Figure 8.

SMAD3 promotes the expression of AR mRNA in human PCa and can be targeted with PROTAC inhibitor. (A) The SMAD3 peak in AR intron 3 overlapped with the H3K27ac peak in human PCa cells. The H3K27ac peaks in AR upstream enhancer are also shown. The indicated datasets of H3K27ac ChIP-seq were download from GEO database and visualized by IGV software. (B) H3K27ac peaks in AR upstream enhancer and AR intron 3 enhancer in a published dataset of 8 CRPC PDX samples. (C) ATAC-seq peaks in AR upstream enhancer and AR intron 3 enhancer in a published dataset of 6 PCa PDX samples. (D) Positive correlation (Pearson correlation, R = 0.29, P-value = 2.9e-11) between AR mRNA and SMAD3 mRNA in the TCGA PCa dataset (n = 492). The correlation analysis was performed using the web server of GEPIA2. (E and F) Upregulation of AR mRNA and SMAD3 mRNA in metastatic PCa (E) or CRPC (F) relative to the primary PCa. The indicated datasets of profiling array studies on human PCa tissues were downloaded from the GEO database. Cai dataset includes 22 primary PCa and 29 metastatic PCa. Chandra dataset includes 207 primary PCa and 13 CRPC. The intensity values of AR and SMAD3 were Log2 transformed, and the mean values of the indicated PCa groups were compared. Quantification was presented as mean ± SD, and t test was used for statistical analysis (**P < 0.01; ***P < 0.001). (G and H) Partial knockdown of SMAD3 reduced the levels of AR mRNA (G) and protein (H) in the enzalutamide-resistant C4-2B cells (EnzR). Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (**P < 0.01; ***P < 0.001). (I and J) A SMAD3 PROTAC inhibitor decreased levels of AR, AR-V7 and AR targets. Rv1 cells were treated with the indicated concentration of SMAD3 PROTAC inhibitors for 24 hours before western blot (I) or real-time RT-PCR analysis (J). Quantification was presented as mean ± SD (n = 3), and ANOVA was used for statistical analysis (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001).