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. 2011 Oct 15;71(20):6503-13.
doi: 10.1158/0008-5472.CAN-11-0532. Epub 2011 Aug 25.

Intratumoral de novo steroid synthesis activates androgen receptor in castration-resistant prostate cancer and is upregulated by treatment with CYP17A1 inhibitors

Changmeng Cai  1 , Sen Chen, Patrick Ng, Glenn J Bubley, Peter S Nelson, Elahe A Mostaghel, Brett Marck, Alvin M Matsumoto, Nicholas I Simon, Hongyun Wang, Shaoyong Chen, Steven P Balk
Affiliations

Intratumoral de novo steroid synthesis activates androgen receptor in castration-resistant prostate cancer and is upregulated by treatment with CYP17A1 inhibitors

Changmeng Cai et al. Cancer Res. .

Abstract

Relapse of castration-resistant prostate cancer (CRPC) that occurs after androgen deprivation therapy of primary prostate cancer can be mediated by reactivation of the androgen receptor (AR). One important mechanism mediating this AR reactivation is intratumoral conversion of the weak adrenal androgens DHEA and androstenedione into the AR ligands testosterone and dihydrotestosterone. DHEA and androstenedione are synthesized by the adrenals through the sequential actions of the cytochrome P450 enzymes CYP11A1 and CYP17A1, so that CYP17A1 inhibitors such as abiraterone are effective therapies for CRPC. However, the significance of intratumoral CYP17A1 and de novo androgen synthesis from cholesterol in CRPC, and the mechanisms contributing to CYP17A1 inhibitor resistance/relapse, remain to be determined. We report that AR activity in castration-resistant VCaP tumor xenografts can be restored through CYP17A1-dependent de novo androgen synthesis, and that abiraterone treatment of these xenografts imposes selective pressure for increased intratumoral expression of CYP17A1, thereby generating a mechanism for development of resistance to CYP17A1 inhibitors. Supporting the clinical relevance of this mechanism, we found that intratumoral expression of CYP17A1 was markedly increased in tumor biopsies from CRPC patients after CYP17A1 inhibitor therapy. We further show that CRPC cells expressing a progesterone responsive T877A mutant AR are not CYP17A1 dependent, but that AR activity in these cells is still steroid dependent and mediated by upstream CYP11A1-dependent intraturmoral pregnenolone/progesterone synthesis. Together, our results indicate that CRPCs resistant to CYP17A1 inhibition may remain steroid dependent and therefore responsive to therapies that can further suppress de novo intratumoral steroid synthesis.

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Figures

Figure 1
Figure 1
VCaP cells have substantial basal AR activity. A, VCaP, LNCaP, or C4-2 cells grown in medium with 5% charcoal-dextran stripped fetal bovine serum (CSS) were treated with 0, 0.1, 1, or 10 nM DHT for 24h and then immunoblotted for PSA and β-actin (loading control). The number on top of each lane is band intensity compared to loading control, which is then normalized to the untreated lane. B, VCaP or LNCaP cells in CSS medium were treated with 10 nM DHT for 0, 0.5 or 4h and then fractionated into nuclear extracts (NE) or cytoplasmic extracts (CE). NE, CE or whole cell lysates were immunoblotted for AR, β-tubulin (cytoplasm control), or β-actin (loading control). Blots shown were from the same gel. C, VCaP or LNCaP cells in CSS medium were treated with 0, 1, or 10 nM DHT for 4h and then subjected to ChIP assay. The DNA fragments were PCR amplified and normalized to input to measure binding to the PSA enhancer ARE or irrelevant site (negative control). D, androgen synthesis pathway. E, LNCaP, C4-2, or VCaP cells were subjected to quantitative real time RT-PCR (qRT-PCR) to measure CYP17A1, AKR1C3, HSD3B2, CYP11A1, SRD5A1, HSD17B3, or HSD17B6 (major enzyme mediating the regeneration of DHT from its reduced metabolite). Equal amounts of cellular RNA were coamplified with 18S RNA as an internal control.
Figure 2
Figure 2
Basal AR activity in VCaP is dependent on CYP17A1. A, VCaP, LNCaP, or C4-2 cells in CSS medium were treated with 0, 2, or 5 μM ketoconazole (Keto) for 24h and then immunoblotted. B, VCaP cells in CSS medium were treated with 0, 1, 10, or 100 nM androstenedione (Ad) with or without 2 μM ketoconazole for 24h. C, VCaP or C4-2 cells in CSS medium were treated with 0, 2, or 5 μM abiraterone (Abir) for 24h. D, VCaP cells in CSS medium were treated with 0 or 100 nM androstenedione with or without 2 μM abiraterone and then immunoblotted or (E) subjected to qRT-PCR. F, VCaP cells grown for 2 days in CSS medium were treated with abiraterone (5 μM) and androstenedione (25 nM) for 24h and then harvested for steroid measurements. Each bar represents a biological replicate. G, tissue samples of recurrent VCaP xenogafts were taken from each mouse pre-treatment and post-treatment with abiraterone (0.5 mg/2d for 8 days) and then subjected to RT-PCR (n=4) or (H) immunohistochemistry. I, testosterone or DHT levels in recurrent VCaP xenograft tumors were measured pre- or post-abiraterone treatment. DHEA and androstenedione levels were below the level of detection pre- and post-treatment.
Figure 3
Figure 3
Basal AR activity in VCaP is AKR1C3 dependent. A, LNCaP, C4-2, or VCaP cells were immunoblotted for AKR1C3 or β-tubulin (loading control). B and C, VCaP cells stably infected with lentivirus expressing either GFP shRNA or AKR1C3 shRNA (Open Biosystems) in CSS medium were treated with 10 nM DHT or 100 nM androstenedione (Ad) for 24h, and then immunoblotted. D, VCaP or C4-2 cells in CSS medium were treated with 0, 20, or 40 μM indomethacin for 24h. E, VCaP cells in CSS medium were treated with 0, 20, or 40 μM indomethacin with or without 10nM DHT and subjected to qRT-PCR. F, mice bearing relapsed VCaP xenografts were treated with indomethacin for 2 weeks (~0.25 mg per day in drinking water) and tissue samples taken pre- and post-therapy from tumors (n=5) were analyzed by RT-PCR as indicated or (G) by immunohistochemistry for PSA, ERG, AR, and Ki67. H, testosterone and DHT levels in recurrent xenograft tumors in transplanted female scid mice (n=3) were measured pre- or post-indomethacin (Indo) treatment.
Figure 4
Figure 4
Inhibition of CYP17A1 and AKR1C3 suppresses AR activity in a catration-resistant VCaP cell line. A, the VCS2 cells generated from a relapsed VCaP xenograft were passaged in culture in 8% CSS/2% FBS medium. VCaP and VCS2 cells were switched to CSS medium for 3 days, then treated for 1 day with 0, 1, or 10 nM DHT, and proteins were then immunoblotted. Long (L) and short (S) exposures are shown for PSA. PSA protein is quantified relative to VCaP with no added DHT. B, VCaP or VCS2 cells were subjected to qRT-PCR to measure CYP17A1, AKR1C3, or HSD3B2. C, VCS2 cells in CSS medium were treated for 24h with 0, 20, or 40 μM indomethacin and then immunoblotted. D, VCS2 cells in CSS medium were transfected with 20 nM AKR1C3 siRNA (Dharmacon) for 2d and then treated for 24h with vehicle (ethanol) or 10 nM DHT and immunoblotted for PSA and AKR1C3. E, VCS2 cells in CSS medium were treated for 24h with 0, 2, or 5 μM abiraterone and then immunoblotted. F, VCS2 cells in CSS medium were treated for 24h with abiraterone (2 μM) and indomethacin (20 or 40 μM) as indicated and then immunoblotted (note longer exposure compared to E).
Figure 5
Figure 5
Basal activity of T877A mutant AR in LNCaP and C4-2 is dependent on CYP11A1. A, COS-7 cells in 5% CSS medium were transfected with an androgen responsive element regulated luciferase reporter (ARE4-Luc) and wild-type AR or T877A mutant AR. Cells were then treated with vehicle (ethanol), DHT, testosterone (T), androstenedione (Ad), pregnenolone (Pn), progesterone (Pg), or 17α-OH-progesterone (17α-OH-Pg) (10 nM for each treatment). Reporter activity was normalized to the cotransfected CMV-Renilla-Luc. B, LNCaP cells in 5% CSS medium were treated with vehicle (−), with 0.1, 1, or 10 nM DHT, progesterone, or testosterone, or with 1 or 10 nM androstenedione and then immunoblotted. C, high-passage number LNCaP cell line (>50, LN-HP) was stably infected with lentivirus expressing either GFP shRNA or CYP11A1 shRNA (Open Biosystems), then treated with vehicle (ethanol), 10 nM DHT, or 10 nM progesterone, and analyzed by qRT-PCR for PSA and CYP11A1. D, LN-HP cells infected with GFP or AKR1C3 shRNA lentivirus were treated with 0, 1, 10 nM DHT and immunoblotted. E, C4-2 cells stably infected with lentivirus expressing either GFP shRNA or CYP11A1 shRNA were treated with 0, 1, 10 nM DHT and analyzed by RT-PCR or (F) treated with 0, 1, 10 nM progesterone and immunoblotted. G, numbers of C4-2-shGFP or C4-2-shCYP11A1 cells cultured in 5% CSS medium and treated with or without DHT were measured using MTT assay after 7 days.
Figure 6
Figure 6
CYP17A1 inhibition in CRPC selects for increased CYP17A1. A, mice bearing recurrent VCaP xenografts were treated with abiraterone for a short period (n=4, 0.5 mg/d for 8 days by i.p. injection) or (B) for extended periods until relapse (n=6, 0.1 mg/ml in drinking water, which was changed every 3 days, for 4–6 weeks). RNA extracted from tumor samples pre- or post-treatment was analyzed by qRT-PCR with GAPDH coamplified as an internal control. The change of gene expression was presented as Log2(fold change). C, Affymetrix microarray expression data for CYP17A1 and CYP11A1 in 27 primary tumors (no hormonal therapy) and 29 CRPC bone marrow metastases. D, expression of CYP17A1, AKR1C3, and CYP11A1 were assessed by qRT-PCR in 29 CRPC bone marrow biopsy tumor samples, 3 relapsed castration resistant VCaP xenografts (VCaP-CR), 3 relapsed castration resistant LNCaP xenografts (LNCR), 2–3 bone marrow biopsy tumor samples each from 6 ketoconazole-treated patient (P1–P6), and 4 bone marrow biopsy samples from CRPC patients that contained only normal bone marrow (NBM), with GAPDH amplified as an internal control.
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