Discovery of a Novel Driver of Radioresistance in Pancreatic Cancer (PC) Using Genome-Wide RNA-Seq

Abstract

<h3>Purpose/Objective(s)</h3> Discovery of genetic drivers of radioresistance in PC is critical for developing novel therapeutic strategies to combine with radiation in this radioresistant disease. In this study, we used genome-wide RNA-seq to identify genes upregulated in radioresistant (RR) generated PC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a promising radiosensitizing target. <h3>Materials/Methods</h3> RR PC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) <i>in vitro</i> over two weeks (total 20 Gy) followed by standard clonogenic assays to calculate the dose enhancement ratio (DER) following one week from the end of IR. Parental and RR cell lines were submitted for RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculate differential expression among genes. Log2 fold change values were calculated for each sample compared to control. Genes with an absolute fold change > 1 were considered significant. Knockdown of target genes via small interfering RNA (siRNA) was confirmed through immunoblotting. Luciferase expressing RR PANC-1 and MIA PaCa-2 with or without shRNA stable knockdown of ID1 were implanted orthotopically in the pancreas of athymic nude mice, and tumors were treated with external beam radiation using a Small Animal Radiation Research Platform to a dose of 20 Gy in 5 daily fractions. Tumor growth was measured weekly via bioluminescence. <h3>Results</h3> Following exposure to two weeks of 2 Gy daily IR <i>in vitro</i>, the two PC cell lines showed significantly greater clonogenic cell survival than parental cell lines (DER 0.4 and 0.5), indicating enhanced RR in these cells. RNA-seq analysis comparing parental and RR cell lines found upregulated seven genes (TNS4, ZDHHC8P1, APLNR, AQP3, SPP1, ID1, ID2) and seven genes downregulated (PTX3, ITGB2, EPS8L1, ALDH1L2, KCNT2, ARHGAP9, IFI16) in both RR cell lines. Western blotting confirmed either increased or decreased expression of these 14 genes in the RR cell lines compared to their parental. siRNA knockdown of each of the seven most significant upregulated genes in the RR MIA PaCa-2 and PANC-1 cells revealed ID1 as the most potent radiosensitizer in the RR cell lines (DER 1.5 and 1.6, respectively). ID1 protein expression was higher in 6 different human PC cell lines than both non-malignant pancreatic and intestinal cell lines. Orthotopic RR tumors with stable knockdown of ID1 showed significantly decreased tumor growth following <i>in vivo IR</i> than the wild-type ID1 tumors (2.5-fold decrease, p<0.001). After the last dose of IR, expression of the DNA damage marker yH2aX in tumors was significantly higher in the ID1 knockdown tumors compared to controls indicating reduced DNA repair in tumors deficient in ID1. <h3>Conclusion</h3> Our analysis indicates a novel role of ID1 in PC radioresistance. Targeting ID1 prior to IR is a promising strategy to overcome the RR of PC, and pre-clinical studies using ID1 inhibitors are ongoing.