Increased expression of CYP17A1 indicates an effective targeting of the androgen receptor axis in castration resistant prostate cancer (CRPC)

Abstract

Recent breakthrough therapies targeting androgen receptor signalling in castration resistant prostate cancer (CRPC) involve multifunctional androgen receptor (AR) blockade and exhaustive androgen deprivation. Nevertheless, limitations to an enduring effectiveness of new drugs are anticipated in resistance mechanisms occurring under such treatments. In this study we used CRPC cell models VCaP and LNCaP as well as AR-negative PC-3- and non-neoplastic epithelial BPH-1-cells treated with 5, 10 or 25 μmol/L abiraterone hydrolyzed from abiraterone acetate (AA). The origin of CYP17A1 up-regulation under AA treatment was investigated in CRPC cell models by qRT-PCR and western-blot procedures. AA treatments of AR positive CRPC cell models led to decreased expression of androgen regulated genes such as PSA. In these cells diminished expression of androgen regulated genes was accompanied by an up-regulation of CYP17A1 expression within short-term treatments. No such effects became evident in AR-negative PC-3 cells. AR directed siRNA (siAR) used in VCaP cells significantly reduced mRNA expression and AR protein abundance. Such interference with AR signalling in the absence of abiraterone acetate also caused a marked up-regulation of CYP17A1 expression. Down-regulation of androgen regulated genes occurs in spite of an elevated expression of CYP17A1, the very target enzyme for this drug. CYP17A1 up-regulation already takes place within such short treatments with AA and does not require adaptation events over several cell cycles. CYP17A1 is also up-regulated in the absence of AA when AR signalling is physically eliminated by siAR. These results reveal an immediate counter-regulation of CYP17A1 expression whenever AR-signalling is inhibited adequately but not a persisting adaptation yielding drug resistance.

Keywords: Abiraterone acetate; Androgen receptor; CRPC; CYP17A1; IGF-2.

Figure 1

Expression profile of PSA and TMPRSS2-ERG: VCaP and LNCaP showing decreased PSA and TMPRSS2-ERG (VCaP) mRNA expression after treatment with with 5, 10 or 25 μM AA (A-C). In addition treatment with 10 μM AA leads to a decreased PSA secretion in VCaP (D); (* = p < 0.05, ** = p < 0.005 and *** = p < 0.0005).

Figure 2

Expression profile of CYP17A1 and AKR1C3: CYP17A1 expression level is significantly increased in VCaP and LNCaP after treatment with 25 μm AA (A + B). In addition AKR1C3 mRNA is decreased in VCaP and increased in LNCaP cell line (C + D); (*= p < 0.05, **= p < 0.005 and ***= p < 0.0005).

Figure 3

CYP17A1 and AKR1C3 expression in BPH-1 and PC3: increased CYP17A1 and decreased AKR1C3 mRNA in BPH-1 after treatment with 25 μM AA (A + B). In PC-3 no significant effects after AA treatment in CYP17A1 and AKR1C3 mRNA expression could be seen (C + D); (*= p < 0.05, **= p < 0.005 and ***= p < 0.0005).

Figure 4

AR targeting siRNA in VCaP: siRNA against AR significantly reduced AR expression and an up-regulation of CYP17A1 expression in the absence of AA. No such effect is seen in AKR1C3 mRNA expression levels (A-D); (*= p < 0.05, **= p < 0.005 and ***= p < 0.0005).

Figure 5

Western Blot analysis of the AR and CYP17A1 after AA treatment with 5 μmol/L. In western blot analyses no significant changes were detected on the AR protein level (A), whereas CYP17A1 protein expression increased markedly (B).

Figure 6

Correlation of AA treatment on IGF-2 expression: IGF-2 is up-regulated in VCaP-CRPC, LNCaP, BPH-1 and PC3 after treatment with 25 μm AA (A, C-E). In contrast IGF-2 expression was not elevated after treatments of increasing AA concentrations in VCaP-CRPCrev (B); (*= p < 0.05, **= p < 0.005 and ***= p < 0.0005).