The androgen receptor (AR) transcription factor plays a key role in the development and progression of prostate cancer. Inhibition of its ligand by androgen deprivation therapy (ADT) is consequently the main medical treatment of invasive prostate cancer. AR is activated and maintained throughout prostate cancer progression even in castration resistant prostate cancer (CRPC). Prostate cancer cells escape from ADT using a variety of mechanisms. The AR and target genes have therefore become even more focused therapeutic targets in aggressive and disseminated prostate cancer. AR and its classical target genes, such as KLK3 (PSA) are, however, efficiently shut off in basal epithelial prostate cells and possibly in prostate cancer stem cells (CSCs). To investigate mechanisms of AR and AR target gene activation in different subpopulations of prostate cancer cells, several androgen response elements (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 recording. The 241B promoter sequence reporter was selected among the constructed ARE reporters on the basis of higher activity in initial screenings. The AR positive and androgen responsive prostate cancer cell line, LNCaP, and the prostate transit amplifying epithelial cell line, EP156T-AR with exogenous AR, were transduced with the lentiviral 241B-mCherry fluorescence reporter. Flow cytometry, multi-well reader and fluorescence microscopy results corresponded when reporter cells were treated with androgen and with the AR antagonist, enzalutamide, and with the anti-androgen, abiraterone. An SV40-promoter GFP reporter element was next cloned into the 241B-mCherry reporter. Constitutive expression of GFP facilitated normalization of ARE driven mCherry fluorescent signals. Furthermore, AR expression vectors were also constructed with the AR open reading frames cloned into lentiviral expression vectors and used in co-transfection and co-transduction experiments in AR negative cell types. The developed ARE reporter system will help us to investigate the role of the AR in differentiation and proliferation of prostate cells and to study the AR activity in two and three dimensional cell cultures. This system can also be useful in screening for drugs with activity against the AR.

Citation Format: Waqas Azeem, Margrete R. Hellem, Jan R. Olsen, Yaping Hua, Kristo Marvyin, Lisha Li, Yi Qu, Biaoyang Lin, Xisong Ke, Anne M. Oyan, Karl-Henning Kalland. Assays for androgen receptor activity using cell based ARE reporter systems. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2902.