Abstract 4100: HuR regulates death receptor-5 (DR5, TRAIL-R2)-targeted treatment of pancreatic cancer cells
Authors: Rittenhouse David W. , Valley Christopher , Cozzitorto Joseph A. , Richards Nathan G. , Talbott Vanessa A. , Yeo Charles J. , Sachs Jonathan N. , Witkiewicz Agnieszka K. , Brody Jonathan R. 
Published: July 2012

Apoptosis has been identified as a core signaling pathway disrupted in pancreatic ductal adenocarcinoma (PDA) tumorigenesis. Death Receptor 5 (DR5, TRAIL-R2) is a membrane bound protein that initiates the extrinsic apoptotic pathway upon ligand exposure and is currently being explored as a ‘druggable’ target in multiple cancers including PDA. Identifying a mechanism that regulates DR5 in the tumor microenvironment (e.g. hypoxia, chemotherapeutic exposure) is critical for optimizing DR5 based-therapies. Human antigen R (HuR), an RNA binding protein, controls post-transcriptional gene expression by binding to specific regions of 3’and 5’ UTRs of mRNA target genes. Previously, HuR, a pro-survival molecule, has been shown to play an important role in the intrinsic apoptotic pathway. We identified DR5 mRNA as a HuR target in PDA cells and explored the significance of HuR's role in functionally regulating the extrinsic apoptotic pathway in PDA cells. We also explored HuR as a modulator of DR5-targeted therapy for the treatment of PDA. Ribonucleoprotein immunoprecipitation (RNP-IP) assays were performed on PDA cells using HuR antibody (Ab) compared to a control (IgG Ab) under stress conditions, 3 hours with 1μM of the standard of care drug for PDA, gemcitabine; and 75 μM of a PARP inhibitor (PARPi). mRNA was converted to cDNA using RT-PCR, and then analyzed by qPCR. DR5 mRNA was validated as a HuR target with a 6-fold greater binding to HuR compared to the control. Strikingly, this binding increases 12- and 24-fold upon treatment with gemcitabine and the PARPi respectively. Silencing HuR expression, through siRNA transfections, leads to an increase of DR5 protein expression at 24 and 48 hours in multiple PDA cell lines. Additionally, silencing of HuR significantly enhances the action of a DR5-specific monoclonal Ab (0.8 mg/mL) against PDA cells within 36 hours (a 20% detected increase in cell death compared to control cells), most likely due to an enhanced availability of the DR5 receptor. Finally, in a training set of PDA clinical specimens, we found a significant inverse correlation between high/low HuR cytoplasmic expression and low/high DR5 levels (p value=0.03). In over 80% (26 of 31) of the specimens HuR cytoplasmic levels inversely correlated with DR5 expression levels, providing further evidence that elevated cytoplasmic HuR is repressing DR5 protein levels in patient tumor cells. In sum, we have shown that ‘activated HuR’ represses DR5 protein expression in PDA cells. Therefore, we conclude that low cytoplasmic HuR levels allow for greater availability of the target DR5, and will thus accordingly enhance the efficacy of DR5-targeted therapy. Thus, manipulating and/or utilizing HuR expression levels may serve as a clinically informative tool for optimizing DR5-targeted therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4100. doi:10.1158/1538-7445.AM2011-4100