Unique bisphenol A transcriptome in prostate cancer: novel effects on ERbeta expression that correspond to androgen receptor mutation status

Abstract

Background: Prostatic adenocarcinomas are dependent on androgen receptor (AR) activity for growth and progression, and therapy for disseminated disease depends on ablation of AR activity. Recurrent tumors ultimately arise wherein AR has been re-activated. One mechanism of AR restoration is via somatic mutation, wherein cells containing mutant receptors become susceptible to activation by alternative ligands, including bisphenol A (BPA). In tumors with specific AR mutations, BPA promotes therapeutic bypass, suggesting significant negative impact to the clinical management of prostate cancer.

Objective: Our goal was to determine the mechanism of BPA action in cancer cells carrying BPA-responsive AR mutants.

Methods: The molecular signature of BPA activity in prostate cancer cells harboring mutant AR was delineated via genetic microarray analysis. Specificity of BPA action was assessed by comparison with the molecular signature elicited by dihydrotestosterone (DHT).

Results: BPA and DHT elicited distinct transcriptional signatures in prostate cancer cells expressing the BPA-responsive mutant AR-T877A. BPA dramatically attenuated estrogen receptor beta (ERbeta) expression; this finding was specific to prostate tumor cells in which BPA induces cellular proliferation.

Conclusions: BPA induces a distinct gene expression signature in prostate cancer cells expressing somatic AR mutation, and a major molecular consequence of BPA action is down-regulation of ERbeta. Since ERbeta functions to antagonize AR function and AR-dependent proliferation, these findings reveal a novel mechanism by which BPA likely regulates cellular proliferation. Future investigation directed at dissecting the importance of ERbeta in the proliferative response to BPA will establish the contribution of this event to adverse effects associated with human exposure.

Keywords: androgen receptor; endocrine disruptor; microarray; prostatic adenocarcinoma; xeno-estrogen.

Figure 1

BPA induces androgen-independent cellular proliferation in cells expressing AR-T877A. (A) LNCaP cells were cultured under conditions of steroid hormone ablation (5% charcoal-/dextran-treated serum, CDT) and subsequently stimulated with either DHT, BPA, or vehicle control [0.1% ethanol (EtOH)]. (B) Cells cultured as in A were pulse labeled with BrdU for the last 16 hr of treatment, fixed, and BrdU incorporation was quantified by indirect immunofluorescence. Percent BrdU positive cells is shown. Error bars represent mean ± SD.

Figure 2

BPA induces a unique transcriptome in prostate cancer cells expressing AR-T877A. Microarray analyses were performed using triplicate biological replicates (n = 9) and statistical analyses performed as described in “Materials and Methods.” (A) Heat map of the fold change estimates, averaged across sample, showing all genes with a statistically significant, > 2-fold alteration over vehicle control. Column 1 is statistically significant changes in averaged gene expression in BPA-treated samples compared with EtOH control; column 2 represents the statistically significant changes in expression after DHT relative to EtOH control. Column 3 is a comparative analysis and represents the genes that were statistically significant for BPA exposure compared with DHT treatments. Green bars indicate reduced expression, and red bars indicate induced gene expression. The bar representing PSA (KLK3) is indicated. Microarray experiment was performed on three independent experiments in triplicate. (B) VENN diagrams highlight the disparity in DHT and BPA effects on gene regulation. C) Gene Ontology was performed as described in “Materials and Methods.” Categories passing statistical significance are shown. Gene annotations are from Unigene (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unigene).

Figure 3

Validation of selected targets reveals that BPA significantly down-regulates ERβ expression in cells expressing the BPA-responsive AR-T877A mutant. A) Validation of alterations in PSA expression was performed by RT-PCR. As expected, both DHT and BPA induced PSA expression in cells expressing AR-T877A (left panel). In agreement with the microarray, it was also noted that BPA induces down-regulation of ERβ. By contrast, marked induction of WISP3 was also noted, and no effect was observed with FKBP5 (right panel). Numbers correspond to the band intensity of each sample set to EtOH (EtOH = 1) and relative to the GAPDH RNA control. B) The impact of BPA on ERβ was quantified by real-time PCR, and relative copy number was determined. Error bars represent mean ± SD; n = 9; *p < 0.05, **p < 0.01. (C) Representative (n = 3) immunoblot of ERβ protein levels after BPA or DHT exposure. As shown, BPA exposure causes a marked reduction in ERβ accumulation.

Figure 4

Specificity of BPA-mediated modulation of ERβ. ERβ expression was monitored using at least two independent biological replicates for each condition and cell line, analyzed in triplicate and quantified by real-time PCR in cells expressing AR-H874Y (22Rv1, A) or wild-type AR (LAPC4, B). Error bars represent mean ± SD.