Inhibiting CBP Decreases AR Expression and Inhibits Proliferation in Benign Prostate Epithelial Cells

Abstract

(1) Background: CREB-binding protein (CBP) is a key transcriptional coactivator of androgen receptors (AR). We conducted this study to investigate the effects of CBP on AR expression and proliferation in benign prostatic hyperplasia (BPH) prostate epithelial cells. (2) Methods: By analyzing a published data set, we found that CBP was closely related to the gene expression of AR in prostate cells. We enrolled 20 BPH patients who underwent transurethral resection of the prostate (TURP) in Peking University First Hospital in 2022, and analyzed the expressions of CBP and AR in BPH prostate tissues. Then, we used ICG-001 and shRNA to inhibit CBP in prostate epithelial cells (BPH-1 cells and RWPE-1 cells), and conducted immunofluorescence, cell viability assay, flow cytometry analysis, and Western blot to analyze the effects of CBP on AR expression and proliferation in prostate epithelial cells. We also studied the interaction between CBP and AR through a co-immunoprecipitation assay. (3) Results: CBP is consistent with AR in expression intensity in prostate tissues. Inhibiting CBP decreases AR expression, and induces proliferation inhibition, apoptosis, and cell cycle arrest in BPH prostate epithelial cells. The co-immunoprecipitation assay showed that CBP binds with AR to form transcription complexes in prostate epithelial cells. (4) Conclusions: Inhibiting CBP decreases AR expression and inhibits proliferation in benign prostate epithelial cells. CBP may be a potential target to affect AR expression and the proliferation of prostate epithelial cells in BPH.

Keywords: CBP; androgen receptor; benign prostatic hyperplasia.

Figure 1

CBP and AR gene expression are closely related in BPH prostate cells. (A) Study design of Love and colleagues (part). SCID mice and human BPH tissues were used to construct PDX animal models; testosterone rods were implanted into mice, and blank control group was set up to observe the effect of androgen on gene expression in BPH prostate cells. (B) Volcano plot of gene expression differences between androgen-treated prostate cells and non-androgen-treated prostate cells. (C) Enrichment analysis results. (D) Immunohistochemical staining of AR and CBP in BPH prostate tissues. (E) Scatter diagram of correlation of expression intensity, and the red area represents the 95% confidence interval of the fitted line. SCID, severe combined immunodeficient; PDX, patient-derived xenograft; GO, gene ontology; BP, biological process; CC, cellular component; MF, molecular function.

Figure 2

Inhibiting CBP decreases AR expression in benign prostate epithelial cells. (A) Western blot was used to detect the change in AR level after shRNA was used to knock down CBP in BPH-1 cells. (B) Western blot was used to detect the change in AR level after ICG-001 was used to inhibit CBP in RWPE-1 cells. (C) Immunofluorescence staining was used to detect changes in AR levels after using ICG-001 or (D) shRNA to inhibit CBP in BPH-1 cells, and blue represents DAPI, red represents AR; (F,G) Quantitative analyses diagrams. (E) Immunofluorescence staining was used to detect the change in AR level after ICG-001 was used to inhibit CBP in RWPE-1 cells, and blue represents DAPI, red represents AR; (H) Quantitative analysis diagram. DMSO, dimethyl sulfoxide; DAPI, 4’,6-diamidino-2-phenylindole; **** p < 0.0001.

Figure 3

Inhibiting CBP inhibits proliferation and induces apoptosis in benign prostate epithelial cells. (A) CCK8 assay was used to detect the effect of different concentrations of CBP inhibitor (ICG-001) on the proliferation of BPH-1 cells and (C) RWPE-1 cells. (B) CCK8 assay was used to detect the effect of knocking down CBP with shRNA on the proliferation of BPH-1 cells. (D) Effects of AR antagonist (enzalutamide) and CBP inhibitor (ICG-001) alone/combined on the proliferation of BPH-1 cells. (E) Flow cytometry was used to detect the effects of inhibiting CBP (using ICG-001 or shRNA) on apoptosis of BPH-1 cells and RWPE-1 cells, as well as (F–H) the quantitative analyses diagrams. DMSO, dimethyl sulfoxide; ns, no significance; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Figure 4

Inhibiting CBP induces cell cycle arrest and protein expression changes in benign prostate epithelial cells. (A) Using flow cytometry to analyze the effect of CBP inhibitor (ICG-001) on the cell cycle of BPH-1 cells and RWPE-1 cells; (B–E) Quantitative analyses diagrams. (F) Western blots were used to detect the effects of different concentrations of CBP inhibitor (ICG-001) on the expressions of apoptosis-related proteins and cell cycle-related proteins in BPH-1 cells and (G) RWPE-1 cells. (H) CoIP assay was used to detect the binding of CBP and AR in BPH-1 cells. DMSO, dimethyl sulfoxide; IP, immunoprecipitation; IB, immunoblot; Ctrl, control; * p < 0.05, ** p < 0.01.

Figure 5

Schematic diagram of the transcription complex formed by CBP and AR in benign prostate epithelial cells. After AR activation, a dimer is formed and enters the nucleus, located in the ARE region of the target gene; it forms a transcription complex with transcriptional coactivators including CBP, and transcribes androgen-related proteins that promote proliferation and decrease apoptosis in benign prostate epithelial cells. The drawing of this schematic refers to other studies, and only transcriptional coactivators mentioned in this study are indicated. DHT, dihydrotestosterone; ARE, androgen response element; CRE, CREB response element.