An androgen response element driven reporter assay for the detection of androgen receptor activity in prostate cells

Abstract

The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer, as is evident from the efficacy of androgen-deprivation therapy, AR is also the most frequently mutated gene, in castration resistant prostate cancer (CRPC). AR has therefore become an even more attractive therapeutic target in aggressive and disseminated prostate cancer. To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, a toolkit of AR expressor and androgen response element (ARE) reporter vectors were developed. Three ARE reporter vectors were constructed with different ARE consensus sequences in promoters linked to either fluorescence or luciferase reporter genes in lentiviral vector backbones. Cell lines transduced with the different vectors expressed the reporters in an androgen-dependent way according to fluorescence microscopy, flow cytometry and multi-well fluorescent and luminescence assays. Interestingly, the background reporter activity in androgen-depleted medium was significantly higher in LNCaP cells compared to the prostate transit amplifying epithelial cell lines, EP156T-AR and 957E/hTERT-AR with exogenous AR. The androgen-induced signal to background was much higher in the latter benign prostate cells than in LNCaP cells. Androgen-independent nuclear localization of AR was seen in LNCaP cells and reduced ARE-signaling was seen following treatment with abiraterone, an androgen synthesis inhibitor. The ARE reporter activity was significantly stronger when stimulated by androgens than by β-estradiol, progesterone and dexamethasone in all tested cell types. Finally, no androgen-induced ARE reporter activity was observed in tumorigenic mesenchymal progeny cells of EP156T cells following epithelial to mesenchymal transition. This underscores the observation that expression of the classical luminal differentiation transcriptome is restricted in mesenchymal type cells with or without AR expression, and presence of androgen.

MiR-205-driven downregulation of cholesterol biosynthesis through SQLE-inhibition identifies therapeutic vulnerability in aggressive prostate cancer

“…To address this question, we used an AR-responsive reporter construct in which a sequence of six copies of the androgen-response element (ARE) had been cloned in front of the luciferase gene 38 . We first confirmed that treatment with dihydrotestosterone (DHT) resulted in a robust activation of this reporter construct in LNCaP and C4-2 cells (Supplementary Fig.…”

Dual Pro- and Anti-Inflammatory Features of Monocyte-Derived Dendritic Cells

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AR-V7 exhibits non-canonical mechanisms of nuclear import and chromatin engagement in Castrate-Resistant Prostate Cancer

“…AR-V7 transcriptional activity has been shown to depend on the D-box domain shown to mediate AR-V7 homodimerization in the nucleus (Centenera et al, 2008; Xu et al, 2015). To determine the potential impact of D-box domain on AR-V7 nuclear import which is a pre-requisite for its transcriptional activity, we generated a construct encoding GFP-AR-V7 containing two established, functionally-inactivating D-box mutations (A596T, S597T) and transiently transfected PC3 cells stably expressing the ARE-mCherry reporter in order to measure ARE activity in single cells (Azeem et al, 2017). Cells transfected with GFP-AR-V7 alone had an average of 71% nuclear AR-V7 with concomitant mCherry expression indicating ARE-transactivation.…”