Application of stable isotopic tracing in an ex-vivo system to reveal heterogeneous patterns of androgen metabolism in localized, hormone-naïve prostate cancer.

Abstract

109 Background: The 5α-androstanedione (5α-dione) pathway is the demonstrated route through which adrenal precursor steroids bypass testosterone (T) to form dihydrotestosterone (DHT) in metastatic castration-resistant prostate cancer (mCRPC). However, accessible pathways of DHT synthesis are less clear in localized, hormone-naïve prostate cancer (HNPCa), where steroidogenic enzyme expression may differ and circulating T and adrenal steroids could act as competing substrates for 5α-reductase. We employed a dual substrate, ex-vivo system with selective stable isotopic labeling of DHT precursors to better understand alterations in androgen synthesis that occur in the transition of HNPCa to mCRPC. Methods: Under IRB approval and patient consent, tumor and adjacent benign prostate tissues were procured from radical prostatectomy specimens of men with HNPCa (n = 16). Tissues were grossly homogenized and incubated ex-vivo in serum-free media spiked with 1 µM of 13C3-AD +/- equimolar unlabeled T. Media was collected over 48 hours and subjected to liquid-liquid extraction to capture free and glucuronidated steroid metabolites for analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Both AD and T were readily 5α-reduced in treated prostate tissues. 13C labeling revealed DHT originating from metabolism of AD. Co-incubation with T did not dramatically alter the efficiency of AD metabolism. Minimal differences were found in metabolism between PZ and TZ tissue. No glucuronidated steroid was detected in media, in contrast to in prostate cancer cell lines and xenograft tumor tissue. Considerable heterogeneity was observed in measurements of reaction efficiency across different patients. Conclusions: AD is an active substrate for 5α-reductase comparable to T in HNPCa. Adrenal steroids may therefore contribute to DHT in HNPCa via the 5α-dione pathway. Metabolic heterogeneity may reflect currently unappreciated genotypic differences in patient tumors.