| In the United States, prostate cancer(PC) remains the most commonly diagnosed cancer in the male population, ranking the second most common cause of cancer deaths in men. Androgen deprivation therapy(ADT) through surgical or chemical procedures has been widely used for the treatment of advanced PC. However, even though ADT initially suppresses PC growth through blocking the AR activities,resistance is eventually inevitable following disease evolution into castration-resistant prostate cancer(CRPC). This might be due to the reactivation of AR pathway and several studies have suggested extra-gonadal androgen and PC cell intracrine could still stimulate the AR pathway even at a very low concentration. The resistance to ADT has led to the development of alternative AR blocking agents with the recent development of the second generation of hormonal therapies including enzalutamide.Enzalutamide is a second-generation anti-androgen for castration-resistant prostate cancer(CPRC) therapy. It has achieved a prolonged overall survival of CRPC patients,but the gain in survival is modest(4.8 months) and by 24 months most patients progressed on enzalutamide. However, the molecular mechanisms underlying enzalutamide resistance remain elusive. Recent advance has shown that e RNA plays an important role in tumorigenesis and anticancer drug resistance in several cancers including PC. However, the role and the mechanism of e RNA in the development of CRPC or enzalutamide resistance is unknown. Regarding the important role of AR in the resistance of PC to enzalutamide, in our study, we explored the alterations of AR-regulated e RNAs and m RNAs in response to the treatment of enzalutamide and their contribution to enzalutamide resistance in PC. Herein, we report enzalutamide treatment-induced alterations of androgen receptor(AR)-regulated enhancer RNAs(AR-e RNAs) and their roles in growth and survival and enzalutamide resistance in CRPC cells. AR chromatin immunoprecipitation and high throughput sequencing(Ch IP-seq) and RNA-seq analyses revealed that 188 and 227 AR-e RNAs were differentially expressed in enzalutamide-resistant LNCa P and C4-2 cells, respectively.The AR-e RNAs upregulated in C4-2 cells and downregulated in LNCa P cells were selected by Meta-analysis. The expression of AR-e RNAs and related m RNAs in the loci of FTO, LUZP2, MARC1 and NCAM were further verified by real-time RT-PCR,which showed that the expression of AR-e RNAs and m RNAs in these four loci were upregulated in C4-2 cells and downregulated or remained unchanged in LNCa P cells.The enhancer-promoter interation were confirmed by 3C(chromosome conformation capture) assay in the loci of NCAM2 and MARC1, thus the target m RNAs have been verified. In the functional study, we found that silencing of LUZP2 inhibited the growth of enzalutamide-resistant C4-2 cells, but silencing of MARC1 increased the growth of these cells. Intriguingly, meta-analysis of database from a previously published literature showed that expression of LUZP2 m RNA was significantly higher in primary tumors compared to normal prostate tissues, but was lower in metastatic CRPC compared to primary PC. We confirmed this in PC and CRPC cells well as in PC and CRPC patient tissues by performing real-time RT-PCR. These results suggest LUZP2 might have the potential as a putative therapeutic target with pharmaceutical value for the treatment of both naive PC and Enzalutamide resistant CRPC, and MARC1 could be a potential indicator for drug sensitivity, prognostic factor and disease surveillance marker, which requires further and more detailed study. Our study suggests e RNA alteration profiling is a viable new approach to identify functional gene loci that not only contribute to survival and enzalutamide-resistance of CRPC, but also could serve as new targets for CRPC therapy. |
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