Abstract A99: Posttranscriptional gene regulation by HuR can explain metabolic reprogramming in acutely stressed pancreatic cancer cells.

Background and objective: Genetic alterations (e.g., somatic mutations) in metabolic enzymes (e.g., IDH1, SDH subunits), protooncogenes (e.g., KRAS, PI3K, MYC), and tumor suppressor genes (e.g., TP53, PTEN) are believed to drive the metabolic program in cancer towards a glycolytic phenotype (i.e., Warburg effect). However, certain aspects of cancer cell metabolism do not fit into this paradigm: while mutations in specific cancer genes are found throughout a tumor in vivo, the tumor microenvironment and metabolic patterns of cancer cells are heterogeneous; furthermore, genetic changes are irreversible events and do not occur in response to acute stress (rather they develop by experimentation over time). HuR is an RNA binding protein that translocates from the nucleus to the cytoplasm (i.e., HuR activation) in response to acute stress and efficiently orchestrates a pro-survival network through its numerous (>1000) mRNA targets. Multiple core-signaling pathways in pancreatic ductal adenocarcinoma (PDA) are regulated by HuR, including cell cycle progression, invasion, antiapoptosis, KRAS signaling and DNA damage repair. We hypothesized that HuR also regulates PDA metabolism through post-transcriptional regulation of metabolic gene transcripts in response to acute glucose deprivation.

Methods: Experiments were performed on MiaPaca2 cells. First, we measured HuR cytoplasmic expression via immuno fluorescence (IF) and Western blotting (WB) of cytoplasmic extracts from PDA cells exposed to 1mM glucose (glucose deprivation) or 25mM (normal culture glucose levels). Descriptively, we then examined the effect of removing HuR (siHuR vs. control) from cells in the setting of glucose deprivation on cell survival and ambient glucose concentrations. To gain mechanistic insights, we screened mRNAs bound to HuR (ribonucleoprotein immunoprecipitation or RNP-IP using an HuR antibody) for metabolic targets. We profiled the metabolome using chromatography and mass spectroscopy in isogenic PDA cells (siHuR vs. control).

Results: Acute glucose deprivation was a potent activator of cytoplasmic HuR expression by IF and WB. Transient HuR knockdown (e.g., siHuR) compared to control cells impaired cell survival at 5 days in low glucose conditions, as determined by picogreen (staining for double-stranded DNA) and soft agar survival assays. HuR expressing cells consumed less glucose in the setting of glucose deprivation, as evidenced by higher glucose concentrations in the media after just 48 hours. An HuR RNP-IP performed after 4 hours of glucose deprivation identified 11 HuR targets (from a focused glucose metabolism mRNA array) with >4-fold binding compared to a control IgG RNP-IP (p<0.05). HuR mRNA targets involved every major branch of glucose metabolism. Five targets have been validated by RIP-SEQ including two enzymes in the PPP; two targets (IDH1 and GPI) were additionally validated by qRT-PCR (decreased mRNA expression) and WB (decreased protein expression) after HuR knockdown. Metabolomics of the HuR knockdown cells at 1 mM glucose for 12 hours revealed a decrease in fatty acid and amino acid catabolism and less oxidative stress compared to controls. The knockdown cells also had a relative increase in the PPP intermediate, sedoheptulose-7-phosphate, under these conditions.

Conclusion: HuR is activated under acute glucose deprivation and orchestrates a pro-survival response through regulation of key metabolic pathways. Specifically, HuR may minimize catabolic activity (e.g., autophagy) and oxidative stress as a protective measure. Post-transcriptional gene regulation of metabolic enzymes is a novel mechanism of metabolic reprogramming under routine metabolic stress and reveals new therapeutic opportunities.

Citation Format: Jordan M. Winter, Rick A. Burkhart, Joseph Cozzitorto, Charles J. Yeo, Jonathan R. Brody. Posttranscriptional gene regulation by HuR can explain metabolic reprogramming in acutely stressed pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr A99.