Abstract 2854: CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype
Authors: Edwin Cheung, Shruti Lal, Mahsa Zarei, Nicole C. Mambelli-Lisboa, Saswati Chand, Carmella Romeo, Kevin O’Hayer, Eric Londin, Joseph A. Cozzitorto, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody
Journal: Cancer Research (2016)

Abstract Pancreatic ductal adenocarcinoma (PDA) is the most prevalent type of pancreatic cancer and will soon become the second leading cause of cancer related deaths in the U.S. Studies show that the nuclear localized mRNA-binding protein HuR (ELAVL1) is activated in PDA cells, with cytoplasmic translocation associated with increased tumor size and poor prognosis. Previous in vitro and in vivo studies have established HuR's role as a PDA cell survival mechanism. Thus, we explored the phenotypic effect of completely eliminating HuR expression from PDA cells through the use of clustered, regularly interspaced, short palindromic repeat (CRISPR)/Cas9 technology to target and disrupt the HuR genomic sequence. Since INDELs are induced randomly, we designed 3 gRNAs to target HuR at different loci. Gene disruption was determined via sequencing and validated through protein and mRNA expression, where homozygous knockouts (HuR−/−) had undetectable HuR expression as compared to wild-type (HuR+/+), heterozygotes (HuR+/−), and CRISPR/Cas9 negative control. Sanger sequencing confirmed homozygous knockouts with a frame shift mutation on both alleles. When HuR knockout cells were exposed to chemotherapeutic stress including mitomycin C, oxaliplatin, and gemcitabine, no HuR expression (nuclear or cytoplasmic) was detected via immunofluorescence. Phenotypically, HuR−/− cells resulted in increased apoptosis and necrosis as measured via trypan blue assay, and accordingly, had increased caspase 3 activity, a marker of a cell death. HuR−/− cells, when treated with mitomycin C, oxaliplatin, gemcitabine, and glucose deprivation exhibited decreased long and short-term cell survival as compared to control cells. HuR−/− cells, pulse-labeled with bromodeoxyurdine (BrdU), had a higher proportion of cells in S phase and fewer cells in G2/M phase. HuR deletion enhanced premature mitotic entry thereby preventing efficient repair of DNA damage, leading to cell death. Importantly, CRISPR knockout of HuR showed marked impairment in tumor growth in mouse xenografts. The differences in median tumor volume with HuR−/- xenografts was significant as compared to xenografts in mice with HuR(+/+) cells (0.0 mm3 vs 378.0 mm3, P &amp;lt; 0.005). Taken together with our past work in patient samples, this pre-clinical model demonstrates that HuR is essential for PDA growth in vivo. Future work will develop strategies to target HuR either as a monotherapy or in combination with other chemotherapies. Citation Format: Edwin Cheung, Shruti Lal, Mahsa Zarei, Nicole C. Mambelli-Lisboa, Saswati Chand, Carmella Romeo, Kevin O’Hayer, Eric Londin, Joseph A. Cozzitorto, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. CRISPR knockout of HuR in pancreatic cancer cells causes a xenograft lethal phenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2854.