Proteasome‐mediated regulation of GATA2 expression and androgen receptor transcription in benign prostate epithelial cells

GATA2 has been shown to be an important transcription factor together with androgen receptor (AR) in prostate cancer cells. Less is known about GATA2 in benign prostate epithelial cells. We have investigated if GATA2 exogenous expression in prostate epithelial basal-like cells could induce AR transcription or luminal differentiation. Prostate epithelial basal-like (transit amplifying) cells were transduced with lentiviral vector expressing GATA2. Luminal differentiation markers were assessed by RT-qPCR, Western blot and global gene expression microarrays. We utilized our previously established AR and androgen-dependent fluorescence reporter assay to investigate AR activity at the single-cell level. Exogenous GATA2 protein was rapidly and proteasome-dependently degraded. GATA2 protein expression was rescued by the proteasome inhibitor MG132 and partly by mutating the target site of the E3 ligase FBXW7. Moreover, MG132-mediated proteasome inhibition induced AR mRNA and additional luminal marker gene transcription in the prostate transit amplifying cells. Different types of intrinsic mechanisms restricted GATA2 expression in the transit amplifying cells. The appearance of AR mRNA and additional luminal marker gene expression changes following proteasome inhibition suggests control of essential cofactor(s) of AR mRNA expression and luminal differentiation at this proteolytic level.publishedVersio

Proteasome‐mediated regulation of GATA2 expression and androgen receptor transcription in benign prostate epithelial cells

GATA2 has been shown to be an important transcription factor together with androgen receptor (AR) in prostate cancer cells. Less is known about GATA2 in benign prostate epithelial cells. We have investigated if GATA2 exogenous expression in prostate epithelial basal-like cells could induce AR transcription or luminal differentiation. Prostate epithelial basal-like (transit amplifying) cells were transduced with lentiviral vector expressing GATA2. Luminal differentiation markers were assessed by RT-qPCR, Western blot and global gene expression microarrays. We utilized our previously established AR and androgen-dependent fluorescence reporter assay to investigate AR activity at the single-cell level. Exogenous GATA2 protein was rapidly and proteasome-dependently degraded. GATA2 protein expression was rescued by the proteasome inhibitor MG132 and partly by mutating the target site of the E3 ligase FBXW7. Moreover, MG132-mediated proteasome inhibition induced AR mRNA and additional luminal marker gene transcription in the prostate transit amplifying cells. Different types of intrinsic mechanisms restricted GATA2 expression in the transit amplifying cells. The appearance of AR mRNA and additional luminal marker gene expression changes following proteasome inhibition suggests control of essential cofactor(s) of AR mRNA expression and luminal differentiation at this proteolytic level

Context dependent regulatory patterns of the androgen receptor and androgen receptor target genes

Background Expression of the androgen receptor (AR) is associated with androgen-dependent proliferation arrest and terminal differentiation of normal prostate epithelial cells. Additionally, activation of the AR is required for survival of benign luminal epithelial cells and primary cancer cells, thus androgen deprivation therapy (ADT) leads to apoptosis in both benign and cancerous tissue. Escape from ADT is known as castration-resistant prostate cancer (CRPC). In the course of CRPC development the AR typically switches from being a cell-intrinsic inhibitor of normal prostate epithelial cell proliferation to becoming an oncogene that is critical for prostate cancer cell proliferation. A clearer understanding of the context dependent activation of the AR and its target genes is therefore desirable. Methods Immortalized human prostate basal epithelial EP156T cells and progeny cells that underwent epithelial to mesenchymal transition (EMT), primary prostate epithelial cells (PrECs) and prostate cancer cell lines LNCaP, VCaP and 22Rv1 were used to examine context dependent restriction and activation of the AR and classical target genes, such as KLK3. Genome-wide gene expression analyses and single cell protein analyses were applied to study the effect of different contexts. Results A variety of growth conditions were tested and found unable to activate AR expression and transcription of classical androgen-dependent AR target genes, such as KLK3, in prostate epithelial cells with basal cell features or in mesenchymal type prostate cells. The restriction of androgen- and AR-dependent transcription of classical target genes in prostate basal epithelial cells was at the level of AR expression. Exogenous AR expression was sufficient for androgen-dependent transcription of AR target genes in prostate basal epithelial cells, but did not exert a positive feedback on endogenous AR expression. Treatment of basal prostate epithelial cells with inhibitors of epigenetic gene silencing was not efficient in inducing androgen-dependent transcription of AR target genes, suggesting the importance of missing cofactor(s). Conclusions Regulatory mechanisms of AR and androgen-dependent AR target gene transcription are insufficiently understood and may be critical for prostate cancer initiation, progression and escape from standard therapy. The present model is useful for the study of context dependent activation of the AR and its transcriptome

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

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 consensus promoter sequences.

<p>ARE consensus promoter sequences.</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

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 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