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

Purpose/Objective(s): Discovery of genetic drivers of radioresistance is critical for developing novel therapeuticstrategies to combine with radiotherapy of radioresistant PDAC. In this study, we used genome-wide RNA-seq toidentify genes upregulated in generated radioresistant PDAC cell lines and discovered the Inhibitor of DNA Binding1 (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 bydelivering daily ionizing irradiation (IR) (2 Gy/day) in vitro over two weeks (total 20 Gy) followed by standardclonogenic assays following one week from the end of IR. The generated RR and parental cell lines were submittedfor RNA-seq analysis to identify differentially expressed genes. The Limma R package was used to calculatedifferential expression among genes. Log2 fold change values were calculated for each sample compared to thecontrol. Genes with an absolute fold change > 1 were considered significant. RNA sequencing expression data fromthe Cancer Genome Atlas (TCGA) database was analyzed through the online databases GEPIA, cBioPortal, and theHuman Protein Atlas.Results: Following exposure to two weeks of 2 Gy daily IR in vitro, the two PDAC cell lines showed significantlygreater clonogenic cell survival than their parental cell lines, indicating enhanced RR in these cells. RNA-seqanalysis 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 bothRR cell lines. Western blotting confirmed increased expression of the ID1 protein in the RR cell lines compared totheir parental cell lines. We found that ID1 mRNA was significantly higher in PDAC tumors compared to matchednormal 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, aknown driver of radioresistance.Conclusion: Our analysis indicates a novel role of ID1 in PDAC radioresistance. ID1 expression is higher in tumortissue compared to normal, and high expression correlates with both worse DFS and association with the TP53mutation, suggesting that targeting ID1 prior to IR is an attractive strategy for overcoming radioresistance in PDAC.