BioProject

Background: Most forms of castration-resistant prostate cancer (CRPC) are dependent on the androgen receptor (AR) for survival. While, enzalutamide provides a substantial survival benefit, it is not curative and many patients develop resistance to therapy. Although not yet fully understood, resistance can develop through a number of mechanisms, such as AR copy number gain, the generation of splice variants such as AR-V7 and mutations within the ligand binding domain (LBD) of the AR. Circular RNAs (circRNAs) are a novel type of non-coding RNA, which can regulate the function of miRNA, and may play a key role in the development of drug resistance. CircRNAs are highly resistant to degradation, are detectable in plasma and, therefore may serve a role as clinical biomarkers. Methods: AR-V7 expression was assessed in an isogenic model of enzalutamide resistance. The model consisted of age matched control cells and two sub-lines displaying varied resistance to enzalutamide. circRNA profiling was performed on the panel using a high throughout microarray assay. Bioinformatic analysis identified a number of differentially expressed circRNAs and predicted five miRNA binding sites for each circRNA. miRNAs were stratified based on known associations with prostate cancer, and targets were validated using qPCR. Results: Overall, circRNAs were more often down regulated in resistant cell lines compared with control (588 vs. 278). Of particular interest was hsa_circ_0004870 (Probe ID ASCRP3009662), which was down-regulated in enzalutamide resistant cells (p≤0.05, vs. sensitive cells), decreased in AR positive cells (p≤0.01, vs. AR negative), and in malignant cells (p≤0.01, vs. benign). The associated parental gene was identified as RBM39, a member of the U2AF65 family of proteins. Both genes were down-regulated in resistant cells (p<0.05, vs. sensitive cells). Conclusion: This is one of the first studies to profile and demonstrate discrete circRNA expression patterns in an enzalutamide resistant cell line model of prostate cancer. Our data suggests that hsa_circ_0004870, through RBM39, may play a critical role in the development of enzalutamide resistance in CRPC. Overall design: The isogenic enzalutamide resistance LNCaP model was gifted from Novartis (13). The panel consisted of an aged match control cell line (drug sensitive), and two sub-lines termed clone 1 and clone 9. Clone 1 was most resistance to the drug, with clone 9 displaying moderate resistance. Cells were cultured in RPMI-1640 media (Merck KGaA, Darmstadt, Germany) with 10% FBS (Merck KGaA) and 1% Penicillin Streptomycin (Merck KGaA). DU145 were cultured in ATCC-formulated Eagle's Minimum Essential Medium (Merck KGaA) with 10% FBS and 1% Penicillin Streptomycin and PC-3 were cultured in ATCC-formulated F-12K Medium containing 10% FBS and 1% Penicillin Streptomycin (Thermo Fisher Scientific, CA, US).