Abstract 628: A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells.
Authors: Lal Shruti , Burkhart Richard A. , Norris Zoe , Beeharry Neil , Bhattacharjee Vikram , Rigoutsos Isidore , Yen Timothy , Yeo Charles J. , Winter Jordan M. , Brody Jonathan R. 
Published: August 2014

Introduction: We explored HuR's (an RNA binding protein) significance in the DNA damage response (DDR) in pancreatic ductal adenocarcinoma (PDA) cells.

Experimental Procedures: We treated PDA cells with DNA damaging agent's mitomycin C (MMC), oxaliplatin, cisplatin, carboplatin, gemcitabine and a PARP inhibitor (ABT-888). HuR subcellular localization was assessed by immunoblotting. Additionally, we measured γH2AX foci, a marker of DNA damage, by immunofluorescence. Drug sensitivity assays were performed in isogenic PDA cells that differed in HuR expression (siRNA oligos against HuR, cDNAs encoding HuR for overexpression, and appropriate controls). A BrdU/PI pulse chase experiment monitored the cell cycle progression after treating cells with low (150nM) and high (1μM) doses of MMC. Ribonucleoprotein-immunoprecipitation (RIP) assays were used to isolate mRNAs bound to HuR to identify HuR targets in the DDR.

Results: Enhanced-HuR cytoplasmic protein expression was observed in PDA cells exposed to all DNA damaging agents. Under high doses of certain DNA damaging agents (e.g., MMC) HuR was cleaved in a caspase-dependent manner. To assess HuR's role in the DDR, isogenic PDA cells lines manipulated with HuR levels were treated with MMC (150nM) for 2 hrs. γH2AX foci assay revealed a 3-fold increase in foci number with MMC treatment in the HuR depleted cells, as compared to the control cells, suggesting that DNA repair was delayed in the absence of HuR. Cell cycle analyses demonstrated that HuR-depleted PDA cells progressed through the G2/M checkpoint and into mitosis upon MMC treatment. A combinatorial approach with RIP-sequencing and an siRNA library screen for genes resistant to DNA damaging agents was performed, and WEE1, a mitotic inhibitor kinase, was identified as a novel HuR target. A separate RIP-qPCR experiment validated WEE1 as an HuR binder with 56-fold more expression upon MMC stress, compared to an IgG control-IP. WEE1 protein expression and Cdk1 (a target of the WEE1 kinase and G2/M phase regulator) phosphorylation was reduced in HuR depleted cells, validating HuR as an upstream post-transcriptional regulator of WEE1 and an indirect inhibitor of the cdk1-cyclin complex in the face of DNA damage.

Conclusions: We demonstrate that HuR-deficiency allows PDA cells to enter the cell cycle more efficiently upon DNA damage. This event is dependent on HuR's regulation of WEE1 leading to activation of the CDK1-cyclin complex. These data support a model where HuR is integral to the G2/M checkpoint, forcing a cell cycle recovery response which is critical for PDA cells to avoid DNA damage. Therefore, HuR promotes survival of PDA cells through its acute activation of the DDR pathway. Disruption of HuR's molecular interaction with WEE1 represents a therapeutic opportunity to enhance clinically utilized chemotherapeutic DNA damaging agents for PDA.

Citation Format: Shruti Lal, Richard A. Burkhart, Zoe Norris, Neil Beeharry, Vikram Bhattacharjee, Isidore Rigoutsos, Timothy Yen, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. A novel chemoresistance mechanism: HuR post-trancriptionally regulates WEE1, the mitotic inhibitor, upon DNA damage in pancreatic adenocarcinoma cells. [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 628. doi:10.1158/1538-7445.AM2013-628