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.
Highlights
Androgen receptor (AR) plays a critical role in the normal development and function of the prostate gland [1]
AR remains critically important in prostate cancer as shown by the fact that castration resistant prostate cancer (CRPC) may be independent of androgen levels, it continues to rely on AR signaling
The pLenti6.3/AR-GC-E2325 androgen receptor expression vector was selected in initial screenings on the basis of high-level exogenous AR expression
Summary
Androgen receptor (AR) plays a critical role in the normal development and function of the prostate gland [1]. AR, named NR3C4 (nuclear receptor subfamily 3, group C, member 4), belongs to the steroid hormone group of nuclear receptors [2]. It has four distinct structural and functional domains, a less conserved N-terminal domain (NTD), the central highly conserved DNA-binding domain (DBD) which is connected to the moderately conserved C-terminal ligand binding domain (LBD) by a flexible hinge region [3]. The AR homodimer recruits a variety of co-regulatory proteins and binds to specific DNA sequences, termed androgen response elements (ARE), in the promoter and enhancer regions of target genes, such as prostate-specific antigen (PSA) and transmembrane protease serine 2 (TMPRSS2), and modulates the transcription of androgen responsive genes [7,8,9,10]. The ARE consists of the consensus sequence for AR binding in two equal, hexameric half-sites organized in direct repeats formation, separated by a three base-pairs spacer (IR3) [11]
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