CRISPR Knockout of the HuR Gene Causes a Xenograft Lethal Phenotype

Abstract

Pancreatic ductal adenocarcinoma (PDA) is the third leading cause of cancer related deaths in the U.S., while colorectal cancer (CRC) is the third most common cancer. The RNA binding protein HuR (ELAVL1), supports a pro-oncogenic network in gastrointestinal (GI) cancer cells through enhanced HuR expression. Using a publically available database, HuR expression levels were determined to be increased in primary PDA and CRC tumor cohorts as compared to normal pancreas and colon tissues, respectively. CRISPR/Cas9 technology was successfully used to delete the HuR gene in both PDA (MIA PaCa-2 and Hs 766T) and CRC (HCT116) cell lines. HuR deficiency has a mild phenotype, in vitro, as HuR-deficient MIA PaCa-2 (MIA.HuR-KO(−/−)) cells had increased apoptosis when compared to isogenic wild-type (MIA.HuR-WT(+/+)) cells. Using this isogenic system, mRNAs were identified that specifically bound to HuR and were required for transforming a 2D culture into 3D (i.e., organoids). Importantly, HuR-deficient MIA PaCa-2 and Hs 766T cells were unable to engraft tumors in vivo compared to control HuR-proficient cells, demonstrating a unique xenograft lethal phenotype. While not as a dramatic phenotype, CRISPR knockout HuR HCT116 colon cancer cells (HCT.HuR-KO(−/−)) showed significantly reduced in vivo tumor growth compared to controls (HCT.HuR-WT(+/+)). Finally, HuR deletion affects KRAS activity and controls a subset of pro-oncogenic genes. Implications The work reported here supports the notion that targeting HuR is a promising therapeutic strategy to treat GI malignancies.

Complex HuR function in pancreatic cancer cells

“…These data support our previous published work that inducible knockdown of HuR using short hairpin RNA-based silencing in PDAC cells in vivo can reduce the tumor size by roughly fourfold (Jimbo et al, 2015). Similarly, we recently demonstrated the efficacy of targeting HuR in GI cancer in vivo by using small molecule inhibitors of HuR function Lal et al, 2017).…”

Posttranscriptional Upregulation of IDH1 by HuR Establishes a Powerful Survival Phenotype in Pancreatic Cancer Cells

“…We observed in vivo that CRISPR deletion of the HuR gene from PDAC cells [Mia.HuR(−/−)] abrogated xenograft transplantation and uptake in the flanks of nude mice (18), underscoring the profound phenotypic impact of HuR expression on tumor growth in an animal model. In this study, stable reconstitution of IDH1 alone by retroviral transduction in these cells [Mia.HuR(−/−).…”

“…CRISPR/Cas9-mediated knockout of HuR in MiaPaCa2 and HS-766T cells was accomplished using a guide RNA targeting HuR, fused with CRISPR/Cas9 and GFP protein (18). Plasmids were designed and purchased from (Sigma-Aldrich) along with the CRISPR Universal Negative Control plasmid (CRISPR06-1EA).…”

Posttranscriptional Regulation of PARG mRNA by HuR Facilitates DNA Repair and Resistance to PARP Inhibitors

“…Fig. S1B) (22); and 3) a doxycycline inducible siHuR cell line MIA.sh290 (18). Dose response curves from cell survival assays in response to several clinically relevant PARPi indicate that CRISPR-knockout of HuR (Table 1) in both MIA PaCa-2 and Hs 766T [HuR (+/+) vs HuR (−/−)] caused a dramatic 20-fold decrease in sensitivity to the PARPi olaparib (Fig.…”

“…Cells were cultured in standard DMEM media supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine and 1% penicillin-streptomycin (Invitrogen) at 37°C and 5%CO 2 . MIA PaCa-2 and Hs 766T with CRISPR/Cas9 knockout of HuR and MIA PaCa-2 cells with doxycycline inducible silencing of HuR were generated and characterized as previously described (18, 22). …”