Abstract
Prostate cancer remains a deadly disease especially when patients become resistant to drugs that target the Androgen Receptor (AR) ligand binding domain. At this stage, patients develop recurring castrate-resistant prostate cancers (CRPCs). Interestingly, CRPC tumors maintain dependency on AR for growth; moreover, in CRPCs, constitutively active AR splice variants (e.g., AR-V7) begin to be expressed at higher levels. These splice variants lack the ligand binding domain and are rendered insensitive to current endocrine therapies. Thus, it is of paramount importance to understand what regulates the expression of AR and its splice variants to identify new therapeutic strategies in CRPCs. Here, we used high throughput microscopy and quantitative image analysis to evaluate effects of selected endocrine disruptors on AR levels in multiple breast and prostate cancer cell lines. Bisphenol AP (BPAP), which is used in chemical and medical industries, was identified as a down-regulator of both full length AR and the AR-V7 splice variant. We validated its activity by performing time-course, dose-response, Western blot and qPCR analyses. BPAP also reduced the percent of cells in S phase, which was accompanied by a ~60% loss in cell numbers and colony formation in anchorage-independent growth assays. Moreover, it affected mitochondria size and cell metabolism. In conclusion, our high content analysis-based screening platform was used to classify the effect of compounds on endogenous ARs, and identified BPAP as being capable of causing AR (both full-length and variants) down-regulation, cell cycle arrest and metabolic alterations in CRPC cell lines.
Highlights
Androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily regulating the expression of genes that are important in development, differentiation, and cancer progression, especially of the prostate [1]
The data indicate that, as expected, AR expression and translocation increased following treatment with DHT to different degrees in all the cell lines except 22Rv1. This is largely due to the high expression of several constitutively nuclear AR splice variants (e.g., AR-V7) lacking the ligand binding domain (LBD) that are recognized by the N-terminal AR antibody
Cytoplasmic/ nuclear translocation was more evident in the breast cancer cell lines, suggesting that these cells may be more useful for screening compounds that affect AR cytoplasmic/nuclear ratio, and further support testing of multiple model systems to gain a clearer picture of the effects of different classes of compounds
Summary
Androgen receptor (AR) is a member of the nuclear receptor (NR) superfamily regulating the expression of genes that are important in development, differentiation, and cancer progression, especially of the prostate [1]. Prostate cancers are generally treated by blocking AR activity using some form of androgen ablation therapy that targets the AR ligand binding domain (LBD); this approach has been shown to be therapeutically very effective, at least initially [1, 4]. CRPC tumors do not lose dependency on the AR for growth. Another layer of complexity in CRPCs is caused by the appearance of constitutively active AR splice variants, which lack the C-terminal LBD, and cannot be targeted by current therapies [6,7,8]. Identifying small molecules that reduce AR levels is an attractive avenue for treatment of CRPC patients, especially since all available therapies leave untouched any of the splice variants
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