FOXA1 Is Essential for Aryl Hydrocarbon Receptor–Dependent Regulation of Cyclin G2

Molecular Cancer Signaling and Regulation Research

Shaimaa Ahmed, Sarra Al-Saigh, and Jason Matthews

Abstract The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recently, AHR has emerged as a potential therapeutic target for breast cancer by virtue of its ability to modulate estrogen receptor-a (ERa) signalling and/or its ability to block cell proliferation. Our previous studies identiﬁed cyclin G2 (CCNG2), an inhibitor of cell- cycle progression, as an AHR target gene; however, the mechanism of this regulation is unknown. Chromatin immunoprecipitation assays in T-47D human breast cancer cells revealed a TCDD-dependent recruitment of AHR, nuclear co-activator 3 (NCoA3) and the transcription factor forkhead box A1 (FOXA1), a key regulator of breast cancer cell signaling, to CCNG2 resulting in increases in CCNG2 mRNA and protein levels. Mutation of the AHR response element (AHRE) and forkhead-binding sites abolished TCDD-induced CCNG2-regulated reporter gene activity. RNA interference–mediated knockdown of FOXA1 prevented the TCDD-dependent recruitment of AHR and NCoA3 to CCNG2 and reduced CCNG2 mRNA levels. Interestingly, knockdown of FOXA1 also caused a marked decrease in ERa, but not AHR protein levels. However, RNA interference–mediated knockdown of ERa, a negative regulator of CCNG2, had no effect on TCDD-dependent AHR or NCoA3 recruitment to or expression of CCNG2. These ﬁndings show that FOXA1, but not ERa, is essential for AHR-dependent regulation of CCNG2, assigning a role for FOXA1 in AHR action. Mol Cancer Res; 10(5); 636–48. 2012 AACR.

Introduction results in the recruitment of coregulators (coactivators and The aryl hydrocarbon receptor (AHR) is a ligand-activated corepressors) causing changes in gene expression. Well- transcription factor that mediates the toxic effects of both studied AHR target genes include the phase I and II drug-metabolizing enzymes, such as cytochrome P450 halogenated and polycyclic aromatic hydrocarbons, such as S 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-meth- 1A1 (CYP1A1), CYP1B1, and glutathione -transferases. ylcholanthrene (3MC). AHR plays important roles in devel- Chromatin immunoprecipitation combined with microar- opment and differentiation, as well as in the immune and the rays (ChIP-chip) and gene expression microarray experi- reproductive systems (1, 2). Moreover, AHR has recently ments revealed that AHR regulates numerous genes involved fl in diverse cellular pathways, including metabolism and cell- emerged as a therapeutic target for in ammatory and auto- – fi immune diseases, as well as for the treatment of breast cancer cycle regulation (8 10). In support of these ndings, previous studies have shown that TCDD inhibits cell prolifer- (3, 4). We and others have shown that AHR activation – inhibits estrogen receptor-a (ERa) signaling through direct ation (11 13). interaction with ERa and through the modulation of ERa Cell proliferation is a highly regulated event mediated by target genes (5–7). After ligand binding, AHR translocates the activation of cyclin-dependent kinases (CDK), which require the binding of cyclins for full activity. Mammalian into the nucleus where it binds to its dimerization partner fi AHR nuclear translocator (ARNT). This heterodimer then cyclins are classi ed into 12 different types cyclins A to I binds to specific DNA response elements termed AHR based on sequence and functional similarities (14). Most cyclins have been shown to facilitate growth by either response elements (AHRE) in the regulatory regions of its – – target genes. DNA binding of the AHR/ARNT heterodimer promoting G0 G1 to S-phase or the G2 M phase transition. The G-type cyclins (cyclin G1, G2, and I) on the other hand are associated with cell-cycle arrest (15). Cyclin G1 is – involved in G2 M phase arrest, whereas cyclin I is thought Authors' Affiliation: Department of Pharmacology and Toxicology, Uni- versity of Toronto, Toronto, Ontario, Canada to play a role in apoptosis (16). Cyclin G2 (CCNG2) has been shown to inhibit cell-cycle progression by preventing Corresponding Author: Jason Matthews, Department of Pharmacology and Toxicology, University of Toronto, Medical Sciences Building, Room G1 to S-phase transition (14, 15, 17). This inhibition is 4336, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8. Phone: achieved by interacting with protein phosphatase 2A 416-946-0851; Fax: 416-978-6395; E-mail: [email protected] (PP2A). The CCNG2-PP2A complex alters PP2A targeting doi: 10.1158/1541-7786.MCR-11-0502 and its substrate specificity leading to cell-cycle arrest (14). 2012 American Association for Cancer Research. CCNG2 also stops cell-cycle progression by preventing the

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phosphorylation of CDK2 and retinoblastoma which are purchased from Wisent Bioproducts. All other chemicals required for progression out of the quiescent state (G0; and biochemicals were of the highest quality available from refs. 14, 18). CCNG2 has been shown to be a critical gene commercial vendors. in human epidermal growth factor receptor 2 (HER2)- and hormone-dependent breast cancers (18–21). Moreover, ChIP assays CCNG2 was among the few genes upregulated following T-47D human breast carcinoma cells were cultured in 1:1 anti-HER2 antibody trastuzumab treatment, indicating that mixture of Dulbecco's Modified Eagle's Medium (DMEM) CCNG2 might play a role in inhibiting HER2-dependent and Ham's F12 nutrient mixture medium supplemented proliferation (22). with 10% (v/v) FBS and 1% penicillin/streptomycin. Cells In estrogen-dependent breast cancer, cell proliferation is were maintained at 37 C and 5% CO2. T-47D cells were facilitated by the binding of estrogen to ERa resulting in subcultured every 2 to 3 days or when they reached 80% to either increased expression of genes associated with prolif- 90% confluency. For the ChIP studies, 3 million T-47D eration or suppression of genes that block cell-cycle progres- cells were seeded in 10-cm dishes in 1:1 mixture of DMEM: sion. ERa represses CCNG2 expression in response to F12 phenol red–free media supplemented with 5% (v/v) estrogen by recruiting a complex containing nuclear co- dextran-coated charcoal (DCC)-stripped serum and 1% repressor (NCoR) and histone deacetylases to the CCNG2 penicillin/streptomycin. After 72 hours, cells were treated promoter region resulting in the displacement of RNA with DMSO (final concentration, 0.1%), 10 nmol/L polymerase II (21). Our previous ChIP-chip studies iden- TCDD, 10 nmol/L E2, or E2 þ TCDD (10 nmol/L) for tified an AHR-bound AHRE-containing sequence in the 0.75 hours. ChIP and sequential ChIP assays were con- upstream regulatory region of CCNG2. In contrast to ducted as previously described (8). Enrichment levels (rel- estrogen, the AHR agonists, TCDD and 3MC, induced ative to 100% total input) were determined by quantitative AHR recruitment to CCNG2 causing an increase in real-time PCR (qPCR) using the following primers: CCNG2 CCNG2 mRNA levels (8, 9). However, the molecular AHRE2 (enhancer): 50-TGGGTTACCAAGGACCAA- mechanisms and characterization of the AHR-dependent GAA-30 50-CCAGAGGTTGTAGTGCTGTTGTTT-30; regulation of CCNG2 remain elusive. CCNG2 AHRE1:50-AACTCTCCCGTGGCTGAAAA Another protein shown to be important in the regulation and 50-CGCGGCGCTTCTCCTAA-30 CCNG2 TATA: of genes in breast cancer is the forkhead box A1 (FOXA1) 50-GGGAGGCCGCGAGAGA-30 and 50-TCCTCCACG- transcription factor, a member of the winged-helix tran- GACTTTAAAAAAGAG-30. scription factors (23). The FOXA family is composed of 3 closely related members, FOXA1, A2, and A3, which have RNA isolation and real-time PCR critical roles in development and differentiation (23). High T-47D cells were seeded in 6-well plates and grown in a expression of FOXA1 is observed in ERa-positive breast 1:1 mixture of DMEM:F12. Cells were treated with either tumors and its expression is a positive prognostic factor 10 nmol/L TCDD or pretreatment for 1 hour with 1 which correlates with sensitivity to endocrine therapy and mmol/L CH223191 for 0.75, 1.5, 3, 6, or 24 hours. RNA inhibition of tumor growth (19, 24). FOXA1 acts as a was isolated using illustra RNAspin mini columns (GE "pioneer factor" by altering chromatin structure and facil- Healthcare) and reverse-transcribed as previously described itating ERa binding to its target genes (25). FOXA1 also (8). All target gene transcripts were normalized to ribosomal modulates androgen receptor function, indicating that it has 18S RNA and fold inductions were calculated using time- C a more general role in regulating gene expression (26, 27). matched DMSO-treated samples and the comparative T DDC The role of FOXA1 in AHR transactivation or cross-talk ( T) method was used for data analysis. qPCR primers between AHR and ERa has not been determined. used in the study have been described elsewhere (8) with AHR activation inhibits breast cancer cell proliferation exception of the following primers: FOXA1 mRNA 50- and induces cell-cycle arrest through a number of different GAAGATGGAAGGGCATGAAA-30 and 50-GCCTGA- pathways (2, 10, 28). We report here that FOXA1, but not GTTCATGTTGCTGA-30. ERa, is essential for AHR-dependent regulation of CCNG2, assigning a role for FOXA1 in AHR action and character- Western blotting izing a new mechanism by which AHR inhibits breast cancer Proteins were resolved by SDS-PAGE and transferred to cell proliferation. nitrocellulose membrane. The membrane was blocked in 2% (w/v) ECL-Advanced blocking agent for 1 hour at room temperature with constant rocking and then incubated with Materials and Methods anti-ERa (HC-20), anti-AHR (H-211), or anti-FOXA1 Chemicals (ab23738) overnight at 4C with constant rocking. The Antibodies used for ChIP and Western blotting experi- membrane was then washed 3 times and incubated with an ments include ERa (HC-20), AHR (H-211), and NCoA3 anti-rabbit secondary antibody for 1 hour. For detection of (M-397) from Santa Cruz Biotechnology and FOXA1 b-actin, a primary mouse anti-b-actin antibody (Sigma) was (ab23738) from Abcam. TCDD was purchased from AccuS- incubated for 2 hours at room temperature followed by a tandard and dimethyl sulfoxide (DMSO) was purchased 1-hour washing with PBS/0.1% Tween-20 before incuba- from Sigma. Cell culture media, FBS, and trypsin were all tion with anti-mouse secondary antibody for 1 hour. After

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washing, the bands were visualized using ECL-Advanced Co-immunoprecipitation (GE Healthcare). For co-immunoprecipitation (co-IP) studies, T-47D cells were seeded in 10-cm dishes in 1:1 mixture of DMEM:F12 Transient transfection and siRNA supplemented with 5% DCC-stripped serum. After 72 siFOXA1 seq2 (SASI_Hs01_00168404), siFOXA1 seq3 hours, cells were treated with either 10 nmol/L TCDD or (SASI_Hs01_00168403), and a universal negative control DMSO (final concentration, 0.1%) for 1 hour and cross- (SIC001) were purchased from Sigma-Aldrich. siERa11 linked using 1% formaldehyde and quenched using 125 (J-003401-11-0050), siERa14 (J-003401-14-0050), mmol/L glycine. Cell lysates were precleared using Protein siCCNG2 (L-003217-00-0005), and DharmaFECT1 A, and protein complexes were immunoprecipitated using transfection reagent were purchased from Dharmacon. 2 mg of rabbit IgG (Sigma), AHR (H-211), FOXA1 (Abcam Briefly, T-47D cells were seeded 300,000 per well in a 6- 23738), or NCoA3 (M-397) for 2 hours. Beads were washed well plate in medium containing 5% DCC-stripped FBS. 4 times with wash buffer (10 mmol/L Tris-HCl, pH 8.0, After 24 hours, 50 nmol/L of siRNA against FOXA1, ERa, 150 mmol/L NaCl, 10% glycerol, 1% NP-40, and 2 mmol/L CCNG2, or universal negative control were transfected EDTA). Eighty microliters of sample buffer was added to using 2 mL of DharmaFECT and 400 mL Opti-MEM. the beads and samples were heated to 70C for 10 minutes. ChIP assays, mRNA isolation, and whole-cell extracts were Samples were loaded on an 8% SDS-PAGE gel, transfer- prepared 48 hours after transfection as described above. red to nitrocellulose membrane, and visualized using the Fluorescence-activated cell-sorting (FACS) analysis for the Clean Blot anti-rabbit HRP (Thermo Scientific) and siCCNG2-treated cells is described below. ECL-Advanced.

Plasmid construction and mutagenesis Flow cytometry The plasmid pGL4-CCNG2 containing 1.402 kb to Cell-cycle analysis by bromodeoxyuridine (BrdUrd) and þ179 of the upstream regulatory region of CCNG2 as well propidium iodide (PI) double staining was completed on as plasmids pGL4-CCNG2 DAHRE1 and DAHRE2 were T-47D cells transiently transfected with universal negative generously provided by Dr. Chun Peng (York University, control or 50 nmol/L of siRNA against CCNG2 (L-003217- Toronto, ON, Canada). The numbering of the cloned 00-0005). Twenty-four hours after transfection, cells were CCNG2 regulatory region was relative to CCNG2 mRNA treated with DMSO (final concentration, 0.1%) or 10 (NM_004354). Site-directed mutagenesis targeting the nmol/L TCDD and left for another 48 hours. Cells were AHRE site in CCNG2 was completed as previously described fi 0 then collected and xed in 70% ethanol for 20 minutes at (29). For mutation of AHRE2 the PCR primers, 5 -CTCAT- 20C. Cells were then rinsed in wash buffer (PBS þ CAGCGCGCTAAGTTTT-30 and 50-AAAACTTAGC 0 0.5% bovine serum albumin) and resuspended in 2N HCl GCGCTGATGAG-3 were used to mutate pGL4-CCNG2; for 20 minutes, washed, incubated with 0.1 mol/L sodium the mutated residues underlined. Mutation of the 2 forkhead- borate, pH 8.5, for 2 minutes, washed again, then incu- binding sites upstream from AHRE2 was done using pGL4- bated with fluorescein isothiocyanate (FITC)-conjugated CCNG2 as a template and the following PCR primers anti-BrdUrd (BD Biosciences) in PBS þ 0.5% bovine for FKH3: 50-TAGGAGGGAG AGAGTCCCAAAA- þ 0 0 serum albumin 0.5% Tween-20 and left in the dark for TAAATGTTCCAG-3 and 5 -CTGGAACATTTATTT 30 minutes. Cells were then washed with wash buffer and TGGGACTCTCTCCCTCCTA-30 and for FKH4 m 0 stained with 50 g/mL PI for another 30 minutes. FACS 5 -CCGTAATTATT AGATTGGGACGTACCCTCAT- analyses and data acquisition were done by the Faculty of CAG-30 and 50-CTGATGAGGGTACGTCCCAATCTA 0 Medicine Flow Cytometry Facility using a FACSCalibur ATAATTACGG-3 . All mutations were verified by DNA flow cytometer (BD Biosciences) and FlowJo software sequencing. (TreeStar). Transient transfection and reporter gene assay Statistical analysis T-47D cells were plated in 12-well dishes in a 1:1 All results were expressed as means SEM. Statistical DMEM:F12 mixture containing 5% DCC-stripped serum. analysis was calculated using GraphPad Prism 5 statistical Twenty-four hours after plating, cells were transfected with software. One-way ANOVA followed by Tukey's multiple 200 ng of luciferase reporter vectors using Lipofectamine comparison tests and the Student two-tailed t tests were used LTX (Invitrogen). All reactions included 100 ng pCH110- when appropriate. Statistical significance was assessed at P < b-gal (GE Healthcare) to normalize for transfection effi- 0.05. ciency. The cells were dosed 24 hours posttransfection with either 0.1% DMSO (vehicle control) or 10 nmol/L TCDD. The following day, cells were lysed and luciferase Results activity was determined using the ONE-Glo system accord- TCDD- and AHR-dependent regulation of CCNG2 ing to manufacturer's recommendations (Promega). The To investigate the mechanism of the AHR-dependent firefly luciferase activity was normalized to b-gal levels and regulation of CCNG2,wefirst conducted time course mRNA the normalized data were presented relative to vehicle expression analysis of TCDD-treated T-47D cells cultured control. for 72 hours in medium containing 5% DCC-treated FBS to

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– synchronize the cells in G0 G1 (30) and to reduce the increases in CCNG2 protein levels after 6 hours of treatment concentration of potential AHR activators in the FBS. As (Fig. 1B). shown in Fig. 1A, CCNG2 mRNA levels were increased To assess the impact of CCNG2 upregulation and its role following 1.5 hours of treatment and remained elevated in the TCDD-dependent cell-cycle inhibition, we con- until 24 hours. TCDD-dependent increases in CCNG2 ducted cell-cycle analysis. T-47D cells transiently transfected mRNA levels were reduced after cotreatment with the selec- with RNA interference (RNAi) targeting CCNG2 (70% tive AHR antagonist CH223191 (31) at all time points knockdown was achieved at the mRNA level, data not examined. Western blotting conﬁrmed TCDD-dependent shown) were double-stained with BrdUrd and PI after 48

Figure 1. TCDD-dependent regulation of CCNG2. A, time course analysis of the TCDD-dependent gene regulation of CCNG2. T-47D breast cancer cells were treated (10 nmol/L TCDD or pretreatment for 1 hour with 1 mmol/L CH223191) for the indicated time period and RNA was isolated and reverse-transcribed. Changes in mRNA expression levels were then determined using quantitative PCR. Data were normalized against time-matched DMSO and to ribosomal 18S levels. Each error bar represents the SEM of 3 independent replicates. , statistical significance compared with time-matched DMSO; #, statistical significance compared with time-matched TCDD treatment (P < 0.05, one-way ANOVA). B, Western blot analysis of CCNG2 protein levels. T-47D cells were treated with DMSO or 10 nmol/L TCDD. Cell extracts were probed with rabbit antibody against CCNG2. b-Actin was used as loading control. C–F, cell-cycle analysis of T-47D cells transiently transfected with universal negative (neg.) control or siCCNG2 were exposed to DMSO or 10 nmol/L TCDD for 48 hours and harvested for FACS analysis. Cells were pulsed with 10 mg/mL of BrdUrd before being collected. For each treatment, BrdUrd-PI bivariate plot with numbers corresponding to the percentage of cells in G1, S, and G2– M phases of the cell cycle were generated. Data shown (A–F) are representative graphs of 3 experiments. The data presented in (G and H) are compiled data from 3 independent experiments and indicate the percentage of cells in each phase of the cell cycle. , statistical significance compared with RNAi-matched DMSO treatment cells; #, statistical significance compared with negative control DMSO treatment (P < 0.05, one-way ANOVA).

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hours of treatment with DMSO or 10 nmol/L TCDD and TCDD-dependent interactions between FOXA1 and analyzed using FACS. We observed that cells transfected AHR with universal negative control and treated with TCDD To determine whether FOXA1 and AHR were present in resulted in an increase in the number of cells in G1 when the same protein complex, we carried out sequential ChIP compared with DMSO (Fig. 1C and E). However, in cells and co-IP experiments. Sequential ChIP analyses revealed transfected with RNAi-targeting CCNG2, there was an that AHR and FOXA1 were recruited simultaneously to increased amount in the S-phase (Fig. 1D) but TCDD CCNG2 (Fig. 3A). Furthermore, AHR and FOXA1 were treatment did not alter the distribution of cells (Fig. 1F). shown to be part of the same protein complex in co-IP assays The ability of TCDD to increase the number of cells in G1 completed in the presence or absence of TCDD (Fig. 3B). and reduce the number of cells in S-phase was lost following knockdown of CCNG2 (Fig. 1G and H), AHR mediates the TCDD-dependent regulation of suggesting that CCNG2 is an important contributor to CCNG2 using FOXA1 the TCDD-dependent cell-cycle inhibition in T-47D To determine how AHR regulates CCNG2 transcription cells. and to identify the key response element(s) involved in this regulation, we conducted promoter deletion and site-direct- TCDD induces the recruitment of AHR and FOXA1 to ed mutagenesis analyses. Treatment with 10 nmol/L TCDD CCNG2 resulted in an approximate 1.5-fold increase in activity of the To identify AHREs and other transcription factor–bind- full-length promoter, pGL4-CCNG2 (Fig. 4A). Deletion of ing sites that might be important in the TCDD-dependent AHRE2 abolished the TCDD-dependent regulation of regulation of CCNG2, we conducted transcription factor– CCNG2 (Fig. 4A). Site-directed mutagenesis of AHRE2 binding site analysis on the 1.4 kb CCNG2 regulatory inhibited the TCDD-mediated luciferase activity (Fig. 4B). region using MatInspector (Genomatix). This analysis iden- These findings suggest that AHRE1 is not required for tified 2 AHRE sequences that were positioned in close AHR-dependent regulation of CCNG2. We then mutated proximity to multiple forkhead (FKH) sites. We designated the FKH3 and FKH4 sites to evaluate the role of FOXA1 in the AHRE sites, AHRE1 and AHRE2, and the FKH sites, modulating the AHR-dependent regulation of CCNG2. FKH1–4 (Fig. 2D). Because FOXA1 is an important tran- Mutation of either FKH site significantly decreased, but scription factor in ERa-positive breast cancer cells, we then did not abolish, the TCDD-dependent increase in luciferase determined the role of FOXA1 as well as each of the activity (Fig. 4B). However, mutation of both FKH sites individual AHREs in the AHR-dependent regulation of prevented the TCDD-dependent increase in luciferase activ- CCNG2. To examine AHR and FOXA1 recruitment to ity. Taken together, these data show that AHRE2 and the CCNG2 promoter in T-47D cells, we conducted ChIP FOXA1 play key roles in the AHR-dependent regulation assays. Cells were treated with DMSO or 10 nmol/L TCDD of CCNG2. The AHR binding observed at AHRE1 in the for 1 hour, cross-linked and protein–DNA complexes were ChIP analysis (Fig. 2A) may just represent larger immuno- immunoprecipitated with antibodies directed against AHR, precipitated DNA fragments containing AHRE2, as the FOXA1, H3K4Me2, or H3K9Ac. H3K9Ac was examined resolution of our ChIP assay is 500 to 800 bp. because this modification correlates with actively transcribed genes (32). H3K4Me2 was tested to identify FOXA1 but not ERa is required for the AHR-dependent functional FOXA1-binding sites, as H3K4Me2 has been regulation of CCNG2 reported to correlate with FOXA1 binding to its FKH site FOXA1 is an important modulator of ERa and androgen (33). As shown in Fig. 2A, TCDD treatment significantly receptor transactivation in breast and prostate cancer cells, and preferentially induced the recruitment of AHR and respectively (25, 26, 34–36). Because FKH sites were found FOXA1 to AHRE2, whereas only a modest, albeit signif- to be important in the TCDD-mediated regulation of the icant, increase in AHR recruitment to AHRE1 was CCNG2 luciferase reporter plasmid, we hypothesized that observed (Fig. 2A). ChIP studies revealed constitutive FOXA1 may have a similar role with AHR–chromatin binding of FOXA1 to the enhancer region, which was interactions at CCNG2. To test this hypothesis, we used increased after ligand treatment. Treatment with TCDD RNAi-mediated knockdown of FOXA1 and measured resulted in increased levels of H3K4Me2 or H3K9Ac at mRNA expression, as well as the recruitment patterns of AHRE2 but not at AHRE1 (Fig. 2B). These findings AHR, FOXA1, and ERa;ERa is known to negatively suggest that AHRE2, but not AHRE1, is the dominant regulate CCNG2 (21). Following transient transfection of AHRE driving the TCDD-dependent regulation of 2 distinct siRNA oligos into T-47D cells, we determined that CCNG2. In agreement with TCDD-dependent increases 48 hours posttransfection, FOXA1 protein levels were great- in CCNG2 mRNA levels, ChIP assays confirmed increases ly reduced (Fig. 5A) and mRNA expression was reduced in the recruitment of RNA polymerase II to the proximal to 20% compared with control cells (data not shown). promoter region of CCNG2 after TCDD treatment (Fig. Interestingly, the loss of FOXA1 caused a marked decrease 2C). Although the expression of FOXA2 was comparable in ERa protein levels, which has been previously reported with FOXA1 levels, FOXA2 was not recruited to CCNG2, (34), but did not cause any changes in AHR protein levels. whereas FOXA3 was not detected in T-47D cells (data not Similar findings were observed in MCF-7 ERa-positive shown). breast carcinoma cells (data not shown). RNAi-mediated

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Figure 2. TCDD induces the recruitment of AHR and FOXA1 to CCNG2. A, quantification of AHR and FOXA1 recruitment to AHRE1 and AHRE2 using the ChIP assay. Briefly, T-47D cells were treated with 10 nmol/L TCDD for 45 minutes and immunoprecipitated using the antibodies indicated. The immunoprecipitated DNA was measured by qPCR with primers designed around each response element. B, determining the relative levels of the histone modifications H3K4Me2 and H3K9Ac following TCDD treatment at AHRE1 and AHRE2 using the ChIP assay. C, recruitment of RNA polymerase (pol) II to the TATA box in the proximal promoter region of CCNG2 after 45 minutes of treatment. D, diagram of CCNG2 regulatory region. Each error bar represents the SEM of 3 independent replicates. All data are relative to 100% total input. , statistically significant differences compared with DMSO control samples using a one-way ANOVA (P < 0.05). knockdown of FOXA1 inhibited the TCDD-dependent regulation of CCNG2, although we cannot exclude the gene expression supporting our promoter deletion and possibility that the reduced ERa protein levels influence mutagenesis results described above (Fig. 5A). As indicated AHR transactivation, as our laboratory and others have by our ChIP studies, treatment with 10 nmol/L of TCDD shown that ERa modulates AHR activity (7, 8, 29). treatment resulted in increased recruitment of FOXA1, We then conducted RNAi-mediated ERa knockdown AHR, and ERa (Fig. 5B–D). Interestingly, we observed studies to determine the role of ERa in AHR-mediated constitutive binding of both FOXA1 and ERa when com- regulation of CCNG2 expression. The loss of ERa had pared with IgG controls. RNA-mediated knockdown of no effect on either FOXA1 or AHR protein levels (Fig. FOXA1 abolished the TCDD-dependent recruitment of 5E). In agreement with our previous findings, the knock- both AHR and ERa (Fig. 5C and D). The reduced recruit- down of ERa significantly increased the constitutive ment of ERa to CCNG2 was most likely due to reduced levels of CCNG2 mRNA levels but did not affect the protein expression levels rather than the loss of FOXA1. TCDD-mediated increase in CCNG2 mRNA levels FOXA1, however, was necessary for the AHR-mediated (ref.8;Fig.5E).ChIPanalysisshowedthatERa was

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Figure 3. FOXA1 and AHR are part of the same protein complex. A, sequential ChIP analyses were conducted with the indicated antibodies. Immunoprecipitated DNA was measured by qPCR using the CCNG2 enhancer primers. Quantiﬁcation of binding was determined as a fold induction above IgG DMSO. Each error bar represents the SEM of 3 independent replicates. , statistically signiﬁcant differences compared with IgG DMSO control samples (P < 0.05, one-way ANOVA). B, co-IP studies were completed in T-47D cells. Cells were treated for 1 hour with either DMSO or TCDD and then cross-linked using formaldehyde. Cell lysate was immunoprecipitated using antibodies against AHR and FOXA1. IgG was used as the negative control. Western blotting was then completed using the reciprocal antibody. recruited to CCNG2 in the absence of ligand, suggesting FOXA1 to CCNG2 (Fig.5GandH).Together,these that under these conditions, ERa modulates CCNG2 data provide evidence that FOXA1 is driving the AHR- gene expression (Fig. 5E and F). Knockdown of ERa did mediated regulation of CCNG2 irrespective of ERa not affect the TCDD-dependent recruitment of AHR or levels.

Figure 4. AHR mediates the TCDD- dependent regulation of CCNG2. A, deletion fragments of pGL4- CCNG2 were tested to deduce the functional signiﬁcance of AHRE1 and AHRE2. Two hundred nanograms of each vector was transfected in T-47D cells and luminescence was measured following a 24-hour 10 nmol/L TCDD treatment. B, site-directed mutagenesis of AHRE2 as well as to the FKH recognition sites was generated in pGL4-CCNG2. T47-D cells were transfected with 200 ng of the single and double response element mutants and treated with either DMSO or 10 nmol/L TCDD for 24 hours. Results represent the mean of 3 independent replicates. , luciferase (luc) activity that was statistically different compared with DMSO pGL4-CCNG2 control; #, luciferase activity statistically different compared with TCDD pGL4-CCNG2 (P < 0.05, one-way ANOVA).

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Figure 5. FOXA1, but not ERa, is essential for the TCDD-dependent recruitment of AHR to CCNG2. A, gene expression profiles were completed on T-47D cells transfected for 48 hours with siRNA and then treated for 6 hours with TCDD. RNA was isolated and reverse-transcribed. mRNA expression was then determined using qPCR. Data were normalized against time-matched DMSO and to ribosomal 18S levels. Data represent the mean of 3 independent replicates and # is compared with TCDD negative (neg.) control (P < 0.05, one-way ANOVA). Inset, analysis of FOXA1 knockdown in T-47D cells after 48 hours. Cell extracts were probed with rabbit antibody against AHR, ERa, and FOXA1. b-Actin was used as loading control. Recruitment of FOXA1 (B), AHR (C), and ERa (D) following siRNA-mediated knockdown of FOXA1 using the ChIP assay. Briefly, T-47D cells were transfected for 48 hours with siRNA and then treated for 45 minutes with TCDD and immunoprecipitated using the antibodies indicated. The immunoprecipitated DNA was measured by qPCR with primers targeting the enhancer region (relative to 100% total input). Each graph represents the mean of 3 independent replicates with indicating statistically significant differences compared with DMSO negative control whereas the # indicates statistically significant differences compared with TCDD negative control (P < 0.05, one-way ANOVA). E, CCNG2 mRNA expression levels were completed on T-47D cells transfected for 48 hours with siERa and then treated for 6 hours with TCDD. Data represent the mean of 3 independent replicates and the are compared with DMSO negative control (P < 0.05, one-way ANOVA). Inset, analysis of ERa knockdown in T-47D cells after 48 hours. Cell extracts were probed with rabbit antibody against AHR, ERa, and FOXA1. b-Actin was used as loading control. Recruitment of ERa (F), FOXA1 (G), and AHR (H) was determined following RNAi-mediated knockdown of ERa using ChIP assays. Each graph represents the mean of 3 independent replicates with the indicating statistically significant differences compared with DMSO negative control (P < 0.05, one-way ANOVA). www.aacrjournals.org Mol Cancer Res; 10(5) May 2012 643

Figure 6. NCoA3 is part of the complex formed at CCNG2. Recruitment of NCoA3 was determined after knockdown of FOXA1 (A) and ERa (B). Briefly, cells were transfected with siRNA targeting both factors followed by treatment with 10 nmol/L TCDD, after which chromatin was immunoprecipitated using an antibody against NCoA3. The immunoprecipitated DNA was measured by qPCR with primers targeting the enhancer region. Each graph represents the mean of 3 independent replicates with the indicating statistically significant differences compared with DMSO negative control whereas the # indicates statistically significant differences compared with TCDD negative (neg.) control (P < 0.05, one-way ANOVA). C, co-IP studies were completed in T-47D cells. Cells were treated for 45 minutes with either DMSO or TCDD and then cross-linked using formaldehyde. Cell lysate was immunoprecipitated using a selective NCoA3 antibody. IgG was used as the negative control. Western blotting was done using antibodies against AHR and FOXA1.

TCDD-dependent recruitment of NCoA3 to CCNG2 leading to increased CCNG2 gene expression and prevent- Previous studies showed that CCNG2 was negatively ing the previous described repressive actions of ERa on regulated by ERa through the recruitment of an NCoR CCNG2 expression (ref. 21; Fig. 8). complex leading to the hypoacetylation of histones and the release of RNA polymerase II (21). On the basis of these results, we hypothesized that the TCDD-dependent pos- Discussion itive regulation of CCNG2 must overcome this inhibition AHR has emerged as an important therapeutic target for through the recruitment of nuclear coactivators to promote breast cancer, as its activation has been reported to inhibit the gene expression. Because NCoA3/AIB1 is overexpressed in growth of ER-positive, ER-negative, and HER2-positive breast cancer, we determined the ability of TCDD, E2, breast cancer cells (7, 8, 28, 38, 39). In the present study, and E2 þ TCDD to induce recruitment of NCoA3 to we show that ligand-activated AHR together with FOXA1 CCNG2 in T-47D in the presence or absence of RNAi- increases the expression of CCNG2 in ERa-positive T-47D mediated knockdown of FOXA1 or ERa.TCDDtreat- breast cancer cells. We also report that activation of AHR ment resulted in increased NCoA3 recruitment to prevented the ability of ERa to repress CCNG2 expression, CCNG2, which was significantly reduced only after knock- providing another example where activation of the AHR down of FOXA1 but not ERa (Fig. 6A and B). The co-IP pathway opposes the actions of ERa. Our findings provide a studies provided further evidence that NCoA3 is part of new mechanism by which AHR can inhibit human breast the activated multiprotein AHR-containing complex, as it cancer cell proliferation. The increase in expression of was found to interact with both AHR and FOXA1 CCNG2 by AHR further supports the notion that targeting (ref. 37; Fig. 6C). AHR might be an effective therapy for breast cancer treatment (4, 38). Although the clinical importance of AHR- AHR prevents the ERa-dependent negative regulation of dependent activation of CCNG2 remains to be investigated, CCNG2 trastuzumab treatment has also been reported to increase In support of a previous report, estrogen-bound ERa CCNG2 levels (22). RNAi-mediated knockdown of inhibited CCNG2 mRNA expression levels (ref. 21; Fig. 7A CCNG2 resulted in a slight reduction in trastuzumab- and B). However, this repression was overcome by co- dependent growth inhibition, suggesting that CCNG2 is treatment with TCDD and required FOXA1 but not ERa required, but not necessary, for the inhibitory action of (Fig. 7A and B). Co-treatment of TCDD þ E2 prevented trastuzumab (18, 40). However, the mechanism of CCNG2 the E2-dependent removal of NCoA3 from the CCNG2 upregulation by trastuzumab most likely does not require resulting in increased recruitment of both AHR and FOXA1 FOXA1, as it is not expressed in ER-negative breast cancer (Fig. 7C–E). The TCDD-induced recruitment of NCoA3 (41). Nonetheless, these findings suggest that modulating was dependent on FOXA1 (Fig. 7C–E). RNAi-mediated CCNG2 expression might be an important mechanism to knockdown of ERa had no effect on the ability of AHR to inhibit cancer cell growth. block the repression caused by E2 treatment (Fig. 7G–I). TCDD-dependent activation of AHR blocks cell-cycle fi Overall, our ndings show that FOXA1 facilitated the progression through the G1 phase in several cell lines binding of TCDD-induced AHR and NCoA3 to CCNG2, and under many different conditions, including mouse

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Figure 7. AHR can overcome the ERa-dependent negative (neg.) regulation of CCNG2. CCNG2 mRNA expression levels were determined from T-47D cells transfected for 48 hours with siFOXA1 (A) or siERa (B) and then treated for 6 hours with 10 nmol/L E2 or 10 nmol/L E2 þ 10 nmol/L TCDD. RNA was isolated and reverse-transcribed, and mRNA expression levels were determined using qPCR. Data were normalized against time-matched DMSO and to ribosomal 18S levels. Data represent the mean of 3 independent replicates with the representing statistically signiﬁcant differences compared with DMSO and the # represents statistically signiﬁcant differences compared with treatment-matched samples (P < 0.05, one-way ANOVA). Cells were treated with 10 nmol/L E2 or 10 nmol/L E2 þ 10 nmol/L TCDD for 45 minutes and the recruitment of AHR (C), FOXA1 (D), NCoA3 (E), and ERa (F) was determined 48 hours post-siFOXA1 transfection using ChIP assays and qPCR. Similar experiments were also carried out 48 hours after siERa transfection using antibodies against AHR (G), FOXA1 (H), NCoA3 (I), and ERa (J).

hepatoma Hepa1c1c7 (42), rat hepatoma 5L cells (43), and toma and displacement of the coactivator p300 in estrogen-induced MCF-7 cell proliferation (44). Potential (10, 12, 45). The TCDD-dependent increase in CCNG2 mechanisms include the AHR-dependent induction of expression reported here provides another mechanism by Kip1 WAF1 p27 and p21 , inhibition of CDK function, inhibi- which ligand-activated AHR regulates cell-cycle progression tion of retinoblastoma phosphorylation, and repression of in the G1 phase, as increases in CCNG2 levels result in G1 E2F-regulated genes through interactions with retinoblas- to S-phase arrest (17). In support of these ﬁndings, we

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Figure 8. Proposed mechanism for the FOXA1- and AHR-dependent regulation of CCNG2. In the absence of ligand, FOXA1 is bound to the FKH recognition sites in the enhancer region of CCNG2. ERa and NCoA3 occupy the upstream regulatory region of CCNG2. Treatment with E2 reduces the constitutive NCoA3 but increases E2-bound ERa occupancy at CCNG2 resulting in transcriptional repression as previously described (21). However, upon TCDD or E2þTCDD treatment, FOXA1 binding is increased facilitating the recruitment of AHR and increased recruitment of NCoA3 leading to transcriptional activation of CCNG2. FOXA1 mediates these effects through direct interactions with AHR. The relative transcriptional activity of CCNG2 is represented by the magnitude of the arrow.

report that the TCDD-dependent increase in the number CCNG2 expression in NIH3T3 mouse embryonic fibro- b of T-47D cells in G1 phase arrest is lost after knockdown of blasts (14). Recently, Nodal, a member of the TGF- family, CCNG2. was found to increase CCNG2 mRNA expression by FOXA1 has been implicated in both ERa and androgen increasing the expression of FOXO3a, which then forms receptor signaling (26, 35). Previously reported ChIP-chip a complex with Smad proteins at the CCNG2 promoter data from our laboratory revealed that in both human cells region (50). They found that the more proximal FKH sites and mouse tissue, FKH sites are significantly enriched in (FKH1 and FKH2 in Fig. 2) were required for FOXO3a- AHR-bound regions supporting a possible role for forkhead mediated induction of CCNG2, rather than the distal FKH proteins in AHR signaling (8, 9, 46). In support of these sites (FKH3 and FKH4 in Fig. 2) that are required for AHR- data, we show that FOXA1 is critical for the AHR-depen- dependent induction of CCNG2 reported here. Interest- dent induction of CCNG2 levels. We observe that FOXA1 ingly, the antiproliferative effect of Nodal on ovarian cells is present at CCNG2 in the absence of AHR activation. was found to be partly mediated by CCNG2 (51). These Following AHR ligand treatment, the levels of FOXA1, findings show the important role of the forkhead protein AHR, NCoA3, and H3K4Me2 are increased at CCNG2, family in the regulation of CCNG2 but reveal that the suggesting that FOXA1 primes the CCNG2 for AHR regulation of CCNG2 by FOXA1 or FOXO3a occurs via recruitment and subsequent transcriptional activation. AHR distinct FKH sites. and FOXA1 interact in co-IP and re-chip experiments, Recent RNAi-mediated knockdown of FOXA1 followed showing that they are part of the same multiprotein complex, by ChIP-sequencing studies revealed that FOXA1 is which agrees with other reports showing that forkhead required for almost all ERa binding to its genomic target protein family members interact with other transcription regions irrespective of the proximity of FKH sites to ERa- factors (26). FOXA1 has been previously reported to interact binding sites (35). Although the RNAi-mediated knock- with androgen receptor (47) and was shown to enhance down of FOXA1 was reported to not affect ERa protein glucocorticoid receptor transactivation (48). Similarly, levels (35), we and others report that knockdown of FOXA1 FOXA3 interacts with glucocorticoid receptor where by the has been shown to decrease ERa mRNA expression and binding of FOXA3 provides a favorable DNA conformation protein levels (34). The reason for the discrepancies between for glucocorticoid receptor binding to its target genes (49). the studies is unclear, but we observe that RNAi-mediated FOXA1 may use both the direct interaction with AHR and knockdown of FOXA1 reduces ERa protein levels in 2 altered DNA conformation to enhance AHR binding to different cell lines (T-47D, Fig. 5; MCF-7, data not shown) CCNG2. We hypothesize that FOXA1 stabilizes the AHR- using 2 unique siRNA oligos targeting FOXA1. We also activated complex at CCNG2 and therefore is required for observe that RNAi-mediated knockdown of FOXA1 maximal gene activation by AHR. reduced the TCDD-dependent induction of CYP1A1 and The regulation of CCNG2 by other members of the CYP1B1. But unlike what was observed for CCNG2, RNAi- forkhead protein family has been previously reported, with mediated knockdown of ERa did reduce the TCDD one group showing that FoxO transcription factors increased responsiveness of both CYP1A1 and CYP1B1 in T-47D

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cells (S. Ahmed and J. Matthews unpublished findings; into the antiproliferative action of AHR in breast cancer ref. 8). This suggests that for certain genes the reduced cells and further support AHR as a therapeutic target for AHR transactivation following RNAi-mediated knockdown breast cancer treatment. of FOXA1, may be due to reduced ERa levels and not reduced FOXA1 expression. Therefore, it will be important Disclosure of Potential Conflicts of Interest to distinguish the effects of FOXA1 knockdown on AHR No potential conflicts of interest were disclosed. transactivation compared with those mediated by ERa. Further studies investigating the recruitment patterns of Acknowledgments The authors thank Dr. Chun Peng for providing materials for this study, all AHR and FOXA1 using ChIP-sequencing and RNAi-medi- members of the Matthews' laboratory for their assistance and helpful discussions ated knockdown approaches will be helpful in determining during the course of this work, and Meghan Larin and Nishani Rajakulendran for their whether FOXA1 is a general modulator of AHR signaling or help with the cell preparation and analysis of the FACS samples. a gene-specific regulator. In summary, we report that the AHR-dependent reg- Grant Support ulation of CCNG2 requires FOXA1 to stabilize the The study was supported by Canadian Institute of Health Research operating grant (MOP-82715) and Canadian Breast Cancer Foundation-Ontario funded this binding of activated AHR and aiding in the recruitment research. S. Ahmed is supported by the Canadian Institute of Health Research of other co-regulatory proteins, such as NCoA3. Our Doctoral Research Award. J. Matthews is the recipient of the Canadian Institute of fi Health Research New Investigator Award and Early Researcher Award from the ndings also show that activated AHR is able to overcome Ontario Ministry of Innovation. the repressive actions of ERa at CCNG2,providing The costs of publication of this article were defrayed in part by the payment of page further evidence supporting the antiestrogenic effects of charges. This article must therefore be hereby marked advertisement in accordance with AHR. Although additional studies are needed to fully 18 U.S.C. Section 1734 solely to indicate this fact. characterize the depth and role of FOXA1 in AHR Received October 24, 2011; revised February 2, 2012; accepted March 13, 2012; signaling, the data presented here provide new insight published online May 17, 2012.

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648 Mol Cancer Res; 10(5) May 2012 Molecular Cancer Research

Shaimaa Ahmed, Sarra Al-Saigh and Jason Matthews

Mol Cancer Res 2012;10:636-648.

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