Discovery of the inhibitor of DNA binding 1 as a novel marker for radioresistance in pancreatic cancer using genome-wide RNA-seq.

Abstract

Purpose/Objective(s): Discovery of genetic drivers of radioresistance is critical for developing novel therapeutic strategies to combine with radiotherapy of radioresistant PDAC. In this study, we used genome-wide RNA-seq to identify genes upregulated in generated radioresistant PDAC cell lines and discovered the Inhibitor of DNA Binding 1 (ID1) gene as a potential regulator of radioresistance in PDAC. Materials/Methods: Radioresistant clones of the PDAC cell lines MIA PaCa-2 and PANC-1 were generated by delivering daily ionizing irradiation (IR) (2 Gy/day) in vitro over two weeks (total 20 Gy) followed by standard clonogenic assays following one week from the end of IR. The generated RR and parental 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 the control. Genes with an absolute fold change > 1 were considered significant. RNA sequencing expression data from the Cancer Genome Atlas (TCGA) database was analyzed through the online databases GEPIA, cBioPortal, and the Human Protein Atlas. Results: Following exposure to two weeks of 2 Gy daily IR in vitro, the two PDAC cell lines showed significantly greater clonogenic cell survival than their parental cell lines, 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 increased expression of the ID1 protein in the RR cell lines compared to their parental cell lines. We found that ID1 mRNA was significantly higher in PDAC tumors compared to matched normal and high ID1 expression correlated with significantly worse disease-free survival (DFS) in PDAC patients (HR = 2.2, log rank P = 0.009). ID1 mRNA expression was also strongly correlated in tumors with TP53 mutation, a known driver of radioresistance. Conclusion: Our analysis indicates a novel role of ID1 in PDAC radioresistance. ID1 expression is higher in tumor tissue compared to normal, and high expression correlates with both worse DFS and association with the TP53 mutation, suggesting that targeting ID1 prior to IR is an attractive strategy for overcoming radioresistance in PDAC.