Fig 2: MYBL2 promotes castration-resistance and bone-metastasis of PCa by activating RhoA and YAP signaling. (A) For the RhoA activity assay, cells were immunoprecipitated with anti-active RhoA antibody and then immunoblotted with anti-RhoA antibody. A portion of the cell lysate (1/30) was also directly immunoblotted with anti-RhoA antibody. Western blotting analysis of the indicated protein expression in the indicated groups of LNCaP cells. a-tubulin was used as a loading control of total protein; P84 was used as a loading control of nuclear protein. Sim, Simvastatin, 5 µM, 24 h. (B) Left panel: Expression and subcellular localization of YAP1 was analyzed in the indicated groups of LNCaP cells with Simvastatin treatment and visualized by fluorescence and laser confocal microscopy. Scale bars: 20 µm. Right panel: Percentage of the subcellular localization of YAP1 in the indicated cells (Nucleus (N); Cytoplasm (C)). (C) Luciferase activity of the YAP/TAZ-TEAD promoter reporter was examined in the indicated groups of LNCaP cells. Simvastatin (5 µM, 24 h); Ver, Verteporfin (10 µM, 3 h). (D) QRT-PCR analysis of YAP1-targeted genes mRNA expression in the indicated groups of LNCaP cells. Gene expression levels were normalized to GAPDH. (E) Representative images (left panel) and quantification (right panel) of colonies formed by the indicated cells with Simvastatin or Verteporfin treatment. (F) Apoptosis assessed in the indicated cells treated in medium supplemented with 0.5% CSS for 4 days and subsequently treated with Simvastatin or Verteporfin. (G) Tumor growth curves of the indicated LNCaP xenograft tumors (n = 8/group). Mice were administered with intraperitoneal injection of Simvastatin (5 mg/kg, five days a week) or Verteporfin (100 mg/kg, every two days). (H) Quantification of xenograft tumor weight at the end of the experiment shown in (F). (I) Growth curve for bone metastasis burden as quantified by BLI in the indicated groups of MDA PCa 2b cells. (J) Numbers of metastatic lesions in bones from each group. (K) Kaplan-Meier curves of bone metastasis-free survival (BMFS) and overall survival (OS) in mice inoculated with the indicated groups of MDA PCa 2b cells (log-rank test). In (C-F, H and J), P-values were determined by one-way ANOVA test. In (G), P-values were determined by two-way ANOVA test. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: MYBL2 confers resistance to androgen-deprivation treatment in PCa cells in vitro. (A and B) Western blotting analysis of MYBL2 expression in the indicated cell lines. α-Tubulin was used as a loading control. (C) Cell viability was assessed in the indicated cells, cultured either in medium with fetal bovine serum (FBS) or with charcoal-stripped serum (CSS), every day for 5 days after seeding (two-way ANOVA test). (D) Representative images (left panel) and quantification (right panel) of colonies formed by the indicated cell lines cultured either in medium with FBS or CSS. (E) Apoptosis assessed by flow cytometry using Annexin V-FITC/PI staining in the indicated cells treated in medium supplemented with 0.5% CSS for 4 days. In (D-E), P-values were determined by two-tailed student's t test when comparing two groups and one-way ANOVA test when comparing three groups. (F) Western blotting analysis of the indicated protein expression in the indicated cells cultured in medium with CSS. α-tubulin was used as a loading control. Data were presented as mean ± SD, **P < 0.01, ***P < 0.001.

Fig 4: Silencing of MYBL2 suppresses castration-resistant growth and bone metastatic capacity of CRPC cells. (A) Schematic illustration of in vivo models of castration therapy performed in the mice bearing tumor xenografts formed by LNCaP-AI/MYBL2-Ri#1-DOX cells, followed by treatment with vehicle or doxycycline (DOX). (B) Tumor growth curves of the indicated xenograft tumors (n = 8/group) during the experiments shown in (A) (two-way ANOVA test). (C) Quantification of the xenograft tumor weight at the end of the experiments shown in (A). (D) Representative images (left panel) and quantification (right panel) of IHC staining of MYBL2 and Ki67, and TUNEL analysis of apoptotic cells in the xenografts tissues. Scale bars: 50 µm. (E) Representative BLI, Micro-CT, and histological images (H&E) of bone lesions from mice inoculated intracardially with PC-3/Ri-vector or PC-3/MYBL2-Ri#1 cells in each experimental group (n = 8/group). T, tumor tissues; N, normal tissues. Scale bars: 400 µm. Quantification of hind-limb osteolysis in each experimental group using Micro-CT analysis (right upper panel). Histomorphometric quantification of the tumor area in hind limbs from each experimental group (right lower panel). In (C-E), P-values were determined by two-tailed student's t test. (F) The growth curve for bone metastasis burden as quantified by BLI in each group (two-way ANOVA test). (G) Numbers of metastatic lesions in bones from each mouse in two groups (Student's t test). (H) Kaplan-Meier curves of bone metastasis-free survival (BMFS) and overall survival (OS) in mice inoculated with PC-3/Ri-vector or PC-3/MYBL2-silenced cells (log-rank test). Data were presented as mean ± SD. *P < 0.05, ***P < 0.001.

Fig 5: Upregulation of MYBL2 contributes to the development of CRPC. (A-C) MYBL2 mRNA levels in PCa tissues were assessed by analyzing the GSE21032 (A), GSE10645 (B), and GSE35988 (C) PCa mRNA datasets (NED control: No evidence of disease, PSA control: PSA recurrence but no evidence of clinical progression within 5 years, SYS: Systemic progression within 5 years after PSA recurrence, CRPC: Castration-resistant prostate cancer). (D) QRT-PCR analysis of MYBL2 expression in the indicated cell lines. GAPDH was used an internal control. In (A-D), P-values were determined by one-way ANOVA test. (E) Western blotting analysis of MYBL2 expression in the indicated cell lines. a-Tubulin was used as a loading control. (F) Left panel: Representative IHC images of MYBL2 in benign prostate tissues (n = 21), primary tissues (n = 132), and CRPC tissues (n = 34). Scale bars: 50 µm. Right panel: Statistical quantification of the MYBL2 IHC score (one-way ANOVA test). (G) Kaplan-Meier analysis of disease-free survival (DFS) curves for patients with PCa with low MYBL2 expression versus high MYBL2 expression (log-rank test, n = 132). (H) Multivariate analysis of DFS by Cox-regression analysis in patients with PCa (n = 132 cases). Data were presented as mean ± SD. *P < 0.05, ***P < 0.001, n.s, no significance.