Restoration of FKBP51 protein promotes the progression of castration resistant prostate cancer

Abstract

Background: As deregulation of androgen receptor (AR) signaling target genes is associated with tumorigenesis and the development of prostate cancer (PCa), AR signaling is the primary therapeutic target for PCa. Although patients initially responses to first-line androgen deprivation therapies (ADTs), most of them with advanced PCa progress to lethal castration-resistant prostate cancer (CRPC). Recent studies have suggested the molecular mechanisms by which AR elicit the robust up-regulation of the FKBP51 gene. We suggest that restored expression of FKBP51 gene, modulated by androgen receptor splicing variant 7 (AR-V7) which replaces full length androgen receptor (AR-FL) in androgen ablation status, promotes CRPC progression through activating NF-κB signaling.

Methods: Immunohistochemistry assays were used to detect the expression of AR-V7, FKBP51 and NF-κB signaling correlated proteins in CRPC tissues. An androgen ablation resistant PCa cell line model established by Long-term culturing in androgen depleted medium, named androgen-independent LNCaP (LNCaP-AI) cells, were used to dynamically monitor FKBP51 expression during the process of androgen dependent PCa cells transforming into androgen-independent cells, as well as its association with NF-κB signal pathway. LNCaP-AI cell line was determined to express AR-V7 protein continuously. Luciferase reporter assays and DNA pull down were used to determine the association between AR-V7 and FKBP51.

Results: Our results suggested that CRPC patients with AR-V7 high expression tend to have higher expression of FKBP51 and enhanced NF-κB signaling compared with AR-V7 negative patients. Knockdown of AR-V7 or FKBP51 in LNCaP-AI cells attenuated the level of p-NF-κB (Ser536) and androgen-resistant cells growth. Luciferase reporter assays and DNA pull down results indicated that FKBP51 was transcriptionally promoted by AR-V7 in absence of androgen, which enhanced NF-κB signaling.

Conclusions: Because of upregulation of AR-V7 in androgen-independent PCa cells, increasing of FKBP51 induced NF-κB signaling, leading to progression of CRPC.

Keywords: AR-V7; Castration resistant prostate cancer; FKBP51; NF-κB.

Figure 1

Restoration of FKBP51 promotes the growth of PCa cells after androgen deprivation. (A) RT-PCR analysis of FKBP51 mRNA during the generation of LNCaP-AI cell line. (B) Western blot analysis of FKBP51 protein expression during the generation of LNCaP-AI cell line. (C) MTT assays in LNCaP-P30 cells infected with lentiviruses overexpressing FKBP51. Upper panel: Western blot detection of FKBP51 expression after overexpression of FKBP51; lower panel: cell growth was assessed daily for 72 hours. (D) MTT assays in LNCaP-AI cells infected with lentiviruses carrying shPCAT1. Upper panel: Western blot detection of FKBP51 expression after knockdown of FKBP51; lower panel: cell growth was assessed daily for 72 hours. (E) TUNEL assays in FKBP51-deficient LNCaP-AI cells. For each group, representative images were randomly chosen under fluorescent microscopy. *, P<0.05; **, P<0.01; ***, P<0.001. P, passage.

Figure 2

FKBP51 enhances activity of NF-κB signaling in the progression of CRPC. (A) Immunofluorescence assays in FKBP51-deficient LNCaP-AI cells. For each group, representative images were randomly chosen under fluorescent microscopy. (B) Western blot detection of indicated proteins in LNCaP-P30 cells with FKBP51 overexpression. GAPDH was used as a loading control. (C) Western blot detection of indicated proteins in LNCaP-AI cells transfected with FKBP51 shRNA. GAPDH was used as a loading control. P, passage; CRPC, castration-resistant prostate cancer.

Figure 3

Transcriptional re-activation of FKBP51 depends on AR-V7, not AR-FL in CRPC. (A) Western blot assays of AR-FL and AR-V7 expression in LNCaP-parental, LNCaP-Hormone Naïve P30 and LNCaP-AI cells. (B) Western blot detection of FKBP51 expression after AR-V7 and AR-FL overexpression in LNCaP-P30 cell, respectively. AR-FL, full length androgen receptor. (C) Western blot detection of FKBP51 expression in LNCaP-AI cell transfected with AR-V7 and AR-FL shRNA, respectively. AR-FL, full length androgen receptor. (D) AR-V7 or AR-FL overexpressed COS-1 cells were transfected with FKBP51 luciferase promoter plasmids, cultured without DHT, then, luciferase values were detected. (E) AR-V7 or AR-FL overexpressed COS-1 cells were transfected with FKBP51 luciferase promoter plasmids, cultured with DHT (10 nM), then, luciferase values were detected. (F) DNA pull down assays were used to determine the interaction of biotin-labeled FKBP51 promoter probe with AR-V7 proteins in LNCaP parental, LNCaP-P30 and LNCaP-AI cells. ***, P<0.001; NS, not significant. P, passage; CRPC, castration-resistant prostate cancer; DHT, double hydrogen testosterone.

Figure 4

AR-V7/FKBP51/NF-κB signaling axis promotes the progression of CRPC. (A) Western blot detection of indicated proteins in LNCaP-P30 cells transfected with indicated plasmids. GAPDH was used as a loading control. (B) Western blot detection of indicated proteins in LNCaP-AI cells transfected with indicated plasmids. GAPDH was used as a loading control. (C) Bioinformatic analysis of RNA-seq data from shAR-V7 and shAR-FL mediated knock-down experiments in LN95 cell lines. The RNA-seq data was retrieved fromGSE106560. Fold change of FKBP51 expression in response to AR-V7 knocked down (y axis), AR-FL knocked down (x axis) and DHT stimulation was presented. Size of green dot showed the concentration of DHT. The scales of x axis and y axis are different. P, passage; CRPC, castration-resistant prostate cancer; DHT, double hydrogen testosterone.

Figure 5

Level of AR-V7, FKBP51 and p-NF-κB are positively correlated in CRPC patients. (A) IHC staining of AR-V7, FKBP51, NF-κB, p-NF-κB p65 (Ser536) and c-Myc in the eleven CRPC specimens. For each sample, three different fields were randomly chosen and imaged under microscopy. Representative images were shown. (B,C) Quantitative analysis of IHC staining of the eleven CRPC tissues with Image J software. Average optical density = integrated density/area. CRPC, castration-resistant prostate cancer. The magnifications of these representative images are 10×, 20× and 40× in magnification order.