A dual stable isotope method by LC-MS/MS to define patterns of androgen metabolism in localized versus advanced prostate cancer.

Abstract

40 Background: Prior work has shown that androstenedione (AD) rather than testosterone (T) is the preferred substrate of 5α-reductase for dihydrotestosterone (DHT) synthesis in castration-resistant prostate cancer. However, patterns of metabolism in hormone-naive prostate cancer are still poorly defined. Previously, we reported on the utility of dual radioisotope labeling of steroid precursors to characterize androgen metabolism in localized prostate tissue. We now describe an alternative approach via stable isotope labeling and analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which has unique advantages over the former method. Methods: LNCaP cell lines and prostate tissue from patients undergoing radical prostatectomy for localized cancer were incubated in serum-free media, spiked with 13C-labeled AD and 2H-labeled T. Media was collected at 7, 24, and 48 hours of incubation. Steroids were extracted, separated, and then analyzed by way of LC-MS/MS to identify labeled metabolites of AD and T. Results: Incubation of labeled AD and T resulted in conversion over time to both 13C-labeled and 2H-labeled downstream metabolites, 5α-dione and DHT. Although both precursors contributed to 5α-dione and DHT formation, the steroid of origin could be determined on the basis of differential labeling. In ex-vivo tissue incubations, unlabeled 5α-dione was also observed, which was distinguishable from its stable isotopic form and most likely represents endogenous steroid. Conclusions: Both cell lines and tissue appear to metabolize labeled AD and T, and formation of DHT occurs through both precursors. Furthermore, the presence of endogenous 5α-dione suggests that alternative pathways of DHT synthesis, which bypass T, may be naturally accessible in the hormone-naïve setting. We propose a robust ex-vivo technique to readily track simultaneous pathways of DHT synthesis in prostate cancer. Future work will focus on defining metabolic phenotypes of localized prostate cancer and specific patterns of flux under different hormonal and pharmacologic conditions.