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. 2019 Jun;79(9):937-948.
doi: 10.1002/pros.23799. Epub 2019 Apr 24.

A bypass mechanism of abiraterone-resistant prostate cancer: Accumulating CYP17A1 substrates activate androgen receptor signaling

Jan M Moll  1 , Jinpei Kumagai  1   2 , Martin E van Royen  3   4   5 , Wilma J Teubel  1 , Robert J van Soest  1 , Pim J French  5   6 , Yukio Homma  2 , Guido Jenster  1 , Ronald de Wit  7 , Wytske M van Weerden  1
Affiliations

A bypass mechanism of abiraterone-resistant prostate cancer: Accumulating CYP17A1 substrates activate androgen receptor signaling

Jan M Moll et al. Prostate. 2019 Jun.

Abstract

Background: Intratumoral steroidogenesis and its potential relevance in castration-resistant prostate cancer (CRPC) and in cytochrome P450, family 17, subfamily A, polypeptide 1 (CYP17A1)-inhibitor treated hormone-naïve and patients with CRPC are not well established. In this study, we tested if substrates for de novo steroidogenesis accumulating during CYP17A1 inhibition may drive cell growth in relevant preclinical models.

Methods: PCa cell lines and their respective CRPC sublines were used to model CRPC in vitro. Precursor steroids pregnenolone (Preg) and progesterone (Prog) served as substrate for de novo steroid synthesis. TAK700 (orteronel), abiraterone, and small interfering RNA (siRNA) against CYP17A1 were used to block CYP17A1 enzyme activity. The antiandrogen RD162 was used to assess androgen receptor (AR) involvement. Cell growth was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. AR-target gene expression was quantified by reverse transcription polymerase chain reaction (RT-PCR). Nuclear import studies using cells with green fluorescent protein (GFP)-tagged AR were performed to assess the potential of precursor steroids to directly activate AR.

Results: Preg and Prog stimulated cell proliferation and AR target gene expression in VCaP, DuCaP, LNCaP, and their respective CRPC sublines. The antiandrogen RD162, but not CYP17A1 inhibition with TAK700, abiraterone or siRNA, was able to block Preg- and Prog-induced proliferation. In contrast to TAK700, abiraterone also affected dihydrotestosterone-induced cell growth, indicating direct AR binding. Furthermore, Prog-induced AR translocation was not affected by treatment with TAK700 or abiraterone, while it was effectively blocked by the AR antagonist enzalutamide, further demonstrating the direct AR activation by Prog.

Conclusion: Activation of the AR by clinically relevant levels of Preg and Prog accumulating in abiraterone-treated patients may act as a driver for CRPC. These data provide a scientific rationale for combining CYP17A1 inhibitors with antiandrogens, particularly in patients with overexpressed or mutated-AR.

Keywords: TAK700; abiraterone resistance; androgen receptor activation; castration-resistant prostate cancer; cytochrome P450, family 17, subfamily A, polypeptide 1 inhibitor.

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Conflict of interest statement

RW receives consultancy and speaker fees from Sanofi, Millennium, Merck, Roche. RS receives speaker fee from Sanofi, Janssen Pharmaceuticals. WW receives grant supports from Sanofi, Millennium, Janssen Pharmaceuticals, Servier.

Figures

Figure 1
Figure 1
AR‐blockade, but not CYP17A1 inhibition reduced Preg‐ and Prog‐induced cell growth in CRPC clones of VCaP and DuCaP. A, VCaP CRPC derivatives BIC‐B and FLU‐D and DuCaP CRPC derivative BIC‐H were plated in DCC medium and incubated with vehicle (ethanol, white), Preg (light gray), Prog (dark gray), or DHT (black) at the indicated levels. Data are expressed as mean ± SE of three independent experiments. DuCaP BIC‐H appeared hypersensitive to androgens, growing even better with Preg or Prog as compared with DHT. B, VCaP BIC‐B and FLU‐D and DuCaP BIC‐H were incubated with 10 nM of Preg or Prog, or with 0.1 nM DHT with or without 1 μM of the antiandrogen RD162. Data shown are mean ± SE of four independent experiments. C, VCaP BIC‐B and FLU‐D and DuCaP BIC‐H were treated with 100 nM of Preg or Prog or with 0.1 nM DHT with or without TAK700 at the indicated concentrations. Data shown are mean ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. AR, androgen receptor; CRPC, castration‐resistant prostate cancer; CYP17A1, cytochrome P450, family 17, subfamily A, polypeptide 1; DCC, dextran‐coated charcoal‐stripped fetal calf serum; DHT, dihydrotestosterone; Preg, pregnenolone; Prog, progesterone
Figure 2
Figure 2
Preg‐ and Prog‐activated AR in VCaP and DuCaP. A, Castration‐naïve VCaP (left) and DuCaP (right) cells were treated with Preg (light gray), Prog (dark gray), or DHT (black) at indicated concentrations (M). Data shown are mean ± SE of three independent experiments. B, Castration‐naïve VCaP (left) and DuCaP (right) cells were treated with Preg or Prog or DHT for 48 hours at indicated concentrations (M), and gene expression was assessed by qPCR with each sample in duplicate. Data shown are mean ± SE of three independent experiments. C, VCaP and DuCaP cells were treated with 100 nM Preg or Prog of 0.1 nM with or without RD162. Cell growth was assessed by MTT assay on day 9. Data shown are mean ± SEM of a minimum of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. AR, androgen receptor; CYP17A1, cytochrome P450, family 17, subfamily A, polypeptide 1; DCC, dextran‐coated charcoal‐stripped fetal calf serum; DHT, dihydrotestosterone; Preg, pregnenolone; Prog, progesterone; qPCR, quantitative polymerase chain reaction
Figure 3
Figure 3
Preg‐ and Prog‐induced cell growth of castration‐naïve VCaP and DuCaP is independent of CYP17A1 activity. Cells were treated with 100 nM of Preg (light gray) or Prog (dark gray), or 0.1 nM DHT (black) with or without TAK700 (A) or abiraterone (B) at indicated concentrations. Data shown are mean ± SE of three independent experiments. C, VCaP cells were transfected with 2.5 or 5 nM CYP17A1‐directed or 5 nM scrambled siRNA (Dharmacon) for 24 hours, after which cells were incubated with vehicle or 100 nM Prog for 48 hours after which CYP17A1 and PSA mRNA expression were assessed by qPCR with each sample in duplicate. D, DuCaP cells were transfected with 2.5 or 5 nM CYP17A1 siRNA or 5 nM scrambled siRNA (Dharmacon) for 24 hours and subsequently incubated with Preg, Prog, or vehicle (ethanol) for 6 days. Cell proliferation was assessed by MTT assay. Data shown are mean ± SE of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. AR, androgen receptor; CYP17A1, cytochrome P450, family 17, subfamily A, polypeptide 1; DCC, dextran‐coated charcoal‐stripped fetal calf serum; DHT, dihydrotestosterone; mRNA, messenger RNA; Preg, pregnenolone; Prog, progesterone; qPCR, quantitative polymerase chain reaction; siRNA, small interfering RNA
Figure 4
Figure 4
CYP17A1 inhibitors TAK700 and abiraterone are unable to inhibit progesterone‐induced AR translocation. Top, representative images of PC346C‐GFP‐AR cells 3 hours after the addition of vehicle control (ethanol) or increasing levels of Preg or Prog, with each row showing representative images after pretreatment with either DMSO, 1 μM TAK700, 1 μM abiraterone or 1 μM enzalutamide. Bottom, representative images 3 hours after addition of negative control (ethanol+DMSO) or positive control: 1 nM R1881+DMSO. Scale bar = 40 μm. The number in the top‐left of each picture indicates average nuclear/total AR signal ratio. Corresponding graph bars with SD are provided in Figure S7. AR, androgen receptor; CYP17A1, cytochrome P450, family 17, subfamily A, polypeptide 1; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; Preg, pregnenolone; Prog, progesterone
Figure 5
Figure 5
Schematic representation of alternative mechanisms of CRPC. Simplified overview of the classical androgen synthesis pathway. Thick arrows indicate the preferred steps in human androgen biosynthesis and subsequent AR activation as reported in the literature. Abiraterone and TAK700 effectively inhibit steroid synthesis, but cannot prevent direct binding of the steroid precursors Preg or Prog to the AR (dotted line). Direct AR‐antagonism by enzalutamide will still block activation by either DHT or Prog. AR, androgen receptor; CRPC, castration‐resistant prostate cancer; CYP17A1, cytochrome P450, family 17, subfamily A, polypeptide 1; DHT, dihydrotestosterone; Preg, pregnenolone; Prog, progesterone
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