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  • br Concrete evidence has proven that Wnt

    2020-08-30


    Concrete evidence has proven that Wnt/β-catenin signaling was in-volved in carcinogenesis and stem-like feature manipulation of CSCs by activating β-catenin and its nucleus translocation [35]. However, the pool of β-catenin in the cytoplasm is usually withered accounting for degradation by the proteasome via phosphorylated at Ser45 [36]. There-fore, we assessed if CDC20 could maintain β-catenin and promote its
    nuclear localization. Western blot assay showed that the expression level of p-β-cateninSer45 was elevated in cytoplasm of CD44+ prostate CSCs after CDC20 knockdown, meanwhile the expression of β-catenin was down-regulated in nucleus (Fig. 4e–f). Thus, these results sug-gested that CDC20 expression is necessary for the stability of β-catenin in the cytosolic and its nuclear translocation.
    Considering that the degradation of cytoplasmic β-catenin is manip-ulated by the “destructive complex” which comprised of Axin1, APC, and glycogen synthase kinase 3β (GSK-3β) [36], we examined the po-tential link between CDC20 and β-catenin or its destructive complex members. Western blot assay indicated that alternations of CDC20 in prostate cancer 363-24-6 influenced β-catenin and Axin1 expression level but not APC and GSK-3β (Supplementary Fig. S2h,i). We next detected the direct interaction between CDC20 and β-catenin or Axin1, the co-immunoprecipitation assay suggested that CDC20 and Axin1 could bond to each other in CD44+ C4–2B (Fig. 4g) and CD44+ DU145 cells (Supplementary Fig. S2j), while there was no interaction between CDC20 and β-catenin (Supplementary Fig. S2k). Given that CDC20 is an E3 Ubiquitination ligase, we next explored whether knockdown CDC20 could impede the degradation of Axin1. As expected, western blot assay revealed that Axin1 was more stable with CDC20 knockdown under the treatment by cycloheximide (CHX), a protein synthesis inhib-itor (Fig. 4h). Meanwhile, the enhanced maintenance of Axin1 was not detected in the presence of MG132 (a proteasome inhibitor) (Supple-mentary Fig. S2l) Thus, these results indicated that CDC20 could pro-mote Axin1 degradation through a proteasomal-dependent pathway
    rather than by protein synthesis, although the concrete interaction mode between CDC20 and Axin1 needs to be further elucidated. Taken together, CDC20 directly binds with Axin1 and to some extent in-tensified Axin1 degradation activities, which may impede the destruc-tive complex, resulting in enhancement of the stability of β-catenin and its translocation to nucleus.
    3.5. Expression of CDC20 and β-catenin predicts malignant clinicopatholog-ical features and prognosis for prostate cancer patients
    Considering the crucial role of CDC20 and β-catenin in the stem-like features of CD44+ prostate CSCs, we next explored the clinical correla-tion between CDC20 and β-catenin in prostate cancer specimens. IHC analysis was performed in prostate cancer specimens from 121 patients (Fig. 5a), positive correlation was identified between CDC20 and β-catenin expression (p b .001) (Fig. 5b). Next, we evaluated whether the combined expression of CDC20 and β-catenin could serve as a predicter for malignant clinicopathological features and prognosis for prostate cancer patients. According to their tumoral CDC20 and β-catenin expression, all 121 patients were divided into 4 groups (Supple-mentary Table S9), the concomitant high expression of CDC20 and β-catenin was associated with worst clinicopathological features and prognosis for prostate cancer patients (Supplementary Table S9, Fig. 5c, d). Thus, the concomitant over-expression of CDC20 and β-catenin can serve as an effective predictor for unsatisfactory prognosis of prostate cancer patients, further supporting the important role of CDC20 and β-catenin in the progression of prostate CSCs.
    4. Discussion
    Despite recent advances in new drugs for the treatment of metasta-tic prostate cancer, current treatments gain unsatisfied survival benefits due to acquired drug resistance and disease progression [3]. Recent studies have proven that CSCs exert a critical role in tumorigenesis and spread of prostate cancer [34]. Thus, targeted elimination of pros-tate CSCs may be an effective option to inhibit malignant biological be-haviors of prostate cancer. For this purpose, the potential molecular mechanisms by which CDC20 manipulates prostate CSCs require to be elucidated. In this study, we reported that CDC20 is required for mainte-nance of CD44+ prostate CSCs via enhancing Axin1 degradation and promoting β-catenin translocation to nuclear and transactivation (Fig. 5e). To our best knowledge, it is the first report that there was a positive correlation between CDC20 and CD44 expression in clinical prostate cancer specimens. Likely, CDC20 is preferentially expressed in parts of spheroids or CD44+ or chemo-resistant prostate cancer cell lines. In addition, lentiviral-based methods of interfering with CDC20 significantly inhibited CSC self-renewal ability and inhibited CSCs-driven in vivo tumorigenicity. Our further studies have also found that Wnt/β-catenin signaling plays a critical role in the maintenance of pros-tate CSC. Mechanically, knockdown of CDC20 expression impairs nu-clear translocation of β-catenin and impedes its transcriptional activity, thereby attenuating activation and expansion of prostate CSCs. These findings also indicated that CDC20 can be used as a thera-peutic target to eradicate CSCs in prostate cancer management.