Published OnlineFirst July 21, 2017; DOI: 10.1158/1078-0432.CCR-17-1313 Biology of Human Tumors Clinical Cancer Research Direct Metabolic Interrogation of Dihydrotestosterone Biosynthesis from Adrenal Precursors in Primary Prostatectomy Tissues Charles Dai1,2, Yoon-Mi Chung2,EvanKovac2,3, Ziqi Zhu2, Jianneng Li2, Cristina Magi-Galluzzi4, Andrew J. Stephenson3, Eric A. Klein1,3, and Nima Sharifi1,2,3,5 Abstract Purpose: A major mechanism of castration-resistant prostate dem mass spectrometry. CRPC cell line models and xenograft cancer (CRPC) involves intratumoral biosynthesis of dihydrotes- tissues were similarly assayed for comparative analysis. A tri- tosterone (DHT) from adrenal precursors. We have previously tium-labeled steroid radiotracing approach was used to validate shown that adrenal-derived androstenedione (AD) is the pre- our findings. ferred substrate over testosterone (T) for 5a-reductase expressed Results: Prostatectomy tissues readily 5a-reduced both T and in metastatic CRPC, bypassing T as an obligate precursor to DHT. AD. Furthermore, 5a-reduction of AD was the major direction- However, the metabolic pathway of adrenal-derived DHT bio- ality of metabolic flux to DHT. However, AD and T were com- synthesis has not been rigorously investigated in the setting of parably metabolized by 5a-reductase in primary prostate tissues, primary disease in the prostate. contrasting the preference exhibited by CRPC in which AD was Experimental Design: Seventeen patients with clinically favored over T. 5a-reductase inhibitors effectively blocked the localized prostate cancer were consented for fresh tissues after conversion of AD to DHT. radical prostatectomy. Prostate tissues were cultured ex vivo in Conclusions: Both AD and T are substrates of 5a-reductase in media spiked with an equimolar mixture of AD and T, and prostatectomy tissues, resulting in two distinctly nonredundant stable isotopic tracing was employed to simultaneously follow metabolic pathways to DHT. Furthermore, the transition to CRPC the enzymatic conversion of both precursor steroids into may coincide with a metabolic switch toward AD as the favored nascent metabolites, detected by liquid chromatography-tan- substrate. Clin Cancer Res; 1–12. Ó2017 AACR. Introduction is invariably followed by the onset of castration-resistant prostate cancer (CRPC). CRPC is largely typified by an inappropriate Androgen deprivation therapy by medical or surgical castration restoration of the AR signaling axis (2–4), and in this setting, is a mainstay of treatment for advanced prostate cancer. The intratumoral biosynthesis of DHT from adrenal precursor steroids primary action of androgen deprivation is attributable to the plays a major role to sustain AR activation (4–6). Thus, a detailed disruption of androgen signaling by depleting gonadal testoster- interrogation of androgen metabolism and enzymology is imper- one (T), a modest androgen receptor (AR) agonist, and canonical ative toward understanding the pathophysiology of this disease. precursor to the more potent agonist dihydrotestosterone (DHT; In the absence of gonadal T, one route of DHT biosynthesis ref. 1). Although initially effective, androgen deprivation therapy involves enzymatic modification of dehydroepiandrosterone/ dehydroepiandrosterone sulfate (DHEA/DHEA-S), a primarily adrenal-derived androgen and the most abundant 19-carbon 1Cleveland Clinic Lerner College of Medicine, Cleveland, Ohio. 2Department of steroid in serum (5, 7). Three enzymatic steps are required to Cancer Biology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio. convert DHEA to DHT, consisting of (1) 3b-hydroxyl oxidation 3Department of Urology, Glickman Urological and Kidney Institute, Cleveland and D5!D4 isomerization by 3b-hydroxysteroid dehydrogenase/ 4 Clinic, Cleveland, Ohio. R.T. Pathology and Laboratory Medicine Institute, isomerase (3b-HSD), (2) 17-keto reduction by 17b-hydroxyster- Cleveland Clinic, Cleveland, Ohio. 5Department of Hematology and Medical oid dehydrogenase (17b-HSD), and (3) 5a-reduction by 5a- Oncology, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio. reductase (7, 8). In normal male physiology, this sequence chiefly Note: Supplementary data for this article are available at Clinical Cancer occurs within the testes and first requires the conversion of DHEA Research Online (http://clincancerres.aacrjournals.org/). to its D4-3-keto derivative androstenedione (AD), which is subse- Current address for E. Kovac: Montefiore Medical Center and the Albert Einstein quently 17-keto reduced to T (8). T is then secreted into circulation, College of Medicine, New York, NY. where it may undergo irreversible 5a-reduction to DHT in the Corresponding Author: N. Sharifi, Cleveland Clinic, 9500 Euclid Avenue, Cleve- prostate (Fig. 1A, left). This pathway (DHEA!AD!T!DHT) was land, OH 44195. Phone: 216-445-9750; Fax: 216-445-6269; E-mail: also surmised to be the metabolic route of DHT biosynthesis from sharifi[email protected] circulating DHEA by CRPC, in which T serves as an obligate doi: 10.1158/1078-0432.CCR-17-1313 precursor. Notably though, AD and T share the same D4-3-keto Ó2017 American Association for Cancer Research. structure that is amenable to 5a-reduction (9, 10). We have www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2017 American Association for Cancer Research. Published OnlineFirst July 21, 2017; DOI: 10.1158/1078-0432.CCR-17-1313 Dai et al. CRPC, the 5a-dione pathway appears to be the major direc- Translational Relevance tionality of flux from adrenal steroids to DHT in this context. We show that dihydrotestosterone (DHT) biosynthesis However, AD and T appear to be comparably metabolized by from adrenal steroids in the prostate of men with clinically 5a-reductase in primary prostate tissues, contrasting the pref- localized prostate cancer primarily occurs through 5a-reduc- erence exhibited by CRPC in which AD is clearly favored over T. tase utilizing androstenedione (AD). Notably, this results in This finding provides direct evidence that the transition of two distinctly nonredundant metabolic pathways to DHT eugonadal primary disease to eventual CRPC may coincide from circulating adrenal steroids versus gonadal T. This obser- with a metabolic switch toward AD as the preferred substrate vation deviates from the general view that 5a-reduction of T is of 5a-reductase to thereby augment adrenal steroid utilization the principal enzymatic step to generate intraprostatic DHT, in the biosynthesis of DHT and enable therapeutic resistance. regardless of the source. In addition, using a novel mixed substrate approach to assay relative substrate utilization, we Materials and Methods provide direct evidence that the transition of eugonadal pri- mary disease to eventual castration-resistant prostate cancer Cells and culture conditions (CRPC) may coincide with a metabolic switch toward AD as Human prostate cancer cell lines LNCaP and 22Rv1 were the preferred substrate of 5a-reductase, thereby augmenting purchased from the American Type Culture Collection (ATCC) adrenal steroid utilization toward DHT biosynthesis and and maintained in RPMI 1640 with 10% FBS. VCaP and DU145 enabling therapeutic resistance. Ultimately, these findings cells were purchased from the ATCC and maintained in DMEM underscore the need to better define steroidogenic pathways with 10% FBS. MDA-PCa-2b cells were purchased from the of prostate cancer in its various stages, in the effort to develop ATCC and cultured in BRFF-HPC1 (Athena ES) containing 20% more effective androgen-directed therapies for CRPC. FBS. RWPE-1 cells were obtained from the ATCC and main- tained in Keratinocyte Growth Medium with 0.05 mg/mL bovine pituitary extract and 5 ng/mL human recombinant epidermal growth factor according to ATCC recommendations. LAPC4 cells were generously provided by Charles Sawyers previously shown that AD is the preferred substrate relative to (Memorial Sloan Kettering Cancer Center, New York, NY) and Tfor5a-reductase expressed in CRPC (11). This results in were cultured in Iscove's Modified Dulbecco's Medium with generation of a 5a-reduced metabolite, 5a-androstanedione 10% FBS. C4-2 cells were generously provided by J. T. Hsieh (5a-dione), which can be 17-keto reduced to DHT to bypass T (University of Texas Southwestern, Dallas, TX) and maintained via a different metabolic route (Fig. 1A, right). Surprisingly, this in 10% FBS. All cell lines were incubated in a 5% CO2 humid- "5a-dione pathway" (DHEA!AD!5a-dione!DHT) appears ified incubator, and 1% penicillin/streptomycin (P/S) was to be the predominant directionality of DHEA metabolism in added to all culture media. FBS and charcoal:dextran-stripped the majority of metastatic CRPC cell lines, as well as in sampled FBS were purchased from Gemini Bio Products. All cell lines metastatic CRPC patient tissues (11–14). We therefore sought were authenticated prior to use and routinely tested for myco- to determine whether the same metabolic phenotype is oper- plasma contamination. ational in the eugonadal setting of clinically localized prostate cancer, where both gonadal T and adrenal steroids are potential Human primary prostate tissue procurement precursors of DHT. Institutional Review Board approval and patient written A number of studies have previously attempted to charac- informed consent were obtained at the Cleveland Clinic prior terize androgen synthesis in clinically localized disease. These to procurement of deidentified human tissue samples from efforts