Steroid Metabolism in Human Prostate Cancer Tissue: Effect of CYP17 Inhibitors

Abstract

De novo steroidogenesis is a mechanism that develops during castration‐resistant prostate cancer (CRPC). There is increasing evidence of utilization of the backdoor pathway in CRPC where precursors in the classical pathway are converted to alternative steroids and eventually dihydrotestosterone (DHT) through a series of catalytic reactions without the need to form intermediary testosterone. CYP17 is an essential enzyme for de novo androgen metabolism in the classical and backdoor steroidogenesis pathways. Abiraterone is an FDA‐approved CYP17A1 inhibitor for CRPC, whereas VT‐464 is a nonsteroidal CYP17 inhibitor currently undergoing clinical trials. Although prostate cancer primarily develops in the peripheral zone (PZ), ~20–30% tumors also originate in the transition zone (TZ). However, it is unknown if PZ and TZ differ in steroidogenic ability. We compared the 17‐hydroxypregnenolone (17‐OH Preg) or progesterone metabolism in PZ and TZ human prostate tissues obtained following radical prostatectomy from patients with localized prostate cancer. The effect of CYP17 inhibitors on steroid metabolism in prostate tissues was also evaluated.Human prostate tissues were obtained from the Vancouver Prostate Centre tissue bank. Ethical approval (certificate #H09‐01628) was procured from the Clinical Research Ethics Board of University of British Columbia. The prostate cancer patients underwent radical prostatectomy, without any neoadjuvant therapy. Prostate tissues were homogenized with Precellys homogenizer using 100 mM potassium phosphate. In vitro mixture containing human prostate homogenate (HPH; 45 mg/ml) was incubated with 17‐OH Preg or progesterone (2 μg/ml), NADPH‐regenerating system in 100 mM potassium phosphate buffer (pH 7.4) at 37°C for 60 min. Steroids were extracted twice with hexane:ethyl acetate (60:40) and derivatized with 50 mM hydroxylamine. Steroid formation by HPH was measured by Waters Acquity UPLC coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C18 column. CYP17 inhibitors (abiraterone and VT‐464 at 1 μM) were co‐incubated with HPH.Following incubation of 17‐OH Preg with HPH, androstenedione, testosterone and DHT levels decreased due to their metabolism, whereas 17‐OH‐progesterone and androsterone formation was significantly increased after incubation. Interestingly, the formation of pregnan‐3,20‐dione, 5‐pregnan‐3‐ol‐20‐one and 5‐pregnan‐17‐ol‐3,20‐dione, which are backdoor pathway steroids, dramatically increased in both PZ and TZ tissue. Similarly, incubation of HPH with progesterone led to decrease in DHT and increase in pregnan‐3,20‐dione and 5‐pregnan‐3‐ol‐20‐one levels. The formation of androstenedione, 17‐OH‐progesterone, testosterone, androsterone and 17‐OH Preg remained unchanged after incubation with progesterone. Comparison of steroid metabolism following incubation with 17‐OH Preg and progesterone in PZ and TZ revealed no significant differences between two prostate zones. Co‐incubation of substrates with abiraterone and VT‐464 led to decrease in the formation of pregnan‐3,20‐dione and 5‐pregnan‐3‐ol‐20‐one, however, both the CYP17 inhibitors had limited effects on the CYP17‐mediated activities in classical pathway.The de novo tissue steroidogenesis is similar in PZ and TZ tissues and responds to the availability of precursors in tumor microenvironment through backdoor pathway in CRPC. VT‐464 or abiraterone‐mediated modulation of steroidogenesis observed in our study suggests that compensatory pathways are possibly activated as a result of CYP17 inhibition. Steroid metabolism in HPH presents a viable model for screening of steroidogenesis inhibitors.Support or Funding InformationA grant‐in‐aid was provided by Glaxo SmithKline (GSK).