A Systematic Comparison of Antiandrogens Identifies Androgen Receptor Protein Stability as an Indicator for Treatment Response.

Tiziana Siciliano,Matthias B Stope,Robert I Seed,Cem Aksoy,Christian Thomas,Holger H H Erb,Celina Ebersbach,Ingo H Simons,Alicia-Marie K Beier

doi:10.3390/life11090874

Abstract

Antiandrogen therapy is a primary treatment for patients with metastasized prostate cancer. Whilst the biologic mechanisms of antiandrogens have been extensively studied, the operating protocols used for the characterization of these drugs were not identical, limiting their comparison. Here, the antiandrogens Bicalutamide, Enzalutamide, Apalutamide, and Darolutamide were systematically compared using identical experimental setups. Androgen-dependent LNCaP and LAPC4 cells as well as androgen-independent C4-2 cells were treated with distinct concentrations of antiandrogens. Androgen receptor (AR)-mediated gene transactivation was determined using qPCR. Cell viability was measured by WST1 assay. Protein stability and AR localization were determined using western blot. Response to the tested antiandrogens across cellular backgrounds differed primarily in AR-mediated gene transactivation and cell viability. Antiandrogen treatment in LNCaP and LAPC4 cells resulted in AR protein level reduction, whereas in C4-2 cells marginal decreased AR protein was observed after treatment. In addition, AR downregulation was already detectable after 4 h, whereas reduced AR-mediated gene transactivation was not observed before 6 h. None of the tested antiandrogens displayed an advantage on the tested parameters within one cell line as opposed to the cellular background, which seems to be the primary influence on antiandrogen efficacy. Moreover, the results revealed a prominent role in AR protein stability. It is one of the first events triggered by antiandrogens and correlated with antiandrogen efficiency. Therefore, AR stability may surrogate antiandrogen response and may be a possible target to reverse antiandrogen resistance.

Highlights

Licensee MDPI, Basel, Switzerland.The androgen receptor (AR) is the main driving force behind the growth and progression of prostate cancer (PC), the most common carcinoma in men [1,2]
TMPRSS2 mRNA is significantly downregulated by Bicalutamide and Enzalutamide treatments to ~0.15 fold of 1 nM R1881 at 10 μM (Figure 1B), whereas Darolutamide only reduced
LAPC4 with the the results revealed that the cell line C4-2 responded with the lowest and LAPC4 with the highest sensitivity to antiandrogen treatment regarding Androgen receptor (AR) protein levels (Figure 5H–K)

Summary

Introduction

The androgen receptor (AR) is the main driving force behind the growth and progression of prostate cancer (PC), the most common carcinoma in men [1,2]. The AR belongs to the nuclear receptor family and mediates the actions of androgens. Its topology includes four functional domains: the N-terminal transactivation domain, the Deoxyribonucleic acid (DNA)-binding domain, the hinge region, and the ligand-binding domain. AR signaling is controlled by androgen binding. Caucasian male [19] C4-2. LNCaP sub-cell line [17] LAPC4. AR wt, Androgen dependent non applicable [20] PC3. Cells express no AR protein, Androgen independent, small cell neuroendocrine carcinoma

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