Dihydrotestosterone synthesis bypasses testosterone to drive castration-resistant prostate cancer

Abstract

In the majority of cases, advanced prostate cancer responds initially to androgen deprivation therapy by depletion of gonadal testosterone. The response is usually transient, and metastatic tumors almost invariably eventually progress as castration-resistant prostate cancer (CRPC). The development of CRPC is dependent upon the intratumoral generation of the potent androgen, dihydrotestosterone (DHT), from adrenal precursor steroids. Progression to CRPC is accompanied by increased expression of steroid-5α-reductase isoenzyme-1 (SRD5A1) over SRD5A2, which is otherwise the dominant isoenzyme expressed in the prostate. DHT synthesis in CRPC is widely assumed to require 5α-reduction of testosterone as the obligate precursor, and the increased expression of SRD5A1 is thought to reflect its role in converting testosterone to DHT. Here, we show that the dominant route of DHT synthesis in CRPC bypasses testosterone, and instead requires 5α-reduction of androstenedione by SRD5A1 to 5α-androstanedione, which is then converted to DHT. This alternative pathway is operational and dominant in both human CRPC cell lines and fresh tissue obtained from human tumor metastases. Moreover, CRPC growth in mouse xenograft models is dependent upon this pathway, as well as expression of SRD5A1. These findings reframe the fundamental metabolic pathway that drives CRPC progression, and shed light on the development of new therapeutic strategies.

Fig. 1.

Pathways of DHT synthesis from adrenal DHEA. DHEA is converted by 3β-hydroxysteroid dehydrogenase/isomerase (3βHSD) to AD. In the conventional pathway, AD is first transformed to T (red arrow), which is then converted by SRD5A to DHT. In the alternative pathway, AD is the principal substrate for SRD5A and is converted to 5α-dione (blue arrow), a necessary precursor to DHT. Both 5α-dione and DHT are reversibly interconvertible by 3α-hydroxysteroid dehydrogenases (3αHSD) to the other 5α-reduced steroids, androsterone (AST) and 5α-androstane-3α,17β-diol (Adiol), respectively.

Fig. 2.

An alternative pathway to the synthesis of DHT bypasses T. (A) All six human CRPC cell lines uniformly transform AD preferentially to 5α-dione over T by HPLC. (B) Freshly collected metastatic CRPC tissue exhibits similar metabolism from AD→5α-dione. (C) AD is a preferred substrate over T for endogenously expressed SRD5A in CRPC cell lines. (D) In patient #2, AD is readily consumed by 5α-reduction to 5α-dione, whereas there is no detectable 5α-reduction of T to DHT and no depletion of T over time. (E) Accumulation of 5α-reduced steroids occurs more robustly in CRPC cell lines from AD compared with T. In A–E, blue arrows, bars, and lines represent flux through the alternative pathway and red indicates the conventional pathway. (F) Alternative analysis by TLC demonstrates the preference for 5α-reduction of AD. (G) HPLC tracings demonstrate the conversion of AD to 5α-dione in LNCaP at 7 h and subsequent conversion to the other 5α-reduced steroids (DHT, AST, and Adiol). (H) Similar HPLC analysis in LAPC4. Error bars in A, C, E and F represent the SD from experiments performed in triplicate.

Fig. 3.

The alternative pathway to DHT synthesis drives CRPC progression. CRPC growth in the LAPC4 (Upper) and LNCaP (Lower) CRPC models have more robust growth in orchiectomized mice supplemented with AD versus T (0.25 mg sustained-release steroid pellets). Control mice underwent orchiectomy and no steroid supplementation. Time from injection of tumor cells to tumor volume ≥ 50 mm³ (Left) and tumor volume ≥ 300 mm³ (Right) is statistically significantly different for both models and both endpoints using a log rank test and a pairwise comparison of AD versus T cohorts.

Fig. 4.

SRD5A1 is required for the conversion of AD to 5α-dione in the alternative pathway to DHT synthesis. (A) LAPC4 and (B) LNCaP cells stably expressing nonsilencing (shCTRL), SRD5A1 silencing (shSRD5A1 #1 and #4) and SRD5A2 silencing (shSRD5A2 #516 and #816) lentiviral constructs were treated with [³H]-AD in triplicate, with HPLC quantitation of the indicated steroids at the designated time points. In both models, AD is depleted over time in control and SRD5A2 silenced cells, but AD is largely preserved and not metabolized in SRD5A1 silenced cells. Synthesis of 5α-dione, DHT and total 5α-reduced steroids are dependent on SRD5A1 but not SRD5A2 expression. Blocking AD→5α-dione by silencing SRD5A1 hastens conversion of AD→T, resulting in elevated T. Error bars represent the SD.

Fig. 5.

SRD5A1 is required for PSA expression and CRPC tumor progression in vivo. (A) Silencing SRD5A1 expression blunts PSA expression in response to AD, which is a substrate for SRD5A1, but not R1881, which does not require metabolism to bind AR. PSA expression is normalized to RPLP0 and vehicle (EtOH) control. Error bars represent the SD of experiments performed in triplicate. (B) Silencing SRD5A1 expression inhibits CRPC growth in orchiectomized mice supplemented with AD, as assessed by time for LNCaP and LAPC4 tumors to reach 50 mm³ and (C) 300 mm³. Only LAPC4 tumors reaching the 50-mm³ endpoint was statistically significantly different, although the trend for all models and time points had a disadvantage for the shSRD5A1 group. (D) Bypassing the requirement for SRD5A1 by supplementing orchiectomized mice with 5α-dione leads to nearly superimposable growth for control and shSRD5A1 LAPC4 cells. Control and shSRD5A1 groups for all xenograft studies were compared using a log rank test.