Activation of the androgen receptor by intratumoral bioconversion of androstanediol to dihydrotestosterone in prostate cancer

Abstract

The androgen receptor (AR) mediates the growth of benign and malignant prostate in response to dihydrotestosterone (DHT). In patients undergoing androgen deprivation therapy for prostate cancer, AR drives prostate cancer growth despite low circulating levels of testicular androgen and normal levels of adrenal androgen. In this report, we demonstrate the extent of AR transactivation in the presence of 5α-androstane-3α,17β-diol (androstanediol) in prostate-derived cell lines parallels the bioconversion of androstanediol to DHT. AR transactivation in the presence of androstanediol in prostate cancer cell lines correlated mainly with mRNA and protein levels of 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), one of several enzymes required for the interconversion of androstanediol to DHT and the inactive metabolite androsterone. Levels of retinol dehydrogenase 5, and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9, which also convert androstanediol to DHT, were lower than 17β-HSD6 in prostate-derived cell lines and higher in the castration-recurrent human prostate cancer xenograft. Measurements of tissue androstanediol using mass spectrometry demonstrated androstanediol metabolism to DHT and androsterone. Administration of androstanediol dipropionate to castration-recurrent CWR22R tumor-bearing athymic castrated male mice produced a 28-fold increase in intratumoral DHT levels. AR transactivation in prostate cancer cells in the presence of androstanediol resulted from the cell-specific conversion of androstanediol to DHT, and androstanediol increased LAPC-4 cell growth. The ability to convert androstanediol to DHT provides a mechanism for optimal utilization of androgen precursors and catabolites for DHT synthesis.

Figure 1. Cell-specific wild-type AR transactivation in the presence of androstanediol

(A) CV1, (B) HeLa, (C) PWR-1E, (D) PC-3, (E) LAPC-4 and (F) CWR-R1 cells were transfected as described in Methods with pCMV5 empty vector (p5) or pCMV-AR and PSA-Enh-Luc and incubated for 24 h in serum-free, phenol red-free medium in the absence and presence of increasing concentrations of testosterone (T), DHT and androstanediol (Diol). Luciferase activity measurements indicating the mean ± S.E. are representative of at least three independent experiments.

Figure 2. Androstanediol metabolism to DHT increases LAPC-4 cell growth

(A) PWR-1E (PW), LAPC-4 (LA), CWR-R1 (CW), PC-3 (PC), HeLa (HL), and CV1 (CV) cells were incubated in serum-free, phenol red-free medium containing 100 nM androstanediol for 24 and 48 h at 37°C. Steroids were extracted from cells and medium and quantitated using mass spectrometry. Shown are the nM concentrations of androstanediol (Diol), DHT and androsterone. (B) LAPC-4 cell growth assays were performed as described in Methods. Cells were incubated without hormone (- -) or with 0.1 nM DHT (O) or 10 nM androstanediol (▲). Assays performed in triplicate are the mean ± SE of duplicate experiments.

Figure 3. Schematic diagram of DHT metabolism

Androstenedione is metabolized to testosterone in peripheral tissues by aldo-keto reductase 1C3 (AKR1C3). 5α-reductase type 2 irreversibly converts testosterone to DHT which is 3-keto reduced reversibly to the inactive metabolite androstanediol by the aldo-keto reductase 1C2 (AKR1C2). Androstanediol is oxidized to DHT by 17β-hydroxysteroid dehydrogenase 6 (17β-HSD6), retinol dehydrogenase 5 (RDH5) and dehydrogenase/reductase short-chain dehydrogenase/reductase family member 9 (DHRS9). Androstanediol is oxidized reversibly to androsterone by 17β-HSD6 and 11.

Figure 4. Cell-specific expression of 17β-HSD6, DHRS9 and RDH5 mRNA and 17β-HSD6 protein expression in different cell lines

(A) RNA was analyzed by quantitative RT-PCR from LNCaP (LN), LNCaP-C4-2 (C4), CWR-R1 (CW), LAPC-4 (LA), PC-3 (PC), DU145 (DU), PWR-1E (PW), RWPE-2 (RW), HeLa (HL) and CV1 cells (CV) for 17β-HSD6, DHRS9 and RDH5. (B) Cell extracts (150 µg protein/lane) were analyzed on immunoblots for CV1 (CV), HeLa (HL), PWR-1E (PW), PC-3 (PC), LAPC-4 (LA), CWR-R1 (CW) and LNCaP (LN) cells. The upper panel was probed with AR32 and AR52 antibodies, and the lower portion with 17β-HSD6 rabbit polyclonal antibody, and stripped and reprobed with mouse β-actin antibody.

Figure 5. 17β-HSD6, DHRS9 and RDH5 mRNA levels in the CWR22 xenograft before and after castration and 17β-HSD6 mRNA levels in benign and malignant prostate

(A) RNA was extracted from CWR22 tumors before castration (0) and 2, 8, 12 and 120 days after castration, and from the castration-recurrent tumor > 120 days after castration (R). mRNA was measured using quantitative PCR for 17β-HSD6, DHRS9 and RDH5 and expressed as mRNA copies relative to peptidylprolyl isomerase A (PPIA) ± S.E. (B) 17β-HSD mRNA levels were determined relative to PPIA for individual patient samples using quantitative PCR for androgen-stimulated benign prostate (AS-BP) (1–6 and 9), androgen-stimulated prostate cancer (AS-CaP) (1 and 3–8), and castration-recurrent prostate cancer (CR-CaP) (1–5).