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

<div><p>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.</p></div

ARE promoter response and sensitivity to AR activity in 293FT cells.

<p>(A) Three different ARE promoter sequences, 241B, 248B and 249B inserted in a Gaussia luciferase reporter vector, were co-transfected with AR expression vector pLenti6.3/AR-GC-E2325 into 293FT cells. Cells were treated with 1 nM R1881 for 24 hours and Gaussia luciferase values were detected. These values were normalized by the MTS assay. (B) The 241B ARE promoter mCherry reporter vector was co-transfected with AR expression vector pLenti6.3/AR-GC-E2325 in 293FT cells. Fluorescence microscopy was performed after stimulating the cells with 1 nM R1881 for 24 hours. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

Comparison of different AR expression vectors in 293FT cells.

<p>293FT cells were co-transfected with ARE reporter CCS-1019L and three separate AR expression vectors. The cells were grown with or without synthetic androgen 1 nM R1881 for 24 hours. The AR expression was detected by measuring Firefly luminescent signals. These values were normalized by Renilla luminescence values. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

ARE reporter response to different steroids.

<p>FACS analysis of mCherry signals in (A) EP156T-AR-241B, (B) 957E/hTERT-AR-241B and (C) LNCaP-241B cells. Cells were grown in androgen free medium and treated with different AR agonists (1 nM R1881 and 1 μM DHT), steroids (10 nM dexamethasone, 10 nM progesterone and 1 μM β-estradiol), AR antagonist (10 μM enzalutamide) and anti-androgen (10 μM abiraterone). The error bars show the standard deviation (SD) from three independent experiments.</p

Internal normalization of ARE reporter signals in prostate cells.

<p>(A) FACS analysis of mCherry fluorescent signals in LNCaP-207-01 and LNCaP-207-02 cells grown in androgen free medium. Cells were treated with different concentrations of androgen R1881, <i>i</i>.<i>e</i>. 0.1 nM, 1 nM and 10 nM, and 10 μM enzalutamide (enza) with 1 nM R1881 for 24 hours. The values were normalized by constitutively expressed GFP fluorescent signals. (B) Multi-well mCherry RFU measurements of LNCaP-207-01 and LNCaP-207-02 cells grown in androgen free medium. The cells were treated with different concentrations of androgen R1881, <i>i</i>.<i>e</i>. 0.1 nM, 1 nM and 10 nM, and 10 μM enzalutamide with 1 nM R1881 for 24 hours. The mCherry RFU values were normalized by GFP RFU. (C) Fluorescence microscopy of LNCaP-207-01 cells treated with ± 1 nM R1881 and different concentrations of enzalutamide, <i>i</i>.<i>e</i>. 5 μM, 10 μM and 20 μM for 24 hours. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.</p

ARE reporter response in mesenchymal type cells.

<p>(A) FACS analysis of mCherry signals in EPT3-PT1-AR-241B cells. Cells were grown in androgen free medium and treated with different AR agonists (1 nM R1881 and 1 μM DHT), steroids (10 nM dexamethasone, 10 nM progesterone and 1 μM β-estradiol), AR antagonist (10 μM enzalutamide) and anti-androgen (10 μM abiraterone). (B) Texas red (Tx-Red) indirect immunofluorescent detection of AR in EPT3-PT1-AR-241B cells. The cells were treated with ± 1 nM R1881 for 24 hours.</p

AR activity response in ARE reporter inserted prostate cells.

<p>(A) FACS analysis of mCherry fluorescent signals in LNCaP-241B cells grown in androgen free or supplemented with R1881 medium. The cells were treated with 10 μM enzalutamide and 10 μM abiraterone for 24 hours. (B) Relative fluorescence units (RFU) measurements of mCherry signals in EP156T-AR-241B cells grown in androgen free growth medium in 96-well plates. Cells were treated with ± 1 nM R1881 and increasing concentrations of enzalutamide, <i>i</i>.<i>e</i>. 5 μM, 10 μM and 20 μM for 24 hours. The RFU values were normalized by the MTS assay. (C) Fluorescence microscopy of EP156T-AR-241B cells treated with ± 1 nM R1881 and different concentrations of enzalutamide (enza), <i>i</i>.<i>e</i>. 5 μM, 10 μM and 20 μM for 24 hours. The error bars show the standard error of the mean (SEM) from three independent experiments. Significance was confirmed by using unpaired two-tailed Student’s t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.</p

AR protein localization in prostate cells.

<p>Texas red (Tx-Red) indirect immunofluorescent detection of AR in (A) EP156T-AR, (B) 957E/hTERT-AR and (C) LNCaP cells. The cells were treated with ± 1 nM R1881 for 24 hours.</p

ARE consensus promoter sequences.

<p>ARE consensus promoter sequences.</p