Direct metabolic interrogation of dihydrotestosterone biosynthesis from adrenal precursors in primary prostatectomy tissues

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 5a-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 5a-reduced both T and AD. Furthermore, 5a-reduction of AD was the major directionality of metabolic flux to DHT. However, AD and T were comparably metabolized by 5a-reductase in primary prostate tissues, contrasting the preference exhibited by CRPC in which AD was favored over T. 5a-reductase inhibitors effectively blocked the conversion of AD to DHT. Conclusions: Both AD and T are substrates of 5a-reductase in prostatectomy tissues, resulting in two distinctly nonredundant metabolic pathways to DHT. Furthermore, the transition to CRPC may coincide with a metabolic switch toward AD as the favored substrate.