Abstract
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. In this study, AR-V7 expression was assessed in an isogenic model of enzalutamide resistance. The model consisted of age matched control cells and two sub-line clones 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. Overall, circRNAs were more often down regulated in resistant cell lines compared with control (588 vs. 278). Of particular interest was hsa_circ_0004870, which was down-regulated in enzalutamide resistant cells (p ≤ 0.05, vs. sensitive cells), decreased in cells that highly express AR (p ≤ 0.01, vs. AR negative), and decreased 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). 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.
Highlights
Prostate cancer (PCa) is the second leading cause of male cancer mortality in Western Europe and the United States[1]
They typically comprise of one to several coding exons of otherwise linear messenger RNAs and range between a few hundreds and thousands of nucleotides in length[22]. Their high abundance, stability and evolutionary conservation between species suggest that they may have an important biological regulatory role19. circular RNA (circRNA) have been identified in a number of cancers including PCa23, suggesting a potential role as a biomarker or therapeutic target. Their role in cancer has yet to be fully elucidated, recent research suggests they can bind RNA-binding proteins (RBPs), translate peptides[24] and confer resistance to therapy25. miRNAs have previously been shown to affect a wide array of biological processes and have an important role in regulating gene expression in cancer, where they act through downstream tumour-suppressive mRNAs26
While it is known that the resistant cell lines used in this study harbour increased F876L, which is an agonist-switch mutation resulting in increased resistance to enzalutamide[12], there is no information on it’s association with androgen receptor (AR)-V7 levels
Summary
Prostate cancer (PCa) is the second leading cause of male cancer mortality in Western Europe and the United States[1]. CircRNAs are RNA molecules with covalently joined 3′- and 5′- ends formed by back-splice events, presenting as closed continuous loops, which makes them highly stable[20,21] They typically comprise of one to several coding exons of otherwise linear messenger RNAs (mRNAs) and range between a few hundreds and thousands of nucleotides in length[22]. CircRNAs have been identified in a number of cancers including PCa23, suggesting a potential role as a biomarker or therapeutic target Their role in cancer has yet to be fully elucidated, recent research suggests they can bind RNA-binding proteins (RBPs), translate peptides[24] and confer resistance to therapy. The goal of this study was to determine if circRNAs were differentially expressed in enzalutamide resistant cells, and to examine the circRNA-mRNA network involved in the development of drug resistance
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