BMX controls 3βHSD1 and sex steroid biosynthesis in cancer
Xiuxiu Li, Michael Berk, Christopher Goins, Mohammad Alyamani, Yoon-Mi Chung, Chenyao Wang, Monaben Patel, Nityam Rathi, Ziqi Zhu, Belinda Willard, Shaun Stauffer, Eric Klein, Nima Sharifi
Xiuxiu Li, Michael Berk, Christopher Goins, Mohammad Alyamani, Yoon-Mi Chung, Chenyao Wang, Monaben Patel, Nityam Rathi, Ziqi Zhu, Belinda Willard, Shaun Stauffer, Eric Klein, Nima Sharifi
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Research Article Oncology

BMX controls 3βHSD1 and sex steroid biosynthesis in cancer

Abstract

Prostate cancer is highly dependent on androgens and the androgen receptor (AR). Hormonal therapies inhibit gonadal testosterone production, block extragonadal androgen biosynthesis, or directly antagonize AR. Resistance to medical castration occurs as castration-resistant prostate cancer (CRPC) and is driven by reactivation of the androgen-AR axis. 3β-hydroxysteroid dehydrogenase-1 (3βHSD1) serves as the rate-limiting step for potent androgen synthesis from extragonadal precursors, thereby stimulating CRPC. Genetic evidence in men demonstrates the role of 3βHSD1 in driving CRPC. In postmenopausal women, 3βHSD1 is required for synthesis of aromatase substrates and plays an essential role in breast cancer. Therefore, 3βHSD1 lies at a critical junction for the synthesis of androgens and estrogens, and this metabolic flux is regulated through germline-inherited mechanisms. We show that phosphorylation of tyrosine 344 (Y344) occurs and is required for 3βHSD1 cellular activity and generation of Δ4, 3-keto-substrates of 5α-reductase and aromatase, including in patient tissues. BMX directly interacts with 3βHSD1 and is necessary for enzyme phosphorylation and androgen biosynthesis. In vivo blockade of 3βHSD1 Y344 phosphorylation inhibits CRPC. These findings identify what we believe to be new hormonal therapy pharmacologic vulnerabilities for sex-steroid dependent cancers.

Authors

Xiuxiu Li, Michael Berk, Christopher Goins, Mohammad Alyamani, Yoon-Mi Chung, Chenyao Wang, Monaben Patel, Nityam Rathi, Ziqi Zhu, Belinda Willard, Shaun Stauffer, Eric Klein, Nima Sharifi

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Figure 2

BMX is required for DHEA metabolism by 3βHSD1.

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BMX is required for DHEA metabolism by 3βHSD1. (A) LNCaP cells were trea...
(A) LNCaP cells were treated with ibrutinib or zanubrutinib for 1 hour and subsequently treated with [3H]-DHEA for 5 hours, followed by steroid extraction from media and steroid separation and quantitation with HPLC. The experiment was done in triplicate and repeated in independent experiments. Mean ± SEM represents 3 replicates in 1 experiment. 3 independent experiments were performed. **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA test with Dunnett’s multiple comparisons test). (B) Cells stably expressing shNT or 2 shRNA sequences against BMX were treated with [3H]-DHEA for 6 hours and analyzed as in (A). Mean ± SEM represents combined data from 3 biological independent replicates performed in technical triplicate. ***P < 0.001, ****P < 0.0001 (1-way ANOVA test with Dunnett’s multiple comparisons test). (C) 293T cells were transiently cotransfected with HA-BMX, EGFR, SRC, or YES and GST-3βHSD1, followed by GST pull-down and Western blot (left). 293T cells were transiently cotransfected with HA-BMX and GST-3βHSD1, followed by HA immunoprecipitation and Western blot (right). (D) LNCaP and C4-2 cells were cultured, protein collected, and immunoprecipitation and Western blot performed for endogenously expressed proteins. WCL blots run in parallel, contemporaneously, using identical samples are shown. (E) LNCaP cells were transiently cotransfected with HA-BMX, and GST-3βHSD1cells were starved with medium containing 10% charcoal-stripped FBS for 24 hours, then treated with steroids for 2 hours, followed by GST-pull down and Western blot to detect interaction of HA-BMX and GST-3βHSD1. (F) LNCaP cells were starved as in (E), then transfected with HA-BMX and treated with steroids for 2 hours; p-BMX was detected by Western blot. Blots run in parallel, contemporaneously, using identical samples are shown. (G) Stable C4-2 cell lines with HSD3B1 gRNA or control gRNA were transfected with HA-BMX, starved as in (E) and treated with DHEA for 2 hours; p-BMX was detected by Western blot.