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Tumor and Stem Cell Biology

Androgen and Estrogen Receptors in Breast Cancer Coregulate Human UDP-Glucuronosyltransferases 2B15 and 2B17

Dong G. Hu, Luke A. Selth, Gerard A. Tarulli, Robyn Meech, Dhilushi Wijayakumara, Apichaya Chanawong, Roslin Russell, Carlos Caldas, Jessica L.L. Robinson, Jason S. Carroll, Wayne D. Tilley, Peter I. Mackenzie and Theresa E. Hickey
DOI: 10.1158/0008-5472.CAN-15-3372 Published October 2016

Abstract

Glucuronidation is an enzymatic process that terminally inactivates steroid hormones, including estrogens and androgens, thereby influencing carcinogenesis in hormone-dependent cancers. While estrogens drive breast carcinogenesis via the estrogen receptor alpha (ERα), androgens play a critical role as prohormones for estrogen biosynthesis and ligands for the androgen receptor (AR). In this study, the expression and regulation of two androgen-inactivating enzymes, the UDP-glucuronosyltransferases UGT2B15 and UGT2B17, was assessed in breast cancer. In large clinical cohorts, high UGT2B15 and UGT2B17 levels positively influenced disease-specific survival in distinct molecular subgroups. Expression of these genes was highest in cases positive for ERα. In cell line models, ERα, AR, and the transcription factor FOXA1 cooperated to increase transcription via tandem binding events at their proximal promoters. ERα activity was dependent on FOXA1, facilitated by AR activation, and potently stimulated by estradiol as well as estrogenic metabolites of 5α-dihydrotestosterone. AR activity was mediated via binding to an estrogen receptor half-site 3′ to the FOXA1 and ERα-binding sites. Although AR and FOXA1 bound the UGT promoters in AR-positive/ERα-negative breast cancer cell lines, androgen treatment did not influence basal transcription levels. Ex vivo culture of human breast tissue and ERα+ tumors provided evidence for upregulation of UGT2B15 and UGT2B17 by estrogen or androgen treatment. ERα binding was evident at the promoters of these genes in a small cohort of primary tumors and distant metastases. Collectively, these data provide insight into sex steroid receptor-mediated regulation of androgen-inactivating enzymes in ERα+ breast cancer, which may have subtype-specific consequences for disease progression and outcomes. Cancer Res; 76(19); 5881–93. ©2016 AACR.

Introduction

Breast and prostate cancer are the most common forms of hormone-dependent cancer and account for a major proportion of cancer-related deaths in women and men, respectively. Breast cancer is predominantly driven by aberrant estrogen receptor alpha (ERα) signaling and prostate cancer by aberrant androgen receptor (AR) signaling. These diseases display multiple similarities of etiology and pathology (1). There has been mounting interest in the role of AR in breast cancer following identification of a subgroup (∼12%) that lacks expression of ERα but is AR+ and displays features reminiscent of prostate cancers (2, 3). However, the large majority (>70%) of breast cancers express both sex hormone receptors, and the role of AR in ERα+ breast cancer may be antiestrogenic and, thereby, tumor suppressive (4).

Inactivation and disposal of steroids regulates target cell exposure to steroid receptor agonists, and derangement of these pathways is evident in the pathology of hormone-dependent cancers (5, 6). Several enzymatic pathways inactivate steroids, but glucuronidation is the only irreversible event. Uridine 5′-diphospho (UDP) glucuronosyltransferases (UGT) create steroid conjugates incapable of binding to cognate receptors. The resulting steroid glucuronides are more hydrophilic and more readily excreted from tissues than unconjugated steroids. Therefore, glucuronidation is a vital mechanism that regulates steroid receptor activity within hormone-responsive tissues. Metabolism of estrogenic hormones has been widely investigated in breast cancer (6), but metabolism of androgenic hormones has yet to be extensively investigated in relation to this disease.

Among 19 human UGTs, only a few glucuronidate sex steroids to any significant extent (7, 8). The most potent natural androgens, testosterone and 5α-dihydrotestosterone (DHT), are glucuronidated by UGT2B15 and UGT2B17. Testosterone is a major precursor for synthesis of 17β-estradiol (E2), the most potent natural ERα ligand, while testosterone and DHT are AR ligands. Hence, the activity of UGT2B15 and UGT2B17 functionally impacts ERα and AR activity, but the significance of UGT2B15 and UGT2B17 expression has not been examined in relation to the different molecular subtypes and clinical outcomes of breast cancer.

Primary breast tissues express UGT2B15 and UGT2B17 (9), with evidence of transcriptional upregulation by ERα signaling in breast cancer cell lines (10, 11). One study also reported that DHT weakly stimulated expression of these genes in an ERα-dependent manner (10). The latter finding is feasibly due to metabolism of DHT into 3α-androstanediol (3α-diol) or 3β-androstanediol (3β-diol), compounds that have some estrogenic activity (12), but this has not been tested. Finally, regulation of UGT2B15 and UGT2B17 by AR signaling has not been thoroughly investigated in an ERα+ context and never in an AR+, ERα− breast cancer context, in which the consequences of androgen-metabolizing capacity may markedly differ. The current study was undertaken to fill these gaps in knowledge by examining expression in large clinical cohorts and elucidating mechanisms of transcriptional regulation by sex steroid receptor signaling in breast cancer, taking into account the molecular complexity of this disease.

Materials and Methods

Chemicals and cell culture

Steroids and steroid receptor antagonists were purchased from Sigma-Aldrich, including E2, DHT, 5α-androstane-3α,17β-diol (3α-diol), 5α-androstane-3β,17β-diol (3β-diol), methyltrienolone (R1881), 4-hydroxy-flutamide (Flut), fulvestrant (ICI 182,780), and bicalutamide. Enobosarm was provided by the manufacturer (GTx, Inc). The MCF7, ZR75-1, and MDA-MB-453 breast cancer cell lines were obtained from the ATCC and the MFM223 line from the German Collection of Microorganisms and Cell Cultures (DMSZ); these were regularly verified for identity via short tandem repeat analyses. MCF7 and ZR75-1 cells were maintained in RPMI1640 medium (Invitrogen), MDA-MB-453 cells in DMEM (Gibco), and MFM223 cells in EMEM (Gibco) containing either 5% (v/v; MCF7, ZR75-1, MDA-MB-453) or 10% (v/v; MFM223) FBS (Gibco) at 37°C and 5% CO2.

Clinical tissues and ex vivo culture

Normal breast tissues from reduction mammoplasties were obtained from Flinders Medical Centre (Adelaide, Australia). Primary ERα+ breast tumor samples were obtained from the Burnside Private Hospital (Adelaide, Australia). All tissues were collected after provision of informed, written consent by the donors. This study was approved by the University of Adelaide Human Research Ethics Committee (approval numbers: H-065-2005; H-169-2011). Tissue pieces were cultured on hydrated gelatin sponges (3–4/sponge) as described previously (13, 14). After a 36-hour preincubation period, hormones were added to the media as indicated and tissues harvested 24 hours later for RNA extraction. Representative pieces of uncultured tissue were fixed in 4% formalin in PBS at 4°C overnight, then processed into paraffin blocks. Sections (2 μm) were (i) stained with hematoxylin and eosin and examined by a pathologist to quantify the relative proportion of tumor cells; and (ii) immunostained for ERα and AR, as described previously (14). Treatment conditions included vehicle (0.01% ethanol), E2 (10 nmol/L), DHT (10 nmol/L), enobosarm (100 nmol/L).

RNA extraction and transcriptional analyses

Cell lines were cultured for 3 days in phenol red-free medium supplemented with 5% dextran-coated charcoal (DCC)-stripped FBS, then seeded under the same conditions into 6-well plates at 5 × 105 cells per well. Three days later, cells were treated for 24 hours with treatments as indicated. RNA extraction, reverse transcription, and qRT-PCR were performed as previously reported (11, 15, 16). For tissue samples, replicate pieces representing one condition were snap frozen in one tube containing RNAlater (Qiagen) and RNA isolated using RNeasy Kits (Qiagen) according to the manufacturer's protocol. Reverse transcription was performed using total RNA (500 ng–1 μg) and an iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was diluted 1:10 and used for transcriptional analysis as above.

Testosterone glucuronidation assay

MCF7 cells were precultured for 72 hours in phenol red-free RPMI1640 supplemented with 5% DCC–FBS in T175 flasks (Gibco), then treated with vehicle control (0.01% ethanol), E2 (10 nmol/L), DHT (10 nmol/L), 3α-diol (100 nmol/L), or 3β-diol (100 nmol/L) for 72 hours. Cells were scraped from flasks and whole-cell lysates prepared in TE buffer (10 nmol/L Tris-HCl and 1 mmol/L EDTA, pH 7.6). Protein concentration was determined by Bradford assay (Bio-Rad), according to the manufacturer's protocol. The testosterone glucuronidation assay was conducted as previously reported (17).

Luciferase reporter assays

MCF7 cells were plated into 96-well dishes in 100 μL of phenol red-free RPMI1640 supplemented with 5% DCC–FBS and cultured for 24 to 48 hours prior to transfection. Cells were transfected with wild-type or mutated UGT2B15 or UGT2B17 promoter constructs, treated with hormones as indicated, and assessed for luciferase activity as described previously (11, 15). Promoter construct details are provided in Supplementary Methods. Transfections were performed in triplicate and experiments repeated at least twice.

Electrophoretic mobility shift and supershift assays

MCF7 cells were cultured in T75 flasks for 6 days in phenol red-free RPMI1640 supplemented with 5% DCC–FBS, then treated overnight (∼16 hours) with 0.1% ethanol (vehicle), E2 (10 nmol/L), DHT (10 nmol/L), 3α-diol (100 nmol/L), or 3β-diol (100 nmol/L). Nuclear extracts were prepared and electrophoretic mobility shift and supershift assays (EMSA) performed as previously reported (11, 18). Probe and antibody information is provided in Supplementary Methods.

siRNA knockdown experiments

ON-TARGETplus SMARTpool siRNAs were purchased from Dharmacon RNA Technologies: (i) FOXA1 (NM_004496); (ii) ERα (NM_000125); (iii) AR (NM_001011645); and (iv) ON-TARGETplus nontargeting pool siRNA (scramble siRNA). Cells were cultured for 3 days in phenol red-free RPMI1640 supplemented with 5% DCC–FBS, then seeded into 6-well plates at 5 × 105 cells per well in 2 mL of fresh medium containing 8 μL of Lipofectamine 2000 (Invitrogen) and 100 nmol/L siRNA. After 24 hours, transfection media were replaced by fresh media supplemented with 5% DCC–FBS. After 48 hours, cells were treated overnight as indicated. Cells were harvested for RNA isolation and transcriptional analyses as above.

Chromatin immunoprecipitation experiments

Standard methodologies were employed as described previously for chromatin immunoprecipitation (ChIP), followed by qRT-PCR (11) and for ChIP followed by exonuclease digestion and deep sequencing (ChIP-exo; ref. 19). Immunoprecipitations were performed with Santa Cruz Biotechnology, Inc. antibodies to ERα (HC-20), FOXA1 (H-120), AR (N-20), or rabbit preimmune IgG control (sc-2027).

Statistical analyses

Log-transformed data met the assumption of equal variance at a significance level of less than 0.01 (Levene test). Analyses of transformed data were conducted by one- or two-way ANOVA with the Tukey post hoc test or independent t test using SPSS software (IBM, version 22). P < 0.05 was considered statistically significant. Recursive partitioning was used to dichotomize the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) transcriptional data to assess the influence of relatively high versus low expression of each gene on patient survival using an R statistical language program as previously implemented (20). This was performed for all probes representing a gene.

Results

Expression of UGT2B15 and UGT2B17 in clinical breast cancers

The METABRIC investigated 2,000 cases of breast cancer and redefined this heterogeneous disease into 10 molecular subgroups with distinct survival outcomes (20). The subgroups, called iClusters, were created by an integration of gene copy number variations and cis-acting transcriptomes (20, 21). Analysis of the microarray probes representing UGT2B15 and UGT2B17 in the METABRIC cohort revealed cases that displayed very high expression of these genes (Fig. 1A). Their expression was positively correlated in this (Supplementary Fig. S1A) as well as an independent breast cancer cohort (n = 825 cases) from The Cancer Genome Atlas (Supplementary Fig. S1B; refs. 22, 23). In the METABRIC cohort, ERα+ cases had significantly higher expression of these genes compared to ERα− cases (Fig. 1B), and highest expression was observed in integrative cluster 1 (iCluster 1), predominantly composed of ERα+, luminal B breast cancers (Fig. 1C). Integrative cluster 10, largely composed of aggressive, triple-negative breast cancers, had the lowest expression (Fig. 1C). Transcript levels of both enzymes significantly decreased with increasing tumor grade (Fig. 1D). Higher levels of UGT2B17 were significantly associated with increased survival outcomes in cases classified into iCluster 5, largely composed of high-grade breast cancers with HER2 amplification and very poor survival outcomes, as well as all cases in the cohort classified as being HER2 amplified by SNP6 array analysis (Fig. 1E). Note that these groups contain both ERα+ and ERα− breast cancers. Higher levels of both UGT2B15 and UGT2B17 were associated with increased survival in iCluster 6 (Fig. 1F), dominated by ERα+, HER2− breast cancers with amplification of the 8p12 gene locus. Conversely, higher levels of UGT2B15 were associated with poor survival in iCluster 9, composed of ERα+ cancers with a luminal B phenotype and a high rate of TP53 mutations (Fig. 1G). We also analyzed the relationship between expression of these genes and patient outcome in another publicly available resource (KM plotter) containing normalized microarray data from multiple studies that collectively represent 3,455 cases of breast cancer (24). At present, this cohort can only be interrogated via the older PAM50 gene classifier. A high level of UGT2B15 expression was associated with improved relapse-free survival in the entire cohort and in all PAM50 subgroups (Supplementary Fig. S2, top row). Although high UGT2B17 expression was also associated with improved relapse-free survival in the entire cohort, this association was only sustained in the luminal B and HER2+ subtypes (Supplementary Fig. S2, bottom row), the latter supporting results in Fig. 1E. Collectively, these data suggest that expression of UGT2B15 or UGT2B17 impacts breast cancer survival in a gene- and disease subtype-specific manner.

Figure 1.
Figure 1.

UGT2B15 and UGT2B17 expression in clinical breast cancers. A, heatmap showing relative expression of UGT2B15 (2 probes) and UGT2B17 (3 probes) in the METABRIC cohort of 2,000 breast cancers. The degree of red or blue color indicates high and low expression, respectively. The 10 integrative clusters (iClusters) defined by the METABRIC study are indicated by colors that correspond to the boxes in C. B, UGT2B15 and UGT2B17 gene expression is significantly higher in ERα+ compared with ERα− breast cancers. C, levels of UGT2B15 and UGT2B17 in the iClusters. D, UGT2B15 and UGT2B17 expression significantly decreases with increasing tumor grade. E, high expression of UGT2B17 is associated with increased disease-specific survival in iCluster 5, representing cases in which HER2 amplification is the key genomic feature (left) and in all cases with HER2 amplification as determined by SNP6 array (right). F, high expression of UGT2B15 (left) and UGT2B17 (right) is associated with better disease-specific survival in iCluster 6. G, high expression of UGT2B15 is associated with decreased disease-specific survival in iCluster 9. Box and whisker plots (B–D), median and interquartile range (25th–75th percentiles; ***, P < 0.0001).

ERα and AR transcriptionally regulate UGT2B15 and UGT2B17 in an estrogen-sensitive context

As expected, E2 significantly increased transcripts of UGT2B15 and UGT2B17 (Fig. 2A and B) and an ERα-regulated positive control gene (GREB1; Supplementary Fig. S3A), effects that were abolished by cotreatment with fulvestrant (ICI), an ERα inhibitor. Treatment with DHT or its metabolites, 3α-diol or 3β-diol, dose dependently increased UGT2B15 and UGT2B17 transcripts (Fig. 2A and B) and the control gene (Supplementary Fig. S3A) to varying degrees. These stimulatory effects were all significantly attenuated by coincubation with fulvestrant, supporting the hypothesis that DHT indirectly stimulates ERα activity via conversion to diols. Individual hormone treatments also significantly increased levels of glucuronidated testosterone (Fig. 2C), indicative of enhanced enzyme activity. Surprisingly, fulvestrant also inhibited the activity of R1881 (Fig. 2A and B), a synthetic androgen that is not metabolized to estrogenic compounds, although direct binding of R1881 to ERα at high doses has been reported (25). While these data collectively suggested lack of direct regulation by AR, subsequent analyses revealed that the stimulatory effects of DHT were also significantly attenuated by treatment with flutamide, an AR antagonist (Supplementary Fig. S3B), and treatment with the ERα and AR inhibitors combined resulted in greater inhibition of steroid-mediated upregulation of UGT2B15 and UGT2B17 than either inhibitor alone (Fig. 2D). These complementary effects were not evident for regulation of GREB1 by DHT, which was completely dependent on ERα (Supplementary Fig. S3C). Consistent with a direct role for AR in upregulating the UGT genes, siRNA-mediated ablation of AR in MCF7 cells significantly reduced both E2- and DHT-mediated transcriptional effects (Fig. 2E). The same patterns of hormone regulation and the influence of AR loss were evident for the UGT2B15 gene in ZR75-1 cells, another model of ERα+AR+ breast cancer (Supplementary Fig. S3D). The UGT2B17 gene was not detected under basal or hormone stimulation in ZR75-1 cells. Collectively, these findings indicate that AR directly regulates UGT2B15 and UGT2B17 in ERα+AR+ breast cancer cells, acting cooperatively with ERα.

Figure 2.
Figure 2.

Regulation of UGT2B15 and UGT2B17 by estrogenic and androgenic steroids. A and B, relative mRNA levels of UGT2B15 (A) and UGT2B17 (B) in MCF7 cells treated for 24 hours with estrogen (E2), androgen (DHT), metabolites of DHT (3α-diol, 3β-diol), or a synthetic androgen (R1881) alone or in combination with the ERα inhibitor fulvestrant (ICI) at concentrations as indicated. C, levels of glucuronidated testosterone, indicative of UGT2B15/17 enzyme activity, in MCF7 cells treated with hormones as indicated. D, influence of ERα inhibition (ICI) and/or AR inhibition with flutamide (Flut) on upregulation of UGT2B15 (top) and UGT2B17 (bottom) by DHT in MCF7 cells. E, influence of siRNA-mediated silencing of AR (siAR) versus a nontarget siRNA (siCtrl) on E2- or DHT-mediated induction of UGT2B15 (top) or UGT2B17 (bottom) in MCF7 cells. All data shown are means ± SEM of three independent experiments. Asterisks above columns, comparisons with the vehicle control, set at 1. Asterisks above a line, comparisons between selected columns (one-way ANOVA followed by Tukey post hoc multiple comparison test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Steroid-induced transactivation of the proximal promoters of UGT2B15 and UGT2B17

Highly conserved estrogen response units (ERU) reside in the proximal promoters of the UGT2B15 and UGT2B17 genes, comprised of three estrogen response element (ERE) half-sites and two AP-1–binding sites, all required for E2-mediated transcriptional regulation (11). A FOXA1-binding site has recently been identified in the region between two of the ERE half-sites (11, 16), but its importance in breast cancer cells is unknown. The prototypical ERU, including the FOXA1-binding site, is depicted in Fig. 3A. As expected, E2 significantly enhanced basal promoter activity in an ERα-dependent manner (Fig. 3B). All other steroids, including DHT, 3α-diol, 3β-diol, and R1881, stimulated UGT2B15 promoter activity at levels similar to or greater than that induced by E2, and these stimulatory effects were all significantly repressed to some degree by the ERα inhibitor. However, the AR inhibitor also significantly reduced DHT-induced promoter activity (Fig. 3C), supporting direct regulation by both sex steroid receptors.

Figure 3.
Figure 3.

Estrogen and androgen regulation of the UGT2B15 and UGT2B17 promoters. A, schematic depiction of highly conserved ERUs in the UGT2B15 and UGT2B17 proximal promoters, positioned between −454 and −172 nucleotides in the UGT2B15 promoter and between nucleotides −396 and −112 in the UGT2B17 promoter relative to their translation start sites (ATG). From 5′ to 3′, the six key binding motifs are 5′-AP-1 and 3′-AP-1 sites, an imperfect ERE, a 5′-ERE half-site, a FOXA1 site, and a 3′-ERE half-site. MCF7 cells were transfected with wild-type (WT) promoter constructs or mutated constructs as indicated by a backslash over the relevant motif. B, stimulation of the WT UGT2B15 promoter by estrogen (E2), androgen (DHT), metabolites of DHT (3α-diol, 3β-diol), or a synthetic androgen (R1881), alone or together with the ERα inhibitor fulvestrant (ICI). C, inhibition of DHT-stimulated WT UGT2B15 promoter activity by ICI and an AR inhibitor, flutamide (Flut). D, influence of UGT2B15 promoter mutations on steroid-mediated transactivation. E, activity of WT and FOXA1-mutant (FOXA1mt) UGT2B17 promoters in response to steroids alone or in combination with ICI. Relative luciferase activities of promoter constructs are expressed as fold induction over that of the empty vector (set to 1) after normalizing transfection efficiency with R. reniformis luciferase. Data shown are means ± SD of three independent experiments performed in triplicate (one-way ANOVA followed by Tukey post hoc multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001).

An ERU-mutant UGT2B15 promoter construct dramatically reduced transactivation by E2 and obliterated transactivation by other tested steroids (Fig. 3D; left), and mutation of the FOXA1 site abolished all steroid-induced promoter activity (Fig. 3D; right). Transactivation of the wild-type (Fig. 3E; left) and FOXA1-mutated (Fig. 3E; right) UGT2B17 promoter constructs by steroids mirrored that of the UGT2B15 constructs, but with more attenuated stimulatory effects.

To confirm the direct involvement of FOXA1, EMSA assays were performed. A major protein/DNA complex formed on a DNA probe containing the UGT2B15 promoter FOXA1 site following incubation with nuclear extracts from vehicle and steroid-treated MCF7 cells (Fig. 4A). Addition of a FOXA1 antibody reduced the intensity of the complex and also induced formation of super-shifted complexes. Furthermore, mutation of the FOXA1 site abolished the formation of FOXA1 protein/DNA complexes. Although the UGT2B15 and UGT2B17 gene promoters are highly conserved, a unique but prevalent −155A/G SNP occurs within the FOXA1-binding site in the UGT2B17 promoter (15). Therefore, a UGT2B17 probe with an A-containing FOXA1 site was tested (Fig. 4B). A major complex was formed on the A-containing UGT2B17 FOXA1 probe, which was supershifted by addition of FOXA1 antibody and abolished by mutating the FOXA1 site or adding a 100-fold molar excess of unlabeled probe. Therefore, endogenous FOXA1 in MCF7 cells can bind the “A-containing” FOXA1-binding site SNP in the UGT2B17 promoter. These results confirm a critical role for FOXA1 in steroid-mediated transactivation of the UGT2B15 and UGT2B17 promoters, even in the context of a common SNP variant in UGT2B17.

Figure 4.
Figure 4.

FOXA1, ERα and AR enrichment at UGT2B15 and UGT2B17 promoters. A and B, EMSA blot to demonstrate FOXA1 binding to DNA fragments representing the UGT2B15 (A) and UGT2B17 (B) promoters. MCF7 cells were treated with vehicle (lanes 1–8), estrogen (E2; lanes 9–12), androgen (DHT; lanes 13–14), DHT metabolites 3α-diol (lanes 15–16), or 3β-diol (lanes 17–18). EMSAs were performed with 50,000 cpm (∼1 ng) of indicated 32P-labeled probes. FOXA1-bound DNA complexes are labeled A for the PSA control probe, B for UGT2B15 or UGT2B17 probes, C for nonspecific complexes, and SS for antibody-induced super-shifted complexes. For supershift assays, FOXA1 antibody was added (lanes 4, 7, 12, 14, 16, and 18). For competition assays, unlabeled probes were added at a 10-fold (lane 11) or 100-fold (lane 10) molar excess. In lane 8, a mutated FOXA1 probe was added. C–E, ChIP–qPCR data showing enrichment of ERα, FOXA1, and AR chromatin binding at UGT2B15 (C), UGT2B17 (D), or pS1/TFF (E) promoters in MCF7 cells following stimulation with indicated steroids. Data are presented as relative fold enrichment over the normal rabbit immunoglobulin (IgG) controls (set to 1). Bars, means ± SEM of three independent experiments (one-way ANOVA followed by Tukey post hoc multiple comparison test. *, P < 0.05; **, P < 0.01;***, P < 0.001 compared with the relevant IgG control).

Recruitment of ERα, FOXA1, and AR to the UGT2B15 and UGT2B17 promoters

ERα occupancy at the UGT2B15 and UGT2B17 proximal promoters and a positive control locus (pS2/TIF1) was significantly stimulated by E2 in MCF7 cells (Fig. 4C–E). Of the other steroids tested, 3β-diol stimulated the highest level of ERα enrichment, a compound with significantly less affinity for ERα than E2 (26). FOXA1 bound to the UGT2B15 and UGT2B17 loci before ligand stimulation, consistent with its role as a pioneer factor (27). Interestingly, FOXA1 binding was significantly increased by all hormone treatments (Fig. 4C–E), contrary to observations at other genomic loci (28). This steroid-induced increase in FOXA1 occupancy is consistent with a recent study showing that FOXA1 binding can be altered by activation of steroid receptors, including ERα, in breast cancer cells (29). The AR did not display a strong enrichment over IgG controls under any treatment condition; maximal AR enrichment (2.6-fold) occurred at the positive control locus for ERα binding (pS2/TIFF1) when cells were stimulated with R1881. Low enrichment of AR may be a technical issue due to low AR protein levels in MCF7 cells. However, the requirement for ERα, FOXA1, and AR in the regulation of UGT2B15 and UGT2B17 by steroids was confirmed by siRNA-mediated ablation (Supplementary Fig. S4). Analysis of genome-wide ERα, FOXA1, and AR chromatin binding in MCF7 and ZR75-1 cells generated by ChIP-exo (19) revealed the presence of all three factors in the proximal promoters of the UGT genes (Fig. 5A and B; magnified views in Supplementary Fig. S5). ChIP-exo generates sharper peaks than ChIP sequencing (ChIP-seq) due to the inclusion of an exonuclease step following immunoprecipitation of protein–DNA complexes, allowing better discernment of relative binding positions. These data suggest that AR binds to or overlaps the ERE half-site located 3′ to the FOXA1 and ERα peaks in the promoters of UGT2B15 and UGT2B17. The tandem pattern of chromatin binding strongly supports the concept that ERα, AR, and FOXA1 interact to regulate transcription of these two androgen-inactivating enzymes in an estrogen-sensitive breast cancer context.

Figure 5.
Figure 5.

AR regulation of UGT2B15 and UGT2B17 promoters in ERα+ breast cancer cells. A and B, representative genome browser images showing enrichment of DNA sequences (peaks) pulled down by immunoprecipitation of FOXA1, ERα, or AR by the process of ChIP-exo, indicative of chromatin binding at the proximal promoters of UGT2B15 (A) and UGT2B17 (B) in two models of ERα+, AR+ breast cancer (MCF7, ZR75-1). C, wild-type (WT) UGT2B15 and UGT2B17 promoter constructs were transfected into MCF7 cells, followed by treatment with the selective AR modulator, enobosarm (Eno), as indicated. D, influence of site-specific mutations (MT) in the UGT2B15 promoter constructs transfected into MCF7 cells followed by treatment with enobosarm (100 nmol/L) alone or in combination with the AR inhibitor, bicalutamide (Bic; 1 μmol/L). NS, not significant. Data in C and D are means ± SD of two independent experiments performed in triplicate. Asterisks above columns, comparisons with the appropriate vehicle (Veh) control, set to 1. Asterisks above lines, comparisons between selected columns (one-way ANOVA, followed by Tukey post hoc multiple comparison test. *, P < 0.05; **, P < 0.01).

Regulation of UGT2B15 and UGT2B17 by enobosarm, a new selective AR modulator

Enobosarm (GTx024) is a selective AR modulator (30) currently being tested for clinical efficacy in women with ERα+AR+ breast cancer (NCT02463032). This compound dose dependently activated an AR reporter construct transfected into MCF7 cells (Supplementary Fig. S6A), upregulated UGT2B15 and UGT2B17 in an AR-dependent manner (Supplementary Fig. S6B), and dose dependently increased activation of the wild-type UGT promoter constructs (Fig. 5C). Using selective ERE-mutant constructs of the UGT2B15 promoter, we show that the previously defined “3′ ERE half-site” is critical for AR-mediated upregulation of promoter activity (Fig. 5D).

AR does not regulate UGT2B15 or UGT2B17 in an estrogen-insensitive context

AR activity sustains a luminal phenotype in MDA-MB-453 cells, a model of AR+ERα− breast cancer, by mimicking the action of ERα in MCF7 cells (31). This type of breast cancer also has similarities to prostate cancer (3). Interrogation of our previously published ChIP-seq data (31) shows that AR and FOXA1 occupy the proximal promoter regions of UGT2B15 and UGT2B17 in MDA-MB-453 cells, similar to ERα and FOXA1 occupancy in MCF7 cells and AR and FOXA1 occupancy in LNCaP prostate cancer cells (Fig. 6A and B). However, treatment with DHT did not upregulate expression of these genes in the MDA-MB-453 or MFM223 breast cancer cell lines (Fig. 6C), which both have a “luminal AR” phenotype (32). UGT2B17 expression was undetectable under any treatment conditions in these AR+ ERα− cell lines. In contrast, DHT potently downregulates both genes in LNCaP cells (33). Thus, although ERα and AR positively coregulate transcription of the two major androgen-metabolizing enzymes in an estrogen-sensitive breast cancer context, AR alone does not appear to have this capacity in an estrogen-insensitive context.

Figure 6.
Figure 6.

Androgen regulation of UGT2B15 and UGT2B17 promoters in ERα− breast cancer cells. A and B, representative genome browser images showing enrichment (peaks) of DNA sequences pulled down by immunoprecipitation of FOXA1 or AR by the process of ChIP-seq, indicative of chromatin binding at the proximal promoters of UGT2B15 (A) and UGT2B17 (B) in MDA-MB-453 breast cancer cells, an AR+, ERα− model of breast cancer. For comparison, ERα and FOXA1 peaks are shown for the same loci in MCF7 breast cancer cells and AR and FOXA1 peaks in prostate cancer cells (LNCaP). C, qRT-PCR data corresponding to indicated cell lines treated with estrogen (E2) or androgen (DHT). Data shown are means ± SEM of three independent experiments. One-way ANOVA followed by Tukey post hoc multiple comparison test (***, P < 0.0001). NS, not significant; Veh, vehicle.

Estrogen and androgen regulation of UGT2B15 and UGT2B17 in clinical tissues

Normal breast tissue from reduction mammoplasties (n = 8) were cultured ex vivo on gelatin sponges to sustain tissue architecture, cellular heterogeneity, and hormone receptor expression as described previously (14). In 3 of 8 cases, all three treatments (E2, DHT, and enobosarm) upregulated transcript levels of UGT2B15 or UGT2B17 compared with the vehicle control (Supplementary Fig. S6C). In other cases, one or two agents demonstrated stimulatory action, and in a few cases, there were no effects, demonstrating the expected heterogeneity of primary tissue responses. This methodology also provides a unique preclinical model for testing drug responses (13, 34). In primary ERα+ breast tumors (n = 13) cultured ex vivo under estrogenic conditions, treatment with enobosarm upregulated UGT2B15 in a total of 7 of 13 cases and UGT2B17 in 8 of 13 cases (Fig. 7A). Two cases demonstrated no stimulatory effect on either gene above baseline, four demonstrated stimulatory effects on both genes, three demonstrated upregulation of UGT2B15 alone, and four demonstrated upregulation of UGT2B17 alone. Collectively, these data indicate that therapeutic activation of AR can significantly upregulate expression of these UGT genes in primary ERα+ breast cancers.

Figure 7.
Figure 7.

Sex hormone regulation of UGT2B15 and UGT2B17 in clinical samples. A, relative mRNA levels of UGT2B15 and UGT2B17 in ex vivo–cultured pieces of ERα+AR+ primary breast tumors (n = 13 independent cases) following treatment with the selective AR modulator, enobosarm (Eno; 100 nmol/L). B and C, representative genome browser images of ERα occupancy at the UGT2B15 (B) and UGT2B17 (C) proximal promoters in clinical samples, representing normal breast tissue (n = 2), good outcome ERα+, PR+, HER2− tumors (n = 3), poor outcome ERα+, PR±, HER2± tumors (n = 3), and ERα+ distant metastases (n = 3).

A previous study investigated genome-wide ERα chromatin binding in primary breast tumors (35). The cohort consisted of ERα+, progesterone receptor (PR)-positive and HER2− tumors associated with good disease outcomes (n = 8); ERα+, PR−, and/or HER2+ tumors associated with poor outcomes (n = 7) and ERα+ distant metastases (n = 3). Interrogation of those data revealed no evidence for ERα occupancy at the promoters of UGT2B15 or UGT2B17 in normal breast tissues (0/2 cases) or ERα+ tumors associated with a good outcome (0/8 cases). However, ERα occupancy was evident at the UGT2B15 (2/7 cases) and UGT2B17 (1/7 cases) promoters in tumors associated with poor outcomes and in 2 of 3 metastases (Fig. 7B and C). Although the study represented a small number of cases, it was the first study to investigate genome-wide ERα–chromatin interactions in clinical breast tissues.

Discussion

Herein, the potential clinical significance and hormone-mediated regulation of two steroid glucuronidation enzymes, UGT2B15 and UGT2B17, was investigated in relation to breast cancer. These enzymes can functionally impact both ERα and AR activity by directly or indirectly reducing levels of their activating ligands in the tissue microenvironment. Although positive regulation of these genes via ERα activity at their proximal promoters has been previously described in breast cancer cell lines (10, 11), we provide the first evidence that this activity is exquisitely dependent on the pioneer factor FOXA1, is facilitated by AR binding to an adjacent ERE half-site, and is potently stimulated by metabolites of DHT normally considered “weak” estrogens. These data are consistent with the fact that UGT2B15 and UGT2B17 levels were highest in clinical ERα+ breast cancers and in low-grade tumors, which are known to be characterized by high levels of both ERα and AR. Importantly, higher expression of one or both of the UGT enzymes was associated with better survival outcomes in a number of molecular subgroups of ERα+ disease, which may have clinical implications. For example, therapeutic activation of AR with enobosarm significantly increased expression of one or both genes in ex vivo–cultured normal human breast tissues and ERα+ breast tumors. Whether this plays a mechanistic role in attenuating the estrogenic signal or determining clinical responses of women treated with this compound for estrogen-sensitive breast cancer (NCT02463032) remains to be determined.

In breast malignancies, the levels of estrogenic or androgenic hormones can greatly exceed levels measured in plasma or in adjacent nonmalignant breast tissues (36, 37). The altered intracrinology involves deregulation of steroid-metabolizing genes, such as 17β-HSDs, sulfotransferases, 5α-reductases, and estrogen-metabolizing UGTs (38–40). Our data indicate that androgen-metabolizing UGTs are also involved, particularly in the context of ERα+ and/or HER2+ clinical breast cancers, in which relative expression of UGT2B15 and/or UGT2B17 could discern cases with distinct survival outcomes. Although it is tempting to speculate on how relative levels of either enzyme influence steroid intracrinology and hence disease outcome in various breast cancer contexts, it is important to note that UGT2B15 and UGT2B17 have different potencies in the inactivation of androgen hormones and that one or both have been shown to inactivate xenobiotics and drugs relevant to breast cancer, including bisphenol AF (41), exemestane (42), the histone deacetylase inhibitor vorinostat (43), and the major active tamoxifen metabolite, 4-OH-tamoxifen (42, 44). Differential activity of the two enzymes in the glucuronidation of androgens or other compounds may explain gene-specific influences on breast cancer outcomes. We recently found that tamoxifen and its metabolites upregulate UGT2B15 via a mechanism involving ERα (45), which may have implications for resistance to this drug. Collectively, these studies indicate that the influence of UGT2B15 and UGT2B17 expression in breast cancer includes, but also has implications that extend beyond, their ability to metabolize androgen hormones.

Both metabolites of DHT (3α- and 3β-diol) could recruit ERα to the proximal promoters of UGT2B15 and UGT2B17 to stimulate transcription, which in part explains the ERα-dependent stimulatory effects of this androgen. Indeed, rapid conversion of DHT into 3α- and 3β-diol has been previously reported in MCF7 cells (46, 47) and all 4 human 3α-hydroxysteroid dehydrogenases capable of this conversion are expressed in breast tissues (48). However, the action of DHT also stimulated AR binding to the promoters at a site 3′ to the ERα-binding site, and ablation or chemical inhibition of AR reduced estrogen- and androgen-mediated transcriptional regulation of these genes. The cooperative activity of AR and ERα at these loci is suggestive of an “assisted loading” mechanism described for the glucocorticoid receptor, a steroid receptor closely related to AR (49). Cooperative regulation of UGT2B15 by ERα and AR was also evident in ZR75-1 cells, another luminal breast cancer model, indicating that the findings are not limited to one cell line.

There has been increasing interest in the capacity of AR signaling to drive breast cancer, particularly in ERα-negative tumors. The MDA-MB-453 cell line has emerged as the prototypical and most studied model of this type of breast cancer, in which AR is expressed at high level, and treatment with DHT can increase proliferation. Indeed, AR signaling in this cell line shares many similarities to AR signaling in LNCaP cells (2, 31). However, we show that AR and FOXA1 occupy the promoter regions of UGT2B15 and UGT2B17 in MDA-MB-453 cells in a similar manner to their occupancy in LNCaP cells, but androgen treatment did not influence their transcription in two models of AR+, ERα− breast cancer. As AR potently downregulates these genes in LNCaP cells (14) and we show that AR upregulates them in ERα+ breast cancer cells, regulation of these genes by AR is highly context dependent.

In summary, the UGT2B15 and UGT2B17 enzymes are transcriptionally regulated by sex hormone signaling in ERα+ breast cancer cells and are highly expressed in a subset of primary breast cancers, which has significance for survival outcomes in distinct genetic subgroups of this disease. Apart from their influence on the level of active steroids in the microenvironment, these enzymes may also determine the level of active xenobiotics and drugs relevant to breast cancer, which could have implications for response to a range of adjuvant hormone or other therapies. Collectively, these observations add new knowledge about the complexity of breast cancer intracrinology that has implications for other hormone-dependent cancers. The findings provide a basis for further exploration of the role of UGTs in moderating the steroid milieu and modulating drug responses in breast and other cancers.

Disclosure of Potential Conflicts of Interest

C. Caldas reports receiving commercial research grants from AstraZeneca, Genentech, and Roche and is a consultant/advisory board member for AstraZeneca. No potential conflicts of interest were disclosed by the other authors.

Authors' Contributions

Conception and design: D.G. Hu, L.A. Selth, R. Meech, W.D. Tilley, P.I. Mackenzie, T.E. Hickey

Development of methodology: D.G. Hu, L.A. Selth, P.I. Mackenzie, T.E. Hickey

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): D.G. Hu, L.A. Selth, D. Wijayakumara, A. Chanawong, C. Caldas, J.L.L. Robinson, J.S. Carroll, W.D. Tilley, T.E. Hickey

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): D.G. Hu, L.A. Selth, R. Russell, C. Caldas, J.L.L. Robinson, W.D. Tilley, T.E. Hickey

Writing, review, and/or revision of the manuscript: D.G. Hu, L.A. Selth, G.A. Tarulli, R. Meech, J.L.L. Robinson, J.S. Carroll, W.D. Tilley, T.E. Hickey

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): L.A. Selth, G.A. Tarulli, C. Caldas, W.D. Tilley, P.I. Mackenzie, T.E. Hickey

Study supervision: D.G. Hu, L.A. Selth, T.E. Hickey

Grant Support

This work was supported by funding from the National Health and Medical Research Council of Australia (ID1020931 to P.I. Mackenzie; ID1085410 to R. Meech and P.I. Mackenzie; ID 1008349 to W.D. Tilley; ID 1084416 to W.D. Tilley and T.E. Hickey), Flinders Medical Centre Foundation (D.G. Hu and P.I. Mackenzie), Cancer Australia (ID 1043497 to W. D. Tilley and T. E. Hickey), and a Prostate Cancer Research Programs Transformative Impact Award from the U.S. Department of Defense (W81XWH-13-2-0093 to W. D. Tilley and L. A. Selth). L. A. Selth was supported by a Young Investigator Award from the Prostate Cancer Foundation (Foundation 14 award). T.E. Hickey held a Postdoctoral Fellowship Award from the U.S. Department of Defense Breast Cancer Research Program (BCRP; W81XWH-11-1-0592).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Acknowledgments

We acknowledge Geraldine Laven-Law for technical assistance and the clinical personnel (particularly Drs. Stephen Birrell and Clive Hoffman) and patients from Adelaide clinics who supported the study.

Footnotes

  • Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

  • Received December 10, 2015.
  • Revision received June 20, 2016.
  • Accepted July 14, 2016.

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Cancer Research: 76 (19)
October 2016
Volume 76, Issue 19

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Androgen and Estrogen Receptors in Breast Cancer Coregulate Human UDP-Glucuronosyltransferases 2B15 and 2B17
Dong G. Hu, Luke A. Selth, Gerard A. Tarulli, Robyn Meech, Dhilushi Wijayakumara, Apichaya Chanawong, Roslin Russell, Carlos Caldas, Jessica L.L. Robinson, Jason S. Carroll, Wayne D. Tilley, Peter I. Mackenzie and Theresa E. Hickey
Cancer Res October 1 2016 (76) (19) 5881-5893; DOI: 10.1158/0008-5472.CAN-15-3372
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