Abstract PR026: A bypass gateway from cholesterol to sex steroid biosynthesis circumnavigates CYP17A1

Abstract The canonical biosynthesis of all androgens from cholesterol in humans and other vertebrates requires two cytochrome P450 enzymes. First, cytochrome P450 11A1 (CYP11A1) cleaves six carbons from the side chain of 27-carbon cholesterol to generate 21-carbon pregnanes. Second, in a two-step process, cytochrome P450 17-hydroxylase/17,20-lyase (CYP17A1) cleaves carbons 20 and 21 from pregnanes to make 19-carbon androgens, e.g., conversion from pregnenolone to dehydroepiandrosterone (DHEA). The identification of this pathway eventually led to the development of CYP17A1 pharmacologic inhibitors for the treatment of prostate cancer. However, CYP17A1 inhibition does not completely abolish androgen biosynthesis. We discovered a CYP17A1-independent pathway for androgen synthesis in prostate cancer cells, including LNCaP, C4-2, and LAPC4. These cells produce androgens even in charcoal-stripped serum (CSS) media, as confirmed by 13C-labeled cholesterol tracing followed by liquid chromatography-mass spectrometry (LC-MS) analysis. The de novo synthesis was entirely abrogated by the knockout of 3β-hydroxysteroid dehydrogenase type 1 (HSD3B1). As 3βHSD1 is an essential enzyme to convert the 3β-OH and Δ5-steroid A/B ring steroids to the 3-keto structures of testosterone and 5α-dihydrotestosterone (DHT), this finding confirms that the alternative pathway converges on the canonical route before the synthesis of androstenedione. No CYP17A1 activity was detectable in C4-2 cells, yet androgen production persisted, strongly indicating a bypass mechanism. Specific CYP17A1 inhibitors, including TAK700 and ASN001, failed to suppress androgen synthesis in C4-2 cells, even at concentrations designed to eliminate any residual activity. Screening a panel of oxysterols identified 17α,20-dihydroxycholesterol (DHC) as a substrate that is converted to DHEA and subsequently to testosterone and dihydrotestosterone (DHT). The conversion was confirmed by deuterium-labeled DHC as a stable isotope tracer and observed in other human reproductive cell lines. We verified that androgen receptor activity was activated by androgens metabolized from DHC. Among 57 human cytochrome P450 enzymes tested, only cytochrome P450 51A1 (CYP51A1), the human ortholog of CYP51, cleaves the DHC to DHEA. Specific CYP17A1 inhibitors, including abiraterone, galeterone, TAK700, and ASN001, failed to block CYP51A1-mediated DHC-to-DHEA conversion. DHEA accumulated in abiraterone and galeterone treated samples because the inhibitors blocked 3βHSD activity. In contrast, the broad-spectrum cytochrome P450 inhibitor ketoconazole effectively inhibited the 17-20 carbon bond cleavage, highlighting CYP51A1's distinct pharmacological role. CYP51 orthologs from vertebrates possess this activity, while invertebrate counterparts do not, suggesting the activity may co-evolved with vertebrate steroidogenesis. Finally, human prostate samples can convert d2-DHC to d2-DHEA and subsequent androgens. The clinical importance of this finding needs to be further investigated. Citation Format: Ziqi Zhu, Yoon-Mi Chung, Mohammad Alyamani, Yijing Dai, Kevin D. McCarty, Evan Roberts, Sunita Sinha, Jianneng Li, Xiuxiu Li, Emad M. Gad, Zhiqun Zhou, Jinyuan Shi, Robert A. Burgess, Tatiana Y. Hargrove, Galina I. Lepesheva, Frederick P. Guengerich, Richard J. Auchus, Nima Sharifi. A bypass gateway from cholesterol to sex steroid biosynthesis circumnavigates CYP17A1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(2_Suppl):Abstract nr PR026.