A KRAS-RAD18-miR-296-3p Regulated Network Mediates Radioresistance in Pancreatic Cancer Through Homologous Recombination

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a relatively radioresistant disease, and inhibition of DNA homologous recombination (HR) repair in combination with radiation therapy (RT) is a potentially attractive strategy to overcome radioresistance. We have found that the expression of the HR protein RAD18 is increased upon induction of KRAS mutation and reduced following KRAS suppression. Therefore, we hypothesized targeting RAD18 in KRAS mutant PDAC with its predicted inhibitor, miR-296-3p, will increase radiosensitization. Standard clonogenic assays, γH2aX foci staining, HR-GFP reporter assay, and western blot analysis of DNA damage response proteins was performed in MIA-PaCa2 (MP2) and PANC-1 cells following knockdown of RAD18 in cells via short hairpin RNA (shRNA). Forced expression of miR-296-3p mimics was performed using lipofectamine transfection. Cells with or without stable knockdown of RAD18 were implanted subcutaneously in the hind limb of athymic nude mice and treated with sham radiation or radiation to a dose of 20 Gy in 5 daily fractions once tumors reached 100-150 mm3. Doxycycline treatment of KRAS inducible human pancreatic ductal epithelial (HPDE) cells increased both RAD18 protein and RNA expression. In contrast, knockdown of KRAS by siRNA reduced RAD18 protein and RNA expression in PANC-1 and MP2 cell lines (3.5 and 4.2-fold, respectively). Stable knockdown of RAD18 in MP2 and PANC-1 resulted in decreased radiation clonogenic survival in vitro (dose enhancement factor (DEF) = 1.52 and 1.51, respectively), decreased DNA repair after radiation as measured by increased number of γH2aX nuclear foci assay at 6, 12, and 24 hours (all p<0.05), decreased HR activation following DNA damage via a HR-GFP reporter assay (p = 0.039), and increased tumor growth delay following radiation in vivo (p<0.001). Overexpression of RAD18 resulted in increased radiation clonogenic survival (DEF = 0.88). We identified that miR-296-3p has a conserved target sequence complimentary to the 3’UTR region of the RAD18 mRNA. MiR-296-3p expression via qRT-PCR was absent in a panel of 6 KRAS mutant PDAC cell lines and highly expressed in 2 normal pancreatic cell lines. MiR-296-3p mimic transfection of the high RAD18 expressing PANC-1 and MP2 cells resulted in efficient knockdown of RAD18, which was confirmed through western blotting, qRT-PCR, and luciferase reporter assays. Following the transfection of miR-296-3p mimics into PANC-1 or MP2 cells, radiosensitization increased (DEF = 1.15 and 1.14, respectively), yH2aX nuclear foci remained elevated at 12 and 24 hours (p<0.05), and HR repair was reduced (p = 0.014). Herein, we show that KRAS mutation in PDAC positively regulates expression of the HR repair protein RAD18, and that modulation of RAD18 expression correlates with in vitro and in vivo radiosensitization through altered HR-mediated DNA repair. MiR-296-3p successfully reduced RAD18 expression and resulted in enhanced radiosensitization.