Direct Metabolic Interrogation of Dihydrotestosterone Biosynthesis from Adrenal Precursors in Primary Prostatectomy Tissues

Abstract

Purpose A major mechanism of castration-resistant prostate cancer (CRPC) involves intratumoral biosynthesis of dihydrotestosterone (DHT) from adrenal precursors. We have previously shown that adrenal-derived androstenedione (AD) is the preferred substrate over testosterone (T) for 5α-reductase expressed in metastatic CRPC, bypassing T as an obligate precursor to DHT. However, the metabolic pathway of adrenal-derived DHT biosynthesis has not been rigorously investigated in the setting of primary disease in the prostate. Experimental Design Seventeen patients with clinically localized prostate cancer were consented for fresh tissues after radical prostatectomy. Prostate tissues were cultured ex vivo in media spiked with an equimolar mixture of AD and T, and stable isotopic tracing was employed to simultaneously follow the enzymatic conversion of both precursor steroids into nascent metabolites, detected by liquid chromatography-tandem mass spectrometry. CRPC cell line models and xenograft tissues were similarly assayed for comparative analysis. A tritium-labeled steroid radiotracing approach was used to validate our findings. Results Prostatectomy tissues readily 5α-reduced both T and AD. Furthermore, 5α-reduction of AD was the major directionality of metabolic flux to DHT. However, AD and T were comparably metabolized by 5α-reductase in primary prostate tissues, contrasting the preference exhibited by CRPC in which AD was favored over T. 5α-reductase inhibitors effectively blocked the conversion of AD to DHT. Conclusions Both AD and T are substrates of 5α-reductase in prostatectomy tissues, resulting in two distinctly non-redundant metabolic pathways to DHT. Furthermore, the transition to CRPC may coincide with a metabolic switch towards AD as the favored substrate.

Serum Sex Steroids as Prognostic Biomarkers in Patients Receiving Androgen Deprivation Therapy for Recurrent Prostate Cancer: A Post Hoc Analysis of the PR.7 Trial

“…Contrary to the trend that occurs with age, increases in DHEA and AST levels during ADT were found on multivariate analysis to predict sooner time to castration resistance and may act as an indicator of more aggressive disease. This is in line with reports that indicate adrenal androgens are the principle source of intratumoral androgens in CRPC (13,14). Furthermore, the more rapid production of androgens during ADT due to genomic variants has also been shown to correspond to poorer prognosis (15)(16)(17).…”

Applying mass spectrometric methods to study androgen biosynthesis and metabolism in prostate cancer

“…We, thus, hypothesize that DHT synthesis in these tumors applies a pathway, being independent of T as an intermediate. Also in other MS-based studies T-independent conversion of A-dione to DHT via androstanedione has been demonstrated to be the predominant route for DHT formation in CRPC (Chang et al 2011, Dai et al 2017, while both T-dependent and independent DHT formation are equally used in the primary tumors (Dai et al 2017). Similarly to that observed in VCaP xenografts, intratumoral DHT concentration was retained or increased, after castration in 80% (14/18) of the LuCaP xenografts tested, while T was decreased, suggesting intratumoral DHT synthesis also in majority of these castration-resistant patientderived xenografts (Nguyen et al 2017).…”

“…Interestingly, SRD5A1 is also expressed in the castration-resistant VCaP tumors, while no SRD5A2 expression was detected in these tumors. It is also worth noting that A-dione and T do not present with substrate competition in a 5α-reductase activity assay applied on cultured prostatic tissue ex vivo (Dai et al 2017).…”

Testosterone accumulation in prostate cancer cells is enhanced by facilitated diffusion

“…In the classical front door mechanism testosterone either acts as a ligand for AR or is converted into the high‐affinity AR ligand DHT. Conversely, in the back door mechanism, DHT is mainly synthetized from 5α‐dione, thus independent of testosterone as a precursor (Figure A) . Pathway utilization depends on the order in which these three enzymes are activated.…”

“…Conversely, in the back door mechanism, DHT is mainly synthetized from 5α-dione, thus independent of testosterone as a precursor ( Figure 5A). 64,66,67 Pathway utilization depends on the order in which these three enzymes are activated. The order of activation, in turn, depends on precursor concentrations, enzyme expression levels, and affinity of the given enzyme for its substrate.…”