Endocrine Abstracts

Prostate cancer (PCa) is a complex and heterogeneous cancer that progresses from slow-growing, tissue-confined lesions, to highly aggressive and metastatic forms. Among the different processes involved in this progression, dysregulation of the alternative splicing mechanism, and, particularly, the generation of the spliced androgen receptor variant-7 (ARv7), plays a critical role in the pharmacological resistance of PCa patients (i.e. Abiraterone or Enzalutamide). In this context, recent studies have shown that Pladienolide-B, an inhibitor of the spliceosome (the molecular machinery that conducts alternative splicing), exhibits important anti-tumor effects in different cancer types; although, its role in PCa remains unknown. Hence, we aimed to determine the direct effects of Pladienolide-B in PCa cell-lines (LNCaP, 22Rv1, DU145, PC-3) and in normal prostate cells (RWPE-1 cell-line and primary cell-cultures obtained from cystoprostatectomies) by analysing different functional parameters such as cell proliferation, cell migration, tumorosphere formation and/or colony formation. Moreover, the expression of 45 splicing machinery components (major and minor spliceosome and splicing factors) was determined in the normal (RWPE-1) and tumoral (LNCaP/22Rv1/PC-3) prostate cell-lines in response to Pladienolide-B treatment by using a microfluidic-based qPCR array. Our results revealed that Pladienolide-B was able to reduce the proliferation of PCa cell lines, at 24-, 48- and 72-h in a dose-dependent manner (100 nM–0.01 nM), being this inhibitory effect significantly greater in PCa cell-lines compared to normal prostate cells (RWPE-1 and primary cell-cultures) at a dose of 100 nM. Interestingly, the antitumoral capacity of Pladienolide-B was corroborated by the inhibition of cell migration and tumorospheres/colonies formation in all PCa cell lines tested. Moreover, treatment with Pladienolide-B dramatically reduced the expression of proliferation markers (Ki67 and PTTG), EMT markers (Vimentin) and markers of PCa agressiveness (PCA3, androgen-receptor and ARv7 splicing variant) in PCa cell lines. Finally, Pladienolide-B was able to markedly alter the expression of numerous spliceosome components and splicing factors, some of them associated to higher PCa aggressiveness, such as SFPQ, KHDRSB1, SRRM4, NOVA1, ESRP1 and ESRP2. Taken together, our results demonstrate that Pladienolide-B clearly reduces PCa aggressiveness features in vitro, by the dysregulation of the expression of several splicing factors and tumor markers, suggesting a potential novel therapeutic role of this compound in PCa.