA cargo in PDA cells and detected that the HuR target mRNAs dCK and HuR were upregulated under conditions of hypoglycemia as compared to normoglycemia. These data are the first to demonstrate that HuR is activated to the cytoplasm in pancreatic cancer cells as a result of metabolic stress. Additionally, we demonstrate that HuR activation during changes in the metabolic mileau affects mRNA levels of HuR targets. Based on these preliminary findings, HuR is a likely regulator of an acute metabolic transformation necessary for pancreatic cancer cells. Ongoing studies will elucidate HuR's role in adapting and maintaining the metabolic phenotype that is characteristic of malignancy. This may additionally lead to the discovery of novel metabolic targets regulated acutely by these post-transcriptional mechanisms. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5144. doi:1538-7445.AM2012-5144