Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer

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Prostate cancer resistance to castration occurs because tumours acquire the metabolic capability of converting precursor steroids to 5α-dihydrotestosterone (DHT), promoting signalling by the androgen receptor and the development of castration-resistant prostate cancer1,2,3. Essential for resistance, DHT synthesis from adrenal precursor steroids or possibly from de novo synthesis from cholesterol commonly requires enzymatic reactions by 3β-hydroxysteroid dehydrogenase (3βHSD), steroid-5α-reductase (SRD5A) and 17β-hydroxysteroid dehydrogenase (17βHSD) isoenzymes4,5. Abiraterone, a steroidal 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitor, blocks this synthetic process and prolongs survival6,7. We hypothesized that abiraterone is converted by an enzyme to the more active Δ4-abiraterone (D4A), which blocks multiple steroidogenic enzymes and antagonizes the androgen receptor, providing an additional explanation for abiraterone’s clinical activity. Here we show that abiraterone is converted to D4A in mice and patients with prostate cancer. D4A inhibits CYP17A1, 3βHSD and SRD5A, which are required for DHT synthesis. Furthermore, competitive androgen receptor antagonism by D4A is comparable to the potent antagonist enzalutamide. D4A also has more potent anti-tumour activity against xenograft tumours than abiraterone. Our findings suggest an additional explanation—conversion to a more active agent—for abiraterone’s survival extension. We propose that direct treatment with D4A would be more clinically effective than abiraterone treatment.

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Figure 1: Structural consequences of the conversion from Abi to D4A that occurs both in mice and in patients, and requires 3βHSD.
Figure 2: D4A inhibits 3βHSD, CYP17A1 and 5α-reductase enzymatic activity in the androgen pathway.
Figure 3: D4A binds to AR, inhibits AR chromatin occupancy, expression of AR-responsive genes and cell growth.
Figure 4: D4A inhibits xenograft steroidogenesis and growth.


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This work has been supported in part by funding from a Howard Hughes Physician-Scientist Early Career Award (to N.S.), by the Prostate Cancer Foundation (to N.S.), by an American Cancer Society Research Scholar Award (12-038-01-CCE; to N.S.), grants from the US Army Medical Research and Materiel Command (PC080193 to N.S. and PC121382 to Z.L.), and additional grants from the National Cancer Institute (R01CA168899, R01CA172382, and R01CA190289; to N.S.).

Author information

Z.L. performed gene expression, metabolism, chromatin immunoprecipitation (ChIP) and mouse xenograft studies. A.B., M.A. and D.B. performed mass spectrometry studies. J.A.G. and R.D. participated in clinical studies. J.L. and S.K.U. performed enzymology studies, and S.K.U. also performed chemical syntheses. Z.L., R.J.A. and N.S. designed the studies and wrote the manuscript. All authors discussed the results and commented on the manuscript.

Correspondence to Nima Sharifi.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 D4A is detectable in patients with prostate cancer treated with Abi acetate.

a, Mass spectrometry tracings of Abi and D4A in the serum of 12 patients treated with Abi acetate. Blood was drawn between 2 and 14 h after the administration of the 1,000 mg daily dose. b, Duration of Abi acetate therapy and time from last dose to blood draw for individual patients.

Extended Data Figure 2 3βHSD converts Abi to D4A.

a, 3βHSD1 expression permits catalysis of DHEA to AD. LAPC4 cells were transfected with 3βHSD1 or vector and treated with [3H]DHEA. Medium was collected 24 h later and androgens were separated and quantified by HPLC. b, 3βHSD1 expression allows conversion of Abi to D4A. LAPC4 cells were transfected with 3βHSD1 or vector and then treated with Abi. Medium was collected after 24 h and D4A and Abi were separated by HPLC. c, 3βHSD enzymatic activity present in the mouse adrenal gland but not prostate gland converts Abi to D4A. Mouse adrenal and prostate glands were harvested and minced before culturing in the presence of media containing Abi. Medium was collected after 24 h for separation and quantitation of D4A and Abi by HPLC.

Extended Data Figure 3 D4A inhibits 3βHSD1 activity.

a, D4A inhibits 3βHSD1 activity in LNCaP. Cells were treated with [3H]DHEA (DHEA: 100nM; [3H]DHEA, 1,000,000 c.p.m. per well) with 0.1, 1 or 10 µM D4A and Abi, for 9 and 24 h. DHEA and AD were separated and quantified by TLC. ImageJ was used to quantify steroids. For ease of comparison, arrows denote AD percentage for 0.1 µM D4A and 1 µM Abi treatment groups. b, VCaP cells were treated with [3H]DHEA and the indicated concentrations of D4A and Abi. The percentages of DHEA and AD were determined by HPLC. Experiments were performed with biological replicates (n = 3) and results are shown as mean ± s.d.

Extended Data Figure 4 D4A has a higher affinity for both mutant-type AR (LNCaP cells) and wild-type AR (LAPC4 cells) than abiraterone (Abi) and bicalutamide (Bic) and inhibits AR chromatin occupancy better than Abi.

a, Competition plots for D4A, Abi and Bic. b, Competition plots for unlabelled R1881 and D4A. Displacement of [3H]R1881 is described in Methods. Experiments were performed with biological replicates (n = 3) and results are shown as mean ± s.d. c, D4A inhibition of AR chromatin occupancy is superior to Abi. LNCaP cells were treated with the indicated concentrations of DHT, D4A, Abi and enzalutamide (Enz) for 3 h. AR chromatin occupancy for PSA, TMPRSS2 and FKBP5 was detected with ChIP. Experiments were performed with technical replicates (n = 3) and results are shown as mean ± s.d. All experiments were repeated independently at least three times.

Extended Data Figure 5 D4A inhibits expression of androgen-responsive genes.

a, D4A inhibits PSA expression in LAPC4 cells. Cells were treated with DHT (0.5 nM), DHEA (40 nM) or R1881 (0.1 nM) with or without Abi or D4A (1 µM) for 24 h. b, D4A inhibits PSA expression in LAPC4 in a dose-dependent manner. c, D4A inhibits AR target gene expression in C4-2 cells. Cells were treated with vehicle control (Ctrl), DHT (0.5 nM) or DHEA (40 nM) with or without Abi (1 µM), D4A (1 µM) or Enz (1 µM) for 24 h. d, D4A is comparable to Enz in inhibiting DHT-induced target gene expression in VCaP cells. Gene expression was assessed in triplicate, detected by qPCR and normalized to RPLP0. Experiments were performed with technical replicates (n = 3) and results are shown as mean ± s.d. All experiments were repeated independently at least three times.

Extended Data Figure 6 D4A impedes VCaP xenograft growth.

*P < 0.05 and **P < 0.01 for the difference between D4A (n = 10 mice) and AA (n = 10 mice) treatment groups. N = 9 mice for the control group.

Extended Data Figure 7 D4A does not increase deoxycorticosterone concentrations.

Serum of mice undergoing long-term treatment with D4A was collected and deoxycorticosterone concentrations were determined by liquid chromatography–mass spectrometry. Compared with control mice injected with vehicle, D4A does not increase deoxycorticosterone concentrations. For both D4A and control groups, n = 9 biological replicates (mice).

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Li, Z., Bishop, A., Alyamani, M. et al. Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer. Nature 523, 347–351 (2015) doi:10.1038/nature14406

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