locally advanced n docetaxel treated n were
= 83), locally advanced (n = 38), docetaxel treated (n = 8) were presented (scale bar = 50 μm). (b) Expression of CDC20 in different stages was evaluated by IRS score, which was listed in Materials and Methods (P value: Wilcoxon test), values represented as the mean ± SD. (c, d) Kaplan-Meier curves for biochemical recurrence (BCR) (c) and disease-free survival (DFS)
could regulate chemoresistance of prostate CSCs. As shown in Fig. 3g, h, CD44+ CSCs sorted from Lv-shCDC20 infection C4–2B or DU145 cells exhibited more vulnerable to docetaxel treatment as compared to the
controls. More importantly, down-regulation of CDC20 significantly inhibited the invasion and migration ability of CD44+ prostate CSCs as compared to control (Supplementary Fig. 1f, g), while no significantly
Fig. 2. CDC20 is predominantly enriched in prostate CSCs. (a–c) The mRNA expression of a series of stemness related genes (CD44, CD133, SOX2, OCT4) was analysed by quantitative real time PCR (qRT-PCR) in adherent and spheroid prostate cancer cell lines C4–2B (a) and DU145 (b); CDC20 mRNA and protein expression were compared between adherent and spheroid cells using qRT-PCR (a, b) and western blot (c) (P value: Wilcoxon test). (d–f) The mRNA expression of a series of stemness related genes (CD44, CD133, SOX2, OCT4) was analysed by qRT-PCR in prostate cancer cell spheroids derived from sorted CD44− and CD44+ of C4–2B (d) and DU145 (e); CDC20 mRNA and protein expression were compared between CD44− and CD44+ cells using qRT-PCR (d, e) and western blot (f) (P value: Wilcoxon test). (g–i) The mRNA expression of a series of stemness related genes (CD44, CD133, SOX2, OCT4) was analysed by qRT-PCR in prostate cancer cell derived from control and docetaxel resistant C4–2B (g) and DU145 (h); CDC20 mRNA and protein expression were compared between control and docetaxel resistant cells (DTX-R) using qRT-PCR (g, h) and western blot (i) (P value: Wilcoxon test). The results were collected from three independent experiments and all data represent Mean ± SD. All p values were defined as: *p b .05, **p b .01 and ***p b .001.
difference was found in Prostaglandin-E2 distribution between the control and Lv-shCDC20 groups (Supplementary Fig. 1h). Previous studies reported that CD44+ prostate cancer cells exhibited superior tumour initiating rate in vivo . To evaluate whether CDC20 could manipulate tumorigenicity in vivo of CD44+ CSCs subset, xeno-graft assay was performed by inoculation of several gradient CD44+ cells sorted from CD44-Lv-shCon or Lv-shCDC20 cells. As shown in Fig. 3i and Supplementary Table S5, 1 × 104 CD44+ Lv-shCon C4–2B cells efficiently initiated larger tumours in 66.7% (4/6) of mice, while the equal numbers of CD44+/Lv-shCDC20 C4–2B cells produced only 1 tumour at 7 weeks after injection among 6 injected nude mice. Consis-tently, only 1 × 103 CD44+/Lv-shCon cells could generate tumours in 1 of 6 injected mice, whereas 1 × 103 CD44+ cells with CDC20 knokdown failed to do so at 7 weeks after transplantation, and IHC assay showed a weakened expression of CDC20 and CD44 in 1 × 104 Lv-shCDC20 cell
group (Fig. 3j), indicating that knockdown of CDC20 attenuated the CD44+ fractions-driven tumorigenicity in vivo. In conclusion, our data suggested CDC20 is required to maintain stem cell-like features of CD44+ prostate CSCs, and may take a part in promoting the tumorige-nicity of CD44+ prostate CSCs during prostate cancer progression.
3.4. CDC20 activates β-catenin signaling via collapsing the destructive com-plex and promoting β-catenin nuclear translocation and transactivation
To examine the underlying mechanism by which CDC20 maintains the stem-like properties of CD44+ prostate CSCs, RNA-sequence was employed to detect differentially expressed genes in CDC20 knockdown CD44+ C4–2B cells compared to that in control cells, a total of 1600 dif-ferentially expressed genes were identified using a 2-fold cut-off with a P-value b.05 (Supplementary Fig. S2a and Supplementary Table S6).
The first top 60 differentially up- and down-regulated genes were shown and we noticed that many stemness related genes or β-catenin/TCF4 target genes exhibited a decreased expression in Lv-shCDC20 CD44+ C4–2B cells in Supplementary Fig. S2b. Consistently, the pathway analysis showed that transcriptional regulation of pluripo-tent stem cells and β-catenin: TCF complex related pathway was differ-entially regulated in Lv-shCDC20 CD44+ C4–2B cells (Fig. 4a and Supplementary Table S7) as well as gene ontology (GO) analysis (Sup-plementary Fig. S2c and Supplementary Table S8). Interestingly, qRT-PCR assay revealed that a set of important β-catenin target genes such as c-Jun, c-Myc, cyclin D1, TCF4 and MMP7 were down-regulated in CDC20 knockdown CD44+ C4–2B cells, whereas overexpression of CDC20 could recue these inhibitory effectors in mRNA levels (Fig. 4b and Supplementary Fig. S2d). Next, we investigated whether β-catenin took a crucial part in the CDC20-regulated stem-like properties of CD44+ prostate CSCs. The expression of stem-related genes was higher in CD44+ prostate CSCs with CDC20 overexpression compared with the control CD44+ prostate CSCs by qRT-PCR, knockdown of β-catenin (Supplementary Fig. S2e) repressed this effect (Fig. 4c and Sup-plementary Fig. S2f). Meanwhile, spheroid formation assay indicated that CDC20 reinforced the self-renewal of CD44+ prostate CSCs, deple-tion of β-catenin abolished this effect of CDC20 (Fig. 4d and Supplemen-tary Fig. S2g). These results suggested that β-catenin is indispensable for the CDC20-dependent stem-like properties of CD44+ prostate CSCs.