Integrative transcriptional analysis of enzalutamide-resistant castration resistant prostate cancer cells using RNA sequencing.

Abstract

284 Background: Enzalutamide (ENZ)-resistance is a clinically challaengeable issue for management of castration-resistant prostate cancer (CRPC) patients. Here, we aimed to investigate the distinct features of transcriptional expression in ENZ-resistant CRPC cells using RNA sequencing, focusing on the histone modification enzymes as AR co-regulators. Methods: We chronically treated various concentration of ENZ (1 to 40 µM) for 6 to 9 months to establish ENZ-resistant CRPC cells by using C4-2B cells. To explore the phenotypic features of ENZ-resistant CRPC cells (ENZ-R), we analyzed the gene expression patterns, particularly AR target genes, following ENZ treatment. We performed cell viability assay, clonogenic assay, apotosis assay and tumor sphere formation assay. To examine the distinct features of transcriptional expression in ENZ-resistant CRPC cells, we conducted RNA sequencing and classified the key differential expression genes (DEG) according to Gene Ontology Database. Results: ENZ-R cells showed a higher cell viability and clonogenic proliferation compared to control cells following ENZ treatment. Also, ENZ-R cells showed higher gene expression of AR target gens (KLK2, KLK3, TTMPRSS) than those in control cells after dihydrotestosterone treatment. Of note, ENZ-R cells significantly expressed full length AR as well as AR splicing variant 7. Moreover, RNA sequencing revealed that 367 up-regulated genes and 223 down-regulated genes were identified as DEG compared to control cells. We further analyzed these DEG of histone modification enzyme as AR co-regulators, and therefore; we finally determined the candidate 28 target genes of histone modification enzyme (23 up-regulated and 5 down-regulated genes). We validated the differential expression of these key histone modification enzyme genes in ENZ-R cells by using real-time PCR. Conclusions: In sum, we established the ENZ-R cells and identified the 28 candidate DEG of histone modification enzyme in ENZ-R cells by performing RNA sequencing analysis. Our study can offer the valuable information for exploring the ENZ-resistant mechanisms, particularly focusing on the imbalance of AR co-regulators.