Simulating androgen receptor selection in designer yeast

Abstract

Androgen receptor (AR) mutation is closely associated with prostate cancer (PCa) and is one of the mechanisms of resistance to PCa therapies such as AR antagonists. Although sequencing technologies like next-generation sequencing (NGS) contributes to the high-throughput and precise detection of AR mutations carried by PCa patients, the lack of interpretations of these clinical genetic variants has still been a roadblock for PCa-targeted precision medicine. Here, we established a designer yeast reporter assay to simulate natural androgen receptor (AR) selection using AR antagonists. Yeast HIS3 gene transactivation was associated with the ligand-induced recruitment of steroid receptor coactivator-1 (SRC-1) by AR mutants, where yeast growth in histidine-free medium was determined as the outcome. This assay is applicable to determine a wide range of clinical AR mutants including those with loss of function relating to androgen insensitivity syndrome (AIS), and those associated with PCa conferring resistance to AR antagonists such as enzalutamide (ENZ), bicalutamide (BIC), and cyproterone acetate (CPA). One clinical AR mutant previously reported to confer ENZ-resistance, F877L, was found to confer partial resistance to CPA as well using designer yeast. Our simple and efficient assay can enable precise one-pot screening of AR mutants, providing a reference for tailored medicine.

Keywords: Androgen receptor antagonists; Androgen receptor mutations; Prostate cancer; Saccharomyces cerevisiae.

Fig. 1

Construction of designer yeast to identify AR mutations. a-b, Schematic representation of designer yeast. c, Designer yeast integrity requires the presence of AR-LBD and SRC-1, and DHT stimulation (n = 3). Bars indicate mean ± s.d. d, The HIS3 transcript was induced ～10-fold higher upon DHT stimulation (n = 3). DHT concentration was 10 nM. Bars indicate mean ± s.d. e, Readouts from five independent tests validate the reliability of designer yeast (n = 3, 10 nM DHT). Bars indicate mean ± s.d. f, DHT dose-response assay in designer yeast using both liquid and solid cultures (n = 3). Bars indicate mean ± s.d. g, Loss-of-function AR mutants identified in AIS patients exhibited severely impaired activity in response to DHT in designer yeast.

Fig. 2

The identification of AR mutants conferring resistance to clinical antagonists. a, Designer yeast can help streamline personalized PCa therapy and serve as a prescription reference. b, As delineated in a, tests of a panel of AR-LBD mutants against clinical AR antagonists (ENZ, BIC, and CPA) in yeast (n = 3). Ligand conditions: 500 μM CPA; 100 μM ENZ; 200 μM BIC; 100 nM DHT (positive control); Vehicle (dimethyl sulfoxide, negative control). OD600 was measured at 36 h for ENZ/CPA/DHT and at 60 h for BIC/Vehicle. Plate pictures were taken at 48 h for CPA/DHT, at 72 h for ENZ, and at 96 h for BIC/Vehicle. Bars indicate mean ± s.d. c, Dose-response assay of AR antagonists in designer yeast (n = 3). Bars indicate mean ± s.d.

Fig. 3

The identification of AR mutants conferring resistance to clinical antagonists. a, An additional T878A mutation amplifies the resistance of the AR-F877L mutant to ENZ (n = 3). Ligand conditions: 500 nM CPA; 50 μM ENZ; 100 μM BIC. Bars indicate mean ± s.d. b, ENZ dose-response assay in plate culture. c, Growth curve of indicated AR mutation upon treatment of 500 nM CPA (n = 3). Bars indicate mean ± s.d. d, Ranking results of the AR-F877L mutant, AR-T878A mutant, and AR-F877L/T878A double mutant against CPA using liquid culture (n = 3). Bars indicate mean ± s.d. e, Ranking results of the AR-F877L mutant, AR-T878A mutant, and AR-F877L/T878A double mutant against CPA using plate culture. f, AR mutations, W742L and W742C, exhibited similar resistance level against BIC in designer yeast (n = 3). Bars indicate mean ± s.d.

Fig. 4

One-pot screening assay to simulate natural AR selection against clinical AR antagonists with high sensitivity. a, Schematic workflow of the pooled screening method in designer yeast. b, Components in the AR-F877-library were first individually assessed in yeast before the library was subjected to the pooled screening method in a. Ligand conditions: 100 nM DHT; 500 nM CPA, 100 μM ENZ; 200 μM BIC. c, High-throughput Sanger sequencing results of the AR-F877-library against DHT, ENZ, and CPA.