Dihydrotestosterone synthesis bypasses testosterone to drive castration-resistant prostate cancer Kai-Hsiung Changa, Rui Lia, Mahboubeh Papari-Zareeib, Lori Watumullc, Yan Daniel Zhaod, Richard J. Auchusb, and Nima Sharifia,1 aDivision of Hematology and Oncology, bDivision of Endocrinology and Metabolism, Department of Internal Medicine, cDepartment of Radiology, and dDepartment of Clinical Sciences, University of Texas Southwestern Medical Center, Dallas, TX 75390-8852 Edited* by Jan-Åke Gustafsson, Karolinska Institutet, Huddinge, Sweden, and approved July 6, 2011 (received for review May 18, 2011) In the majority of cases, advanced prostate cancer responds initially the testes (17) and T to DHT in the normal prostate (18), and to androgen deprivation therapy by depletion of gonadal testos- furthermore implies the requirement for T as an obligate pre- terone. The response is usually transient, and metastatic tumors cursor of DHT. The existence of an alternative pathway to DHT almost invariably eventually progress as castration-resistant pros- synthesis in peripheral tissue cell lines has been described (19). tate cancer (CRPC). The development of CRPC is dependent upon the However, the role and requirement for this pathway in driving fi intratumoral generation of the potent androgen, dihydrotestoster- CRPC has never been directly de ned. one (DHT), from adrenal precursor steroids. Progression to CRPC Here, we show that the dominant pathway to DHT synthesis is accompanied by increased expression of steroid-5α-reductase from adrenal precursors in CRPC follows an alternative route that bypasses T and requires steroid 5α-reductase isoenzyme-1 isoenzyme-1 (SRD5A1) over SRD5A2, which is otherwise the domi- Δ4 α nant isoenzyme expressed in the prostate. DHT synthesis in CRPC (SRD5A1). The , 3-keto structure of T makes it susceptible 5 - reduction by SRD5A isoenzymes. Similar to T, AD also has a Δ4, is widely assumed to require 5α-reduction of testosterone as the 3-keto structure, permitting it to be 5α-reduced to 5α-androsta- obligate precursor, and the increased expression of SRD5A1 is α fl nedione (5 -dione) (6, 20), which suggests an alternative pathway thought to re ect its role in converting testosterone to DHT. Here, to DHT synthesis (Fig. 1). This alternative pathway, therefore, we show that the dominant route of DHT synthesis in CRPC by- α α circumvents T and instead requires 5 -dione as a necessary pre- passes testosterone, and instead requires 5 -reduction of andros- cursor to DHT. tenedione by SRD5A1 to 5α-androstanedione, which is then MEDICAL SCIENCES converted to DHT. This alternative pathway is operational and dom- Results inant in both human CRPC cell lines and fresh tissue obtained from Alternative Pathway to DHT Synthesis in CRPC. AD may either be human tumor metastases. Moreover, CRPC growth in mouse xeno- 17-keto–reduced to T or alternatively 5α-reduced to 5α-dione. graft models is dependent upon this pathway, as well as expression To establish the dominant pathway to DHT synthesis, six of SRD5A1. These findings reframe the fundamental metabolic established human prostate cancer cell lines originally obtained pathway that drives CRPC progression, and shed light on the de- and derived from patients with CRPC were treated with [3H]-AD velopment of new therapeutic strategies. (Fig. 2A). In all six cell lines, flux from AD→5α-dione occurs earlier and more rapidly than flux from AD→T, as evidenced hormonal therapy | 5-alpha-androstanedione | tumor metabolism | by 5α-dione and T detection by HPLC. To establish proof-of- abiraterone acetate | hormone resistance principle for the preferred route of AD metabolism in clinical samples, metastatic tumors were biopsied under radiologic- ndrogen deprivation therapy with depletion of gonadal tes- guidance from two men with CRPC under an institutional review board-approved protocol (treatment history in Methods). Fresh Atosterone (T) is the frontline treatment for advanced pros- 3 tate cancer and is usually initially effective (1). Metastatic disease tumor collected by 18-G core biopsy was incubated with [ H]- almost always eventually acquires resistance to gonadal T de- AD. In patient #2, from whom tissue obtained was sufficient for “ ” two incubations, an equal portion of tumor was incubated with pletion and is termed castration-resistant prostate cancer 3 (CRPC). A critical mechanism in driving CRPC tumor pro- [ H]-T. As in the six CRPC cell lines, the dominant route of AD gression is a gain-of-function in the androgen receptor (AR) (2). metabolism is 5α-reduction to 5α-dione (Fig. 2B). In patient #2, Multiple clinical studies have shown that intratumoral concen- there was no conversion from AD→T. To determine the pre- 4 trations of T and 5α-dihydrotestosterone (DHT) sufficient to ferred Δ , 3-keto–steroid substrate for endogenously expressed activate AR-dependent transcription are maintained in CRPC SRD5A isoenzymes, flux from AD→5α-dione was compared despite suppression of serum T (3–6). The requirement for the with flux from T→DHT (Fig. 2C). In all models, flux from intratumoral generation of androgen ligand is exemplified by AD→5α-dione is uniformly dominant. In patient #2, no DHT frequent and often sustained responses to AR antagonists (7, 8), as synthesis was observed from the incubation with [3H]-T (Fig. well as an unprecedented prolongation of survival in chemother- 2D). The four 5α-reduced 19-carbon steroids [5α-dione, DHT, apy-refractory metastatic CRPC conferred by abiraterone acetate, androsterone, and Adiol] are interconvertible through reversible a potent inhibitor of CYP17A1, which is required for the synthesis reactions (Fig. 1). Comparison of the accumulation of total 5α- of adrenal 19-carbon steroid precursors of T and DHT (9). reduced steroids with [3H]-AD and [3H]-T treatments further Although T is a modest AR agonist, DHT is more potent (10) shows that AD→5α-dione is the dominant entryway to 5α-re- and is the principal androgen bound to AR in the prostate cell nucleus (11). In the setting of CRPC and the absence of gonadal T, dehydroepiandrosterone (DHEA) and its sulfate, the chief Author contributions: K.-H.C., L.W., R.J.A., and N.S. designed research; K.-H.C., R.L., adrenal 19-carbon steroid in circulation (12), undergoes conver- M.P.-Z., and L.W. performed research; K.-H.C., Y.D.Z., R.J.A., and N.S. analyzed data; sion to Δ4-androstenedione (AD) in prostate cancer (Fig. 1) by and K.-H.C. and N.S. wrote the paper. 3β-hydroxysteroid dehydrogenase/isomerase (13–15). Intra- Conflict of interest statement: N.S. and R.J.A. have been compensated as consultants for tumoral synthesis of DHT in CRPC is generally thought to re- Ortho Biotech. quire 17-keto reduction of AD to T, followed by 5α-reduction of *This Direct Submission article had a prearranged editor. T to DHT (2, 6, 16), thereby following the synthesis and pro- 1To whom correspondence should be addressed. E-mail: Nima.Sharifi@UTSouthwestern. cessing dictated by gonadal T physiology. This “conventional” edu. pathway (AD→T→DHT) in CRPC has been generally surmised This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. by genetic deficiencies in enzymes required to convert AD to T in 1073/pnas.1107898108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1107898108 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 DHEA AD 5 dione AST no participation of this SRD5A isoenzyme in the alternative 3 HSD SRD5A 3 HSD pathway. Representative HPLC plots are shown in Fig. S3. Similar experiments done with a SRD5A2 selective concentration of finasteride (24) and a clinically relevant concentration of dutas- teride (25) corroborate these findings (Fig. S4). 17 HSD 17 HSD 17 HSD Blocking SRD5A1 Inhibits AR-Responsive Gene Expression and CRPC Progression. To determine the effect of blocking flux from AD→5α- SRD5A 3 HSD dione by silencing SRD5A1 expression on AR-dependent tran- scription, cells with silenced SRD5A1 expression were treated with AD, and prostate-specific antigen (PSA) expression was assessed T DHT Adiol (Fig. 5A). AD-induced PSA expression is muted in the absence of SRD5A1. To determine if the role of SRD5A1 expression in Fig. 1. Pathways of DHT synthesis from adrenal DHEA. DHEA is converted by conducting the alternative pathway is critical to drive CRPC 3β-hydroxysteroid dehydrogenase/isomerase (3βHSD) to AD. In the conven- tional pathway, AD is first transformed to T (red arrow), which is then con- growth, surgically orchiectomized mice supplemented with sus- verted by SRD5A to DHT. In the alternative pathway, AD is the principal tained-release AD pellets were injected subcutaneously with α cells expressing an SRD5A1 shRNA or control nonsilencing substrate for SRD5A and is converted to 5 -dione (blue arrow), a necessary 3 precursor to DHT. Both 5α-dione and DHT are reversibly interconvertible by shRNA (Fig. 5 B and C). Time-to-tumor volume ≥ 50 mm was 3α-hydroxysteroid dehydrogenases (3αHSD) to the other 5α-reduced steroids, significantly decreased for SRD5A1-silenced LAPC4 cells (P = androsterone (AST) and 5α-androstane-3α,17β-diol (Adiol), respectively. 0.0364). Although not statistically significant, the trend for the LNCaP model and the volume ≥ 300-mm3 endpoint all consis- tently suggested disadvantaged growth for cells lacking SRD5A1 duced steroids (Fig. 2E). In the VCaP model, which has the lowest expression. SRD5A1 Western blot in tumors collected at the end difference between [3H]-AD and [3H]-T treatments, this finding of study showed that SRD5A1 expression is increased in the was attributable in part because of the conversion of T to AD, knockdown tumors compared with the original cells injected with which then more readily undergoes 5α-reduction. Analysis by silenced SRD5A1 expression (Fig. S5). This finding suggests there TLC in LNCaP confirms that AD is rapidly consumed to 5α-re- is selection for higher SRD5A1-expressing cells, which likely duced steroids, whereas T is not readily metabolized under sim- underestimates the true requirement for SRD5A1 expression in ilar conditions (Fig.