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 3

BMX directly binds 3βHSD1 and phosphorylates Y344.

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BMX directly binds 3βHSD1 and phosphorylates Y344. (A) LNCaP cells overe...
(A) LNCaP cells overexpressing 3βHSD1 were treated with ibrutinib for 1.5 hours with or without DHEA for 0.5 hours. Pan-phospho-tyrosine (pTyr) was detected by immunoprecipitation and Western blot. (B and C) Cells overexpressing 3βHSD1 were treated with ibrutinib or zanubrutinib for 3 hours, and DHEA for 1 hour (B) or 2 hours (C). Phospho-3βHSD1-Y344 was detected by immunoprecipitation and Western blot. Blots run in parallel, contemporaneously, using identical samples are shown. (D) Cells with cooverexpression of GST-3βHSD1 and HA-BMX or vehicle were treated with DHEA for 1 hour. Phospho-3βHSD1-Y344 was detected by immunoprecipitation and Western blot. Actin blots, serving as loading controls, were run in parallel, contemporaneously using identical samples with other blots. (E) Cells overexpressing GST-3βHSD were transfected with siNT or 1 of 2 siRNA sequences against BMX; phospho-3βHSD1-Y344 was detected by GST pull-down and Western blot. 3βHSD1 blots, serving as loading controls, were run in parallel, contemporaneously using identical samples with other blots. (F) Cells with cooverexpression of GST-3βHSD and HA-BMX or HA-BMX-KD (kinase dead) were treated with DHEA for 1 hour. Phospho-3βHSD1-Y344 was detected by immunoprecipitation and Western blot. (G) GST-3βHSD or HA-BMX was purified from 293T cells; 3βHSD1-GST was dephosphorylated using phosphatase in vitro, followed by a kinase assay and Western blot. (H–J) 293T cells were transfected with 3βHSD1 or Y344F mutant with or without cooverexpressed HA-BMX. 3βHSD1 or 3βHSD1-Y344F mutant was immunopurified, and an NAD+ turnover assay was performed. Mean ± SEM represents combined data from 3 independent experiments. *P < 0.05, ***P < 0.001 (1-way ANOVA with Bonferroni’s multiple comparisons test). (K) GST-tagged and flag-tagged WT or Y344F-3βHSD1 were transfected into C4-2 cells, GST pull-down was performed, and flag-tagged 3βHSD1 was detected by Western blot. Cells were treated with DHEA for 2 hours. (L) GST-tagged and flag-tagged WT or Y344F-3βHSD1 were transfected into C4-2 cells, GST pull-down was performed, and flag-tagged 3βHSD1 was detected by Western blot. Cells were treated with DHEA for 2 hours; zanubrutinib (10 μM) treatment was 24 hours.