br Given that the crucial role of CSCs in recurrence
Given that the crucial role of CSCs in recurrence and aggressive pro-gression of prostate cancer , we further investigated the potential role of CDC20 in regulating prostate CSCs. Many studies showed that CD44 positive 58-60-6 can be recognised as putative prostate CSC subpop-ulation in prostate cancer [13–17], we therefore assessed the relation-ship between CDC20 and CD44 expression in 121 prostate cancer patients using immunohistochemical (IHC) analysis, high levels of CDC20 indeed exhibited elevated CD44 staining in prostate cancer
tissues (Fig. 1e, f), suggesting a positive correlation between CDC20 and CD44 expression. We next explored if co-expression of CDC20 and CD44 exerts aggressive biological behaviors of prostate cancer. All 121 patients were divided into four groups according to the expression level of CDC20 and CD44 in prostate cancer samples. As expected, the patients with CDC20high/CD44high were associated with more aggressive characteristics such as Gleason score, T-stage (Supplementary Table S3), and suffered worse BCR (P = .0063) (Fig. 1g) and inferior DFS (P =
.047) (Fig. 1h), compared with other groups. Taken together, CDC20 may correlate with expansion of prostate CSCs, and co-expression of CDC20 and CD44 could serve as an effective predictor for the prognosis of prostate cancer.
3.2. CDC20 is predominantly enriched in prostate CSCs
Given that CDC20 expression is correlated with CD44 in prostate cancer tissues, whether it is required for maintenance of prostate cancer stem-like cells (CSCs) needed to be addressed. We first established three CSC enrichment model: spheroid cells, CD44+ subpopulation (Supplementary Fig. S1b) and chemo-resistant cell lines (Supplemen-tary Fig. S1c). The expression level of CDC20 in these three CSCs enrich-ment model was determined. CDC20 expression level was higher in spheres from C4–2B and DU145 cells than in their corresponding adher-ent cells, which was similar as some representative stemness associated genes such as CD44, CD133, SOX2, OCT4 (Fig. 2a–c). Next, we examined the CDC20 expression in CD44 positive and negative cells from C4–2B and DU145, as anticipated, CDC20 expression level was higher in CD44 positive cells than that in negative cells, and so as some represen-tative stemness associated genes (Fig. 2d–f). It is acknowledged that chemo-resistant cells are enriched in CSCs , we then detected the CDC20 expression in docetaxel (first-line chemotherapy for metastatic CRPC patients) resistant and control cells from C4–2B and DU145, CDC20 expression level was higher in docetaxel resistant cells than that in control cells, which is analogous to some representative stemness associated genes (Fig. 2g–i), and this founding was accor-dance to the previous studies [32,33]. In conclusion, CDC20 is predomi-nantly enriched in prostate CSCs, suggesting a putative role of CDC20 in the maintenance of prostate CSCs.
3.3. CDC20 is required to maintain stem cell-like features of CD44+ prostate CSCs
To further investigate the functional role of CDC20 in regulating the stem-like properties of CD44+ prostate CSCs, we establish Lv-shCDC20-stable transfectants of prostate cells (C4–2B and DU145) via a lentivirus-based knockdown approach. In addition, a wide-type (WT) or mutant type (MT) with a modified codon of CDC20 escaping from shRNA in Lv-shCDC20-infected prostate cancer cell lines to test if up-regulation of CDC20 could rescue these inhibitory effects. The knockdown and rescue efficiency were verified by western blot (Sup-plementary Fig. S1d). As presented in Fig. 3a, b and Supplementary Fig. S1d, e, CD44+ cells magnetic sorted from Lv-shCDC20 infection C4–2B or DU145 cells exhibited down-regulated mRNA and protein levels of CDC20 and some crucial stemness-related genes (including CD44, SOX2, OCT4, MYC, KLF4). Considering that strengthened self-renewal capacity is one of the most crucial features of CSCs, the spheroid formation and in vitro limiting dilution assays were performed to inves-tigate if CDC20 could affect the self-renewal ability of prostate CSCs. Consistently, both quantity and size of the primary and secondary spheroids of CD44+ prostate cancer cells were decreased due to the knockdown of CDC20 (Fig. 3c, d). Meanwhile, decreased expression of CDC20 led to a lower spheroid initiating frequency (SIF) which further supported that the silence of CDC20 impeded self-renewal ability of CSCs in vitro (Fig. 3e, f and Supplementary Table S4). Since drug resis-tance of CSCs is considered to be involved in chemotherapeutic failure and tumour progression , we next determined whether CDC20
Fig. 1. Up-regulated CDC20 in advanced prostate cancer tissues was associated with poor prognosis, and combination of CDC20 and CD44 expression is a significant prognostic factor in prostate cancer. (a) Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining for CDC20 in prostate cancer tissues ranked by different disease stages as localised (n