Genomic and Transcriptomic Profiling of Radioresistant (RR) Cancer Models Revealed Hallmarks of Tumor Aggression

Abstract

<h3>Purpose/Objective(s)</h3> Radiotherapy (RT) is an integral treatment modality of several human cancers, being utilized in 50% of all patients. Resistance to RT is thus an important unmet need, but therapeutic strategies against radioresistance remain suboptimal. This is in part because mechanisms underpinning cancer radioresistance are multifactorial, including DNA repair and microenvironment factors e.g., hypoxia and immune dysregulation. We hypothesized that comprehensive molecular profiling of isogenic wildtype (WT) and radioresistant (RR) cancer models will provide a detailed molecular landscape that is associated with radioresistance. <h3>Materials/Methods</h3> We generated four RR head and neck (HNC) and prostate cancer (PCa) models (FaDu, HK-1, DU145, and 22Rv1) by exposure to 90 Gy of X-irradiation (IR) (45 fr x 2 Gy/fr), and validated with clonogenic assays. Whole exome sequencing (WES) and variant calling using RR-WT cell line pairs were performed to determine mutations that were enriched in RR models. Gene expression was compared between RR and WT pairs at baseline and post-IR (4 Gy at 1, 6, 24, and 48 h). Protein analyses were performed using western blot analyses and immunofluorescence (IF) staining. Pathways were validated by treatment with small molecule inhibitors. <h3>Results</h3> Radioresistance in our RR HNC and PCa cells was confirmed by clonogenic assay (surviving fraction ratio at 2 Gy [SF_2Gy,RR/SF_2Gy,WT] ranged from 1.32 [HK-1] to 1.40 [22Rv1]). Genomic profiling revealed an overall increased tumor mutational burden (TMB) in the RR models relative to the WT cells (TMB range: 6±3 [HK-1] to 387±64 [22Rv1] variants). We observed a significant variation in TMB between the different tumor models, despite the presence of dominant driver mutations in the DNA damage response genes (<i>TP53, BRCA1/2</i>) in the WT cancer cells. Gene expression analyses uncovered two key findings: (1) downregulation of the androgen response pathway in RR tumors; and (2) upregulation of TGFβ- and TNFα-related pathways both at baseline (net enrichment score, NES_TGF-B=1.60 [<i>P</i>_adj=0.045]; NES_TNF-A=1.73 [<i>P</i>_adj=0.0002]) and post-4 Gy IR (NES_TGF-B=1.68 [<i>P</i>_adj=0.014]; NES_TNF-A=1.33 [<i>P</i>_adj=0.049]). In-depth interrogation of the DDR pathways did not reveal significant transcriptional dysregulation between RR and WT tumor-pairs. However, RR HN and PCa cells manifested an enhanced efficiency of end-joining repair of DNA double-strand breaks post-IR compared with their WT pairs, based on western blot and IF; this could have accounted for the RR phenotype, which was reversed following DNA-PKcs inhibition with NU7441. <h3>Conclusion</h3> Herein, we highlighted the molecular complexity of RR tumors generated using a fractionated IR schedule that mimics clinical RT. Upstream mechanistic studies are ongoing to decipher the genome-transcriptome interactions leading to enhanced DNA repair phenotype in our RR tumor models.