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Signaling and Regulation

FOXA1 Is Essential for Aryl HydrocarbonReceptor–Dependent Regulation of Cyclin G2

Shaimaa Ahmed, Sarra Al-Saigh, and Jason Matthews

AbstractThe 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 apotential therapeutic target for breast cancer by virtue of its ability tomodulate estrogen receptor-a (ERa) signallingand/or its ability to block cell proliferation.Our previous studies identified cyclinG2 (CCNG2), an inhibitor of cell-cycle progression, as an AHR target gene; however, the mechanism of this regulation is unknown. Chromatinimmunoprecipitation assays in T-47D human breast cancer cells revealed a TCDD-dependent recruitment ofAHR, nuclear co-activator 3 (NCoA3) and the transcription factor forkhead box A1 (FOXA1), a key regulator ofbreast cancer cell signaling, toCCNG2 resulting in increases inCCNG2mRNA and protein levels.Mutation of theAHR response element (AHRE) and forkhead-binding sites abolishedTCDD-inducedCCNG2-regulated reportergene activity. RNA interference–mediated knockdown of FOXA1 prevented the TCDD-dependent recruitment ofAHR and NCoA3 to CCNG2 and reduced CCNG2 mRNA levels. Interestingly, knockdown of FOXA1 alsocaused a marked decrease in ERa, but not AHR protein levels. However, RNA interference–mediated knockdownof ERa, a negative regulator of CCNG2, had no effect on TCDD-dependent AHR or NCoA3 recruitment to orexpression ofCCNG2. These findings show that FOXA1, but not ERa, is essential for AHR-dependent regulationof CCNG2, assigning a role for FOXA1 in AHR action. Mol Cancer Res; 10(5); 636–48. �2012 AACR.

IntroductionThe aryl hydrocarbon receptor (AHR) is a ligand-activated

transcription factor that mediates the toxic effects of bothhalogenated and polycyclic aromatic hydrocarbons, such as2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-meth-ylcholanthrene (3MC). AHR plays important roles in devel-opment and differentiation, as well as in the immune and thereproductive systems (1, 2). Moreover, AHR has recentlyemerged as a therapeutic target for inflammatory and auto-immune diseases, as well as for the treatment of breast cancer(3, 4). We and others have shown that AHR activationinhibits estrogen receptor-a (ERa) signaling through directinteraction with ERa and through the modulation of ERatarget genes (5–7). After ligand binding, AHR translocatesinto the nucleus where it binds to its dimerization partnerAHR nuclear translocator (ARNT). This heterodimer thenbinds to specific DNA response elements termed AHRresponse elements (AHRE) in the regulatory regions of itstarget genes. DNA binding of the AHR/ARNT heterodimer

results in the recruitment of coregulators (coactivators andcorepressors) causing changes in gene expression. Well-studied AHR target genes include the phase I and IIdrug-metabolizing enzymes, such as cytochrome P4501A1 (CYP1A1), CYP1B1, and glutathione S-transferases.Chromatin immunoprecipitation combined with microar-rays (ChIP-chip) and gene expression microarray experi-ments revealed that AHR regulates numerous genes involvedin diverse cellular pathways, including metabolism and cell-cycle regulation (8–10). In support of these findings, pre-vious studies have shown that TCDD inhibits cell prolifer-ation (11–13).Cell proliferation is a highly regulated event mediated by

the activation of cyclin-dependent kinases (CDK), whichrequire the binding of cyclins for full activity. Mammaliancyclins are classified into 12 different types cyclins A to Ibased on sequence and functional similarities (14). Mostcyclins have been shown to facilitate growth by eitherpromoting G0–G1 to S-phase or the G2–Mphase transition.The G-type cyclins (cyclin G1, G2, and I) on the other handare associated with cell-cycle arrest (15). Cyclin G1 isinvolved in G2–M phase arrest, whereas cyclin I is thoughtto play a role in apoptosis (16). Cyclin G2 (CCNG2) hasbeen shown to inhibit cell-cycle progression by preventingG1 to S-phase transition (14, 15, 17). This inhibition isachieved by interacting with protein phosphatase 2A(PP2A). The CCNG2-PP2A complex alters PP2A targetingand its substrate specificity leading to cell-cycle arrest (14).CCNG2 also stops cell-cycle progression by preventing the

Authors' Affiliation: Department of Pharmacology and Toxicology, Uni-versity of Toronto, Toronto, Ontario, Canada

Corresponding Author: Jason Matthews, Department of Pharmacologyand Toxicology, University of Toronto, Medical Sciences Building, Room4336, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8. Phone:416-946-0851; Fax: 416-978-6395; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-11-0502

�2012 American Association for Cancer Research.

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phosphorylation of CDK2 and retinoblastoma which arerequired for progression out of the quiescent state (G0;refs. 14, 18). CCNG2 has been shown to be a critical genein human epidermal growth factor receptor 2 (HER2)- andhormone-dependent breast cancers (18–21). Moreover,CCNG2 was among the few genes upregulated followinganti-HER2 antibody trastuzumab treatment, indicating thatCCNG2 might play a role in inhibiting HER2-dependentproliferation (22).In estrogen-dependent breast cancer, cell proliferation is

facilitated by the binding of estrogen to ERa resulting ineither increased expression of genes associated with prolif-eration or suppression of genes that block cell-cycle progres-sion. ERa represses CCNG2 expression in response toestrogen by recruiting a complex containing nuclear co-repressor (NCoR) and histone deacetylases to the CCNG2promoter region resulting in the displacement of RNApolymerase II (21). Our previous ChIP-chip studies iden-tified an AHR-bound AHRE-containing sequence in theupstream regulatory region of CCNG2. In contrast toestrogen, the AHR agonists, TCDD and 3MC, inducedAHR recruitment to CCNG2 causing an increase inCCNG2 mRNA levels (8, 9). However, the molecularmechanisms and characterization of the AHR-dependentregulation of CCNG2 remain elusive.Another protein shown to be important in the regulation

of genes in breast cancer is the forkhead box A1 (FOXA1)transcription factor, a member of the winged-helix tran-scription factors (23). The FOXA family is composed of 3closely related members, FOXA1, A2, and A3, which havecritical roles in development and differentiation (23). Highexpression of FOXA1 is observed in ERa-positive breasttumors and its expression is a positive prognostic factorwhich correlates with sensitivity to endocrine therapy andinhibition of tumor growth (19, 24). FOXA1 acts as a"pioneer factor" by altering chromatin structure and facil-itating ERa binding to its target genes (25). FOXA1 alsomodulates androgen receptor function, indicating that it hasa more general role in regulating gene expression (26, 27).The role of FOXA1 in AHR transactivation or cross-talkbetween AHR and ERa has not been determined.AHR activation inhibits breast cancer cell proliferation

and induces cell-cycle arrest through a number of differentpathways (2, 10, 28). We report here that FOXA1, but notERa, is essential for AHR-dependent regulation ofCCNG2,assigning a role for FOXA1 in AHR action and character-izing a newmechanism by which AHR inhibits breast cancercell proliferation.

Materials and MethodsChemicalsAntibodies used for ChIP and Western blotting experi-

ments include ERa (HC-20), AHR (H-211), and NCoA3(M-397) from Santa Cruz Biotechnology and FOXA1(ab23738) fromAbcam.TCDDwas purchased fromAccuS-tandard and dimethyl sulfoxide (DMSO) was purchasedfrom Sigma. Cell culture media, FBS, and trypsin were all

purchased from Wisent Bioproducts. All other chemicalsand biochemicals were of the highest quality available fromcommercial vendors.

ChIP assaysT-47D human breast carcinoma cells were cultured in 1:1

mixture of Dulbecco's Modified Eagle's Medium (DMEM)and Ham's F12 nutrient mixture medium supplementedwith 10% (v/v) FBS and 1% penicillin/streptomycin. Cellswere maintained at 37�C and 5% CO2. T-47D cells weresubcultured every 2 to 3 days or when they reached 80% to90% confluency. For the ChIP studies, 3 million T-47Dcells were seeded in 10-cm dishes in 1:1 mixture of DMEM:F12 phenol red–free media supplemented with 5% (v/v)dextran-coated charcoal (DCC)-stripped serum and 1%penicillin/streptomycin. After 72 hours, cells were treatedwith DMSO (final concentration, 0.1%), 10 nmol/LTCDD, 10 nmol/L E2, or E2 þ TCDD (10 nmol/L) for0.75 hours. ChIP and sequential ChIP assays were con-ducted as previously described (8). Enrichment levels (rel-ative to 100% total input) were determined by quantitativereal-time PCR (qPCR) using the following primers:CCNG2AHRE2 (enhancer): 50-TGGGTTACCAAGGACCAA-GAA-30 50-CCAGAGGTTGTAGTGCTGTTGTTT-30;CCNG2 AHRE1: 50-AACTCTCCCGTGGCTGAAAAand 50-CGCGGCGCTTCTCCTAA-30 CCNG2 TATA:50-GGGAGGCCGCGAGAGA-30 and 50-TCCTCCACG-GACTTTAAAAAAGAG-30.

RNA isolation and real-time PCRT-47D cells were seeded in 6-well plates and grown in a

1:1 mixture of DMEM:F12. Cells were treated with either10 nmol/L TCDD or pretreatment for 1 hour with 1mmol/L CH223191 for 0.75, 1.5, 3, 6, or 24 hours. RNAwas isolated using illustra RNAspin mini columns (GEHealthcare) and reverse-transcribed as previously described(8). All target gene transcripts were normalized to ribosomal18S RNA and fold inductions were calculated using time-matched DMSO-treated samples and the comparative CT(DDCT) method was used for data analysis. qPCR primersused in the study have been described elsewhere (8) withexception of the following primers: FOXA1 mRNA 50-GAAGATGGAAGGGCATGAAA-30 and 50-GCCTGA-GTTCATGTTGCTGA-30.

Western blottingProteins were resolved by SDS-PAGE and transferred to

nitrocellulose membrane. The membrane was blocked in2% (w/v) ECL-Advanced blocking agent for 1 hour at roomtemperature with constant rocking and then incubated withanti-ERa (HC-20), anti-AHR (H-211), or anti-FOXA1(ab23738) overnight at 4�C with constant rocking. Themembrane was then washed 3 times and incubated with ananti-rabbit secondary antibody for 1 hour. For detection ofb-actin, a primary mouse anti-b-actin antibody (Sigma) wasincubated for 2 hours at room temperature followed by a1-hour washing with PBS/0.1% Tween-20 before incuba-tion with anti-mouse secondary antibody for 1 hour. After

FOXA1 Interacts with AHR to Regulate CCNG2

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washing, the bands were visualized using ECL-Advanced(GE Healthcare).

Transient transfection and siRNAsiFOXA1 seq2 (SASI_Hs01_00168404), siFOXA1 seq3

(SASI_Hs01_00168403), and a universal negative control(SIC001) were purchased from Sigma-Aldrich. siERa11(J-003401-11-0050), siERa14 (J-003401-14-0050),siCCNG2 (L-003217-00-0005), and DharmaFECT1transfection reagent were purchased from Dharmacon.Briefly, T-47D cells were seeded 300,000 per well in a 6-well plate in medium containing 5% DCC-stripped FBS.After 24 hours, 50 nmol/L of siRNA against FOXA1, ERa,CCNG2, or universal negative control were transfectedusing 2 mL of DharmaFECT and 400 mL Opti-MEM.ChIP assays, mRNA isolation, and whole-cell extracts wereprepared 48 hours after transfection as described above.Fluorescence-activated cell-sorting (FACS) analysis for thesiCCNG2-treated cells is described below.

Plasmid construction and mutagenesisThe plasmid pGL4-CCNG2 containing �1.402 kb to

þ179 of the upstream regulatory region of CCNG2 as wellas plasmids pGL4-CCNG2 DAHRE1 and DAHRE2 weregenerously provided by Dr. Chun Peng (York University,Toronto, ON, Canada). The numbering of the clonedCCNG2 regulatory region was relative to CCNG2 mRNA(NM_004354). Site-directed mutagenesis targeting theAHRE site inCCNG2was completed as previously described(29). Formutation of AHRE2 the PCR primers, 50-CTCAT-CAGCGCGCTAAGTTTT-30 and 50-AAAACTTAGCGCGCTGATGAG-30 were used to mutate pGL4-CCNG2;themutated residues underlined.Mutation of the 2 forkhead-binding sites upstream from AHRE2 was done using pGL4-CCNG2 as a template and the following PCR primersfor FKH3: 50-TAGGAGGGAG AGAGTCCCAAAA-TAAATGTTCCAG-30 and 50-CTGGAACATTTATTTTGGGACTCTCTCCCTCCTA-30 and for FKH450-CCGTAATTATT AGATTGGGACGTACCCTCAT-CAG-30 and 50-CTGATGAGGGTACGTCCCAATCTAATAATTACGG-30. All mutations were verified by DNAsequencing.

Transient transfection and reporter gene assayT-47D cells were plated in 12-well dishes in a 1:1

DMEM:F12 mixture containing 5%DCC-stripped serum.Twenty-four hours after plating, cells were transfected with200 ng of luciferase reporter vectors using LipofectamineLTX (Invitrogen). All reactions included 100 ng pCH110-b-gal (GE Healthcare) to normalize for transfection effi-ciency. The cells were dosed 24 hours posttransfection witheither 0.1% DMSO (vehicle control) or 10 nmol/LTCDD. The following day, cells were lysed and luciferaseactivity was determined using the ONE-Glo system accord-ing to manufacturer's recommendations (Promega). Thefirefly luciferase activity was normalized to b-gal levels andthe normalized data were presented relative to vehiclecontrol.

Co-immunoprecipitationFor co-immunoprecipitation (co-IP) studies, T-47D cells

were seeded in 10-cm dishes in 1:1 mixture of DMEM:F12supplemented with 5% DCC-stripped serum. After 72hours, cells were treated with either 10 nmol/L TCDD orDMSO (final concentration, 0.1%) for 1 hour and cross-linked using 1% formaldehyde and quenched using 125mmol/L glycine. Cell lysates were precleared using ProteinA, and protein complexes were immunoprecipitated using2 mg of rabbit IgG (Sigma), AHR (H-211), FOXA1 (Abcam23738), or NCoA3 (M-397) for 2 hours. Beads were washed4 times with wash buffer (10 mmol/L Tris-HCl, pH 8.0,150mmol/LNaCl, 10%glycerol, 1%NP-40, and2mmol/LEDTA). Eighty microliters of sample buffer was added tothe beads and samples were heated to 70�C for 10 minutes.Samples were loaded on an 8% SDS-PAGE gel, transfer-red to nitrocellulose membrane, and visualized using theClean Blot anti-rabbit HRP (Thermo Scientific) andECL-Advanced.

Flow cytometryCell-cycle analysis by bromodeoxyuridine (BrdUrd) and

propidium iodide (PI) double staining was completed onT-47D cells transiently transfected with universal negativecontrol or 50 nmol/L of siRNA against CCNG2 (L-003217-00-0005). Twenty-four hours after transfection, cells weretreated with DMSO (final concentration, 0.1%) or 10nmol/L TCDD and left for another 48 hours. Cells werethen collected and fixed in 70% ethanol for 20 minutes at�20�C. Cells were then rinsed in wash buffer (PBS þ0.5% bovine serum albumin) and resuspended in 2N HClfor 20 minutes, washed, incubated with 0.1 mol/L sodiumborate, pH 8.5, for 2 minutes, washed again, then incu-bated with fluorescein isothiocyanate (FITC)-conjugatedanti-BrdUrd (BD Biosciences) in PBS þ 0.5% bovineserum albumin þ 0.5% Tween-20 and left in the dark for30 minutes. Cells were then washed with wash buffer andstained with 50 mg/mL PI for another 30 minutes. FACSanalyses and data acquisition were done by the Faculty ofMedicine Flow Cytometry Facility using a FACSCaliburflow cytometer (BD Biosciences) and FlowJo software(TreeStar).

Statistical analysisAll results were expressed as means � SEM. Statistical

analysis was calculated using GraphPad Prism 5 statisticalsoftware. One-way ANOVA followed by Tukey's multiplecomparison tests and the Student two-tailed t tests were usedwhen appropriate. Statistical significance was assessed at P <0.05.

ResultsTCDD- and AHR-dependent regulation of CCNG2To investigate the mechanism of the AHR-dependent

regulation ofCCNG2, wefirst conducted time coursemRNAexpression analysis of TCDD-treated T-47D cells culturedfor 72 hours in medium containing 5%DCC-treated FBS to

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synchronize the cells in G0–G1 (30) and to reduce theconcentration of potential AHR activators in the FBS. Asshown in Fig. 1A, CCNG2 mRNA levels were increasedfollowing 1.5 hours of treatment and remained elevateduntil 24 hours. TCDD-dependent increases in CCNG2mRNA levels were reduced after cotreatment with the selec-tive AHR antagonist CH223191 (31) at all time pointsexamined. Western blotting confirmed TCDD-dependent

increases in CCNG2 protein levels after 6 hours of treatment(Fig. 1B).To assess the impact of CCNG2 upregulation and its role

in the TCDD-dependent cell-cycle inhibition, we con-ducted cell-cycle analysis. T-47Dcells transiently transfectedwith RNA interference (RNAi) targeting CCNG2 (�70%knockdown was achieved at the mRNA level, data notshown) were double-stained with BrdUrd and PI after 48

Figure 1. TCDD-dependentregulation of CCNG2. A, time courseanalysis of the TCDD-dependentgene regulation of CCNG2. T-47Dbreast cancer cells were treated (10nmol/L TCDD or pretreatment for 1hourwith 1mmol/LCH223191) for theindicated time period and RNA wasisolated and reverse-transcribed.Changes in mRNA expression levelswere then determined usingquantitative PCR. Data werenormalized against time-matchedDMSO and to ribosomal 18S levels.Each error bar represents the SEM of3 independent replicates.�, statistical significance comparedwith time-matched DMSO;#, statistical significance comparedwith time-matched TCDD treatment(P < 0.05, one-way ANOVA). B,Western blot analysis of CCNG2protein levels. T-47D cells weretreated with DMSO or 10 nmol/LTCDD. Cell extracts were probedwith rabbit antibody against CCNG2.b-Actin was used as loading control.C–F, cell-cycle analysis of T-47Dcells transiently transfected withuniversal negative (neg.) control orsiCCNG2 were exposed to DMSO or10 nmol/L TCDD for 48 hours andharvested for FACS analysis. Cellswere pulsedwith 10mg/mLof BrdUrdbefore being collected. For eachtreatment, BrdUrd-PI bivariate plotwith numbers corresponding to thepercentage of cells in G1, S, and G2–

M phases of the cell cycle weregenerated. Data shown (A–F) arerepresentative graphs of 3experiments. The data presented in(G and H) are compiled data from 3independent experiments andindicate the percentage of cells ineach phase of the cell cycle.�, statistical significance comparedwith RNAi-matched DMSOtreatment cells; #, statisticalsignificance compared with negativecontrol DMSO treatment (P < 0.05,one-way ANOVA).





hours of treatment with DMSO or 10 nmol/L TCDD andanalyzed using FACS. We observed that cells transfectedwith universal negative control and treated with TCDDresulted in an increase in the number of cells in G1 whencompared with DMSO (Fig. 1C and E). However, in cellstransfected with RNAi-targeting CCNG2, there was anincreased amount in the S-phase (Fig. 1D) but TCDDtreatment did not alter the distribution of cells (Fig. 1F).The ability of TCDD to increase the number of cells in G1and reduce the number of cells in S-phase was lostfollowing knockdown of CCNG2 (Fig. 1G and H),suggesting that CCNG2 is an important contributor tothe TCDD-dependent cell-cycle inhibition in T-47Dcells.

TCDD induces the recruitment of AHR and FOXA1 toCCNG2To identify AHREs and other transcription factor–bind-

ing sites that might be important in the TCDD-dependentregulation of CCNG2, we conducted transcription factor–binding site analysis on the �1.4 kb CCNG2 regulatoryregion using MatInspector (Genomatix). This analysis iden-tified 2 AHRE sequences that were positioned in closeproximity to multiple forkhead (FKH) sites. We designatedthe AHRE sites, AHRE1 and AHRE2, and the FKH sites,FKH1–4 (Fig. 2D). Because FOXA1 is an important tran-scription factor in ERa-positive breast cancer cells, we thendetermined the role of FOXA1 as well as each of theindividual AHREs in the AHR-dependent regulation ofCCNG2. To examine AHR and FOXA1 recruitment tothe CCNG2 promoter in T-47D cells, we conducted ChIPassays. Cells were treated with DMSO or 10 nmol/L TCDDfor 1 hour, cross-linked and protein–DNA complexes wereimmunoprecipitated with antibodies directed against AHR,FOXA1, H3K4Me2, or H3K9Ac. H3K9Ac was examinedbecause this modification correlates with actively tran-scribed genes (32). H3K4Me2 was tested to identifyfunctional FOXA1-binding sites, as H3K4Me2 has beenreported to correlate with FOXA1 binding to its FKH site(33). As shown in Fig. 2A, TCDD treatment significantlyand preferentially induced the recruitment of AHR andFOXA1 to AHRE2, whereas only a modest, albeit signif-icant, increase in AHR recruitment to AHRE1 wasobserved (Fig. 2A). ChIP studies revealed constitutivebinding of FOXA1 to the enhancer region, which wasincreased after ligand treatment. Treatment with TCDDresulted in increased levels of H3K4Me2 or H3K9Ac atAHRE2 but not at AHRE1 (Fig. 2B). These findingssuggest that AHRE2, but not AHRE1, is the dominantAHRE driving the TCDD-dependent regulation ofCCNG2. In agreement with TCDD-dependent increasesin CCNG2 mRNA levels, ChIP assays confirmed increasesin the recruitment of RNA polymerase II to the proximalpromoter region of CCNG2 after TCDD treatment (Fig.2C). Although the expression of FOXA2 was comparablewith FOXA1 levels, FOXA2 was not recruited to CCNG2,whereas FOXA3 was not detected in T-47D cells (data notshown).

TCDD-dependent interactions between FOXA1 andAHRTo determine whether FOXA1 and AHR were present in

the same protein complex, we carried out sequential ChIPand co-IP experiments. Sequential ChIP analyses revealedthat AHR and FOXA1 were recruited simultaneously toCCNG2 (Fig. 3A). Furthermore, AHR and FOXA1 wereshown to be part of the same protein complex in co-IP assayscompleted in the presence or absence of TCDD (Fig. 3B).

AHR mediates the TCDD-dependent regulation ofCCNG2 using FOXA1To determine how AHR regulates CCNG2 transcription

and to identify the key response element(s) involved in thisregulation, we conducted promoter deletion and site-direct-edmutagenesis analyses. Treatment with 10 nmol/L TCDDresulted in an approximate 1.5-fold increase in activity of thefull-length promoter, pGL4-CCNG2 (Fig. 4A). Deletion ofAHRE2 abolished the TCDD-dependent regulation ofCCNG2 (Fig. 4A). Site-directed mutagenesis of AHRE2inhibited the TCDD-mediated luciferase activity (Fig. 4B).These findings suggest that AHRE1 is not required forAHR-dependent regulation of CCNG2. We then mutatedthe FKH3 and FKH4 sites to evaluate the role of FOXA1 inmodulating the AHR-dependent regulation of CCNG2.Mutation of either FKH site significantly decreased, butdid not abolish, the TCDD-dependent increase in luciferaseactivity (Fig. 4B). However, mutation of both FKH sitesprevented the TCDD-dependent increase in luciferase activ-ity. Taken together, these data show that AHRE2 andFOXA1 play key roles in the AHR-dependent regulationof CCNG2. The AHR binding observed at AHRE1 in theChIP analysis (Fig. 2A) may just represent larger immuno-precipitated DNA fragments containing AHRE2, as theresolution of our ChIP assay is 500 to 800 bp.

FOXA1 but not ERa is required for the AHR-dependentregulation of CCNG2FOXA1 is an important modulator of ERa and androgen

receptor transactivation in breast and prostate cancer cells,respectively (25, 26, 34–36). Because FKH sites were foundto be important in the TCDD-mediated regulation of theCCNG2 luciferase reporter plasmid, we hypothesized thatFOXA1 may have a similar role with AHR–chromatininteractions at CCNG2. To test this hypothesis, we usedRNAi-mediated knockdown of FOXA1 and measuredmRNA expression, as well as the recruitment patterns ofAHR, FOXA1, and ERa; ERa is known to negativelyregulate CCNG2 (21). Following transient transfection of2 distinct siRNAoligos intoT-47Dcells, we determined that48 hours posttransfection, FOXA1 protein levels were great-ly reduced (Fig. 5A) and mRNA expression was reducedto 20% compared with control cells (data not shown).Interestingly, the loss of FOXA1 caused a marked decreasein ERa protein levels, which has been previously reported(34), but did not cause any changes in AHR protein levels.Similar findings were observed in MCF-7 ERa-positivebreast carcinoma cells (data not shown). RNAi-mediated

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knockdown of FOXA1 inhibited the TCDD-dependentgene expression supporting our promoter deletion andmutagenesis results described above (Fig. 5A). As indicatedby our ChIP studies, treatment with 10 nmol/L of TCDDtreatment resulted in increased recruitment of FOXA1,AHR, and ERa (Fig. 5B–D). Interestingly, we observedconstitutive binding of both FOXA1 and ERa when com-pared with IgG controls. RNA-mediated knockdown ofFOXA1 abolished the TCDD-dependent recruitment ofboth AHR and ERa (Fig. 5C and D). The reduced recruit-ment of ERa to CCNG2 was most likely due to reducedprotein expression levels rather than the loss of FOXA1.FOXA1, however, was necessary for the AHR-mediated

regulation of CCNG2, although we cannot exclude thepossibility that the reduced ERa protein levels influenceAHR transactivation, as our laboratory and others haveshown that ERa modulates AHR activity (7, 8, 29).We then conducted RNAi-mediated ERa knockdown

studies to determine the role of ERa in AHR-mediatedregulation of CCNG2 expression. The loss of ERa hadno effect on either FOXA1 or AHR protein levels (Fig.5E). In agreement with our previous findings, the knock-down of ERa significantly increased the constitutivelevels of CCNG2 mRNA levels but did not affect theTCDD-mediated increase in CCNG2 mRNA levels(ref. 8; Fig. 5E). ChIP analysis showed that ERa was

Figure 2. TCDD induces the recruitment of AHR and FOXA1 toCCNG2. A, quantification of AHR and FOXA1 recruitment to AHRE1 and AHRE2 using the ChIPassay. Briefly, T-47D cellswere treatedwith 10 nmol/L TCDD for 45minutes and immunoprecipitated using the antibodies indicated. The immunoprecipitatedDNA was measured by qPCR with primers designed around each response element. B, determining the relative levels of the histone modificationsH3K4Me2andH3K9Ac following TCDD treatment at AHRE1 andAHRE2using theChIP assay. C, recruitment of RNApolymerase (pol) II to the TATAbox in theproximal promoter region of CCNG2 after 45 minutes of treatment. D, diagram of CCNG2 regulatory region. Each error bar represents the SEM of 3independent replicates. All data are relative to 100% total input. �, statistically significant differences compared with DMSO control samples using a one-wayANOVA (P < 0.05).





recruited to CCNG2 in the absence of ligand, suggestingthat under these conditions, ERa modulates CCNG2gene expression (Fig. 5E and F). Knockdown of ERa didnot affect the TCDD-dependent recruitment of AHR or

FOXA1 to CCNG2 (Fig. 5G and H). Together, thesedata provide evidence that FOXA1 is driving the AHR-mediated regulation of CCNG2 irrespective of ERalevels.

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 aboveIgG 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-linkedusing formaldehyde. Cell lysate was immunoprecipitated using antibodies against AHR and FOXA1. IgG was used as the negative control. Western blottingwas then completed using the reciprocal antibody.

Figure 4. AHR mediates the TCDD-dependent regulation of CCNG2.A, deletion fragments of pGL4-CCNG2 were tested to deduce thefunctional significance of AHRE1and AHRE2. Two hundrednanograms of each vector wastransfected in T-47D cells andluminescence was measuredfollowing a 24-hour 10 nmol/LTCDD treatment. B, site-directedmutagenesis of AHRE2 as well asto the FKH recognition sites wasgenerated in pGL4-CCNG2. T47-Dcells were transfected with 200 ngof the single and double responseelement mutants and treated witheither DMSO or 10 nmol/L TCDDfor 24 hours. Results represent themean of 3 independent replicates.�, luciferase (luc) activity that wasstatistically different comparedwith DMSO pGL4-CCNG2 control;#, luciferase activity statisticallydifferent compared with TCDDpGL4-CCNG2 (P < 0.05, one-wayANOVA).

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Figure 5. FOXA1, but not ERa, is essential for the TCDD-dependent recruitment of AHR toCCNG2. A, gene expression profiles were completed on T-47D cellstransfected for 48 hours with siRNA and then treated for 6 hours with TCDD. RNA was isolated and reverse-transcribed. mRNA expression was thendetermined using qPCR. Data were normalized against time-matched DMSO and to ribosomal 18S levels. Data represent the mean of 3 independentreplicates and # is comparedwith TCDDnegative (neg.) control (P < 0.05, one-wayANOVA). 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), andERa (D) following siRNA-mediated knockdown of FOXA1 using theChIP assay. Briefly, T-47D cells were transfected for 48 hours with siRNA and then treatedfor 45 minutes with TCDD and immunoprecipitated using the antibodies indicated. The immunoprecipitated DNA was measured by qPCR with primerstargeting the enhancer region (relative to 100% total input). Each graph represents the mean of 3 independent replicates with � indicating statisticallysignificant differences compared with DMSO negative control whereas the # indicates statistically significant differences compared with TCDD negativecontrol (P < 0.05, one-way ANOVA). E, CCNG2mRNA expression levels were completed on T-47D cells transfected for 48 hours with siERa and then treatedfor 6 hours with TCDD. Data represent themean of 3 independent replicates and the � are comparedwith 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 wasused 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).





TCDD-dependent recruitment of NCoA3 to CCNG2Previous studies showed that CCNG2 was negatively

regulated by ERa through the recruitment of an NCoRcomplex leading to the hypoacetylation of histones and therelease of RNA polymerase II (21). On the basis of theseresults, we hypothesized that the TCDD-dependent pos-itive regulation of CCNG2 must overcome this inhibitionthrough the recruitment of nuclear coactivators to promotegene expression. Because NCoA3/AIB1 is overexpressed inbreast cancer, we determined the ability of TCDD, E2,and E2 þ TCDD to induce recruitment of NCoA3 toCCNG2 in T-47D in the presence or absence of RNAi-mediated knockdown of FOXA1 or ERa. TCDD treat-ment resulted in increased NCoA3 recruitment toCCNG2, which was significantly reduced only after knock-down of FOXA1 but not ERa (Fig. 6A and B). The co-IPstudies provided further evidence that NCoA3 is part ofthe activated multiprotein AHR-containing complex, as itwas found to interact with both AHR and FOXA1(ref. 37; Fig. 6C).

AHRprevents the ERa-dependent negative regulation ofCCNG2In support of a previous report, estrogen-bound ERa

inhibited CCNG2mRNA expression levels (ref. 21; Fig. 7Aand B). However, this repression was overcome by co-treatment with TCDD and required FOXA1 but not ERa(Fig. 7A and B). Co-treatment of TCDD þ E2 preventedthe E2-dependent removal of NCoA3 from the CCNG2resulting in increased recruitment of both AHR and FOXA1(Fig. 7C–E). The TCDD-induced recruitment of NCoA3was dependent on FOXA1 (Fig. 7C–E). RNAi-mediatedknockdown of ERa had no effect on the ability of AHR toblock the repression caused by E2 treatment (Fig. 7G–I).Overall, our findings show that FOXA1 facilitated thebinding of TCDD-induced AHR and NCoA3 to CCNG2,

leading to increased CCNG2 gene expression and prevent-ing the previous described repressive actions of ERa onCCNG2 expression (ref. 21; Fig. 8).

DiscussionAHR has emerged as an important therapeutic target for

breast cancer, as its activation has been reported to inhibit thegrowth of ER-positive, ER-negative, and HER2-positivebreast cancer cells (7, 8, 28, 38, 39). In the present study,we show that ligand-activated AHR together with FOXA1increases the expression of CCNG2 in ERa-positive T-47Dbreast cancer cells. We also report that activation of AHRprevented the ability of ERa to repress CCNG2 expression,providing another example where activation of the AHRpathway opposes the actions of ERa. Our findings provide anew mechanism by which AHR can inhibit human breastcancer cell proliferation. The increase in expression ofCCNG2 by AHR further supports the notion that targetingAHR might be an effective therapy for breast cancer treat-ment (4, 38). Although the clinical importance of AHR-dependent activation of CCNG2 remains to be investigated,trastuzumab treatment has also been reported to increaseCCNG2 levels (22). RNAi-mediated knockdown ofCCNG2 resulted in a slight reduction in trastuzumab-dependent growth inhibition, suggesting that CCNG2 isrequired, but not necessary, for the inhibitory action oftrastuzumab (18, 40). However, the mechanism of CCNG2upregulation by trastuzumab most likely does not requireFOXA1, as it is not expressed in ER-negative breast cancer(41). Nonetheless, these findings suggest that modulatingCCNG2 expression might be an important mechanism toinhibit cancer cell growth.TCDD-dependent activation of AHR blocks cell-cycle

progression through the G1 phase in several cell linesand under many different conditions, including mouse

Figure6. NCoA3 is part of the complex formedatCCNG2. Recruitment ofNCoA3wasdetermined after knockdownof FOXA1 (A) andERa (B). Briefly, cellsweretransfected with siRNA targeting both factors followed by treatment with 10 nmol/L TCDD, after which chromatin was immunoprecipitated using an antibodyagainst NCoA3. The immunoprecipitated DNA was measured by qPCR with primers targeting the enhancer region. Each graph represents the mean of3 independent replicates with the � indicating statistically significant differences compared with DMSO negative control whereas the # indicates statisticallysignificant differences compared with TCDD negative (neg.) control (P < 0.05, one-way ANOVA). C, co-IP studies were completed in T-47D cells. Cells weretreated for 45 minutes with either DMSO or TCDD and then cross-linked using formaldehyde. Cell lysate was immunoprecipitated using a selective NCoA3antibody. IgG was used as the negative control. Western blotting was done using antibodies against AHR and FOXA1.

Ahmed et al.




hepatoma Hepa1c1c7 (42), rat hepatoma 5L cells (43), andin estrogen-inducedMCF-7 cell proliferation (44). Potentialmechanisms include the AHR-dependent induction ofp27Kip1 and p21WAF1, inhibition of CDK function, inhibi-tion of retinoblastoma phosphorylation, and repression ofE2F-regulated genes through interactions with retinoblas-

toma and displacement of the coactivator p300(10, 12, 45). The TCDD-dependent increase in CCNG2expression reported here provides another mechanism bywhich ligand-activated AHR regulates cell-cycle progressionin the G1 phase, as increases in CCNG2 levels result in G1to S-phase arrest (17). In support of these findings, we

Figure 7. AHR can overcome theERa-dependent negative (neg.)regulation ofCCNG2. CCNG2mRNAexpression levels were determinedfrom T-47D cells transfected for 48hours with siFOXA1 (A) or siERa (B)and then treated for 6 hours with 10nmol/L E2 or 10 nmol/L E2 þ 10nmol/L TCDD. RNAwas isolated andreverse-transcribed, and mRNAexpression levels were determinedusing qPCR. Data were normalizedagainst time-matched DMSO and toribosomal 18S levels. Data representthemean of 3 independent replicateswith the � representing statisticallysignificant differences comparedwith DMSO and the # representsstatistically significant differencescompared with treatment-matchedsamples (P < 0.05, one-way ANOVA).Cells were treated with 10 nmol/L E2or 10 nmol/L E2 þ 10 nmol/L TCDDfor 45 minutes and the recruitment ofAHR (C), FOXA1 (D), NCoA3 (E), andERa (F) was determined 48 hourspost-siFOXA1 transfection usingChIP assays and qPCR. Similarexperiments were also carried out 48hours after siERa transfection usingantibodies against AHR (G), FOXA1(H), NCoA3 (I), and ERa (J).





report that the TCDD-dependent increase in the numberof T-47D cells in G1 phase arrest is lost after knockdown ofCCNG2.FOXA1 has been implicated in both ERa and androgen

receptor signaling (26, 35). Previously reported ChIP-chipdata from our laboratory revealed that in both human cellsand mouse tissue, FKH sites are significantly enriched inAHR-bound regions supporting a possible role for forkheadproteins in AHR signaling (8, 9, 46). In support of thesedata, we show that FOXA1 is critical for the AHR-depen-dent induction of CCNG2 levels. We observe that FOXA1is present at CCNG2 in the absence of AHR activation.Following AHR ligand treatment, the levels of FOXA1,AHR, NCoA3, and H3K4Me2 are increased at CCNG2,suggesting that FOXA1 primes the CCNG2 for AHRrecruitment and subsequent transcriptional activation. AHRand FOXA1 interact in co-IP and re-chip experiments,showing that they are part of the samemultiprotein complex,which agrees with other reports showing that forkheadprotein family members interact with other transcriptionfactors (26). FOXA1 has been previously reported to interactwith androgen receptor (47) and was shown to enhanceglucocorticoid receptor transactivation (48). Similarly,FOXA3 interacts with glucocorticoid receptor where by thebinding of FOXA3 provides a favorable DNA conformationfor glucocorticoid receptor binding to its target genes (49).FOXA1 may use both the direct interaction with AHR andaltered DNA conformation to enhance AHR binding toCCNG2. We hypothesize that FOXA1 stabilizes the AHR-activated complex at CCNG2 and therefore is required formaximal gene activation by AHR.The regulation of CCNG2 by other members of the

forkhead protein family has been previously reported, withone group showing that FoxO transcription factors increased

CCNG2 expression in NIH3T3 mouse embryonic fibro-blasts (14). Recently,Nodal, amember of theTGF-b family,was found to increase CCNG2 mRNA expression byincreasing the expression of FOXO3a, which then formsa complex with Smad proteins at the CCNG2 promoterregion (50). They found that the more proximal FKH sites(FKH1 and FKH2 in Fig. 2) were required for FOXO3a-mediated induction of CCNG2, rather than the distal FKHsites (FKH3 and FKH4 in Fig. 2) that are required for AHR-dependent induction of CCNG2 reported here. Interest-ingly, the antiproliferative effect of Nodal on ovarian cellswas found to be partly mediated by CCNG2 (51). Thesefindings show the important role of the forkhead proteinfamily in the regulation of CCNG2 but reveal that theregulation of CCNG2 by FOXA1 or FOXO3a occurs viadistinct FKH sites.Recent RNAi-mediated knockdown of FOXA1 followed

by ChIP-sequencing studies revealed that FOXA1 isrequired for almost all ERa binding to its genomic targetregions irrespective of the proximity of FKH sites to ERa-binding sites (35). Although the RNAi-mediated knock-down of FOXA1 was reported to not affect ERa proteinlevels (35), we and others report that knockdown of FOXA1has been shown to decrease ERa mRNA expression andprotein levels (34). The reason for the discrepancies betweenthe studies is unclear, but we observe that RNAi-mediatedknockdown of FOXA1 reduces ERa protein levels in 2different cell lines (T-47D, Fig. 5; MCF-7, data not shown)using 2 unique siRNA oligos targeting FOXA1. We alsoobserve that RNAi-mediated knockdown of FOXA1reduced the TCDD-dependent induction of CYP1A1 andCYP1B1.But unlike whatwas observed forCCNG2,RNAi-mediated knockdown of ERa did reduce the TCDDresponsiveness of both CYP1A1 and CYP1B1 in T-47D

Figure 8. Proposed mechanism for the FOXA1- and AHR-dependent regulation of CCNG2. In the absence of ligand, FOXA1 is bound to the FKH recognitionsites in the enhancer regionofCCNG2.ERa andNCoA3occupy the upstream regulatory regionofCCNG2. Treatmentwith E2 reduces the constitutiveNCoA3but increases E2-bound ERa occupancy at CCNG2 resulting in transcriptional repression as previously described (21). However, upon TCDD or E2þTCDDtreatment, FOXA1 binding is increased facilitating the recruitment of AHR and increased recruitment of NCoA3 leading to transcriptional activation ofCCNG2.FOXA1 mediates these effects through direct interactions with AHR. The relative transcriptional activity of CCNG2 is represented by the magnitude of thearrow.

Ahmed et al.




cells (S. Ahmed and J. Matthews unpublished findings;ref. 8). This suggests that for certain genes the reducedAHR transactivation following RNAi-mediated knockdownof FOXA1, may be due to reduced ERa levels and notreduced FOXA1 expression. Therefore, it will be importantto distinguish the effects of FOXA1 knockdown on AHRtransactivation compared with those mediated by ERa.Further studies investigating the recruitment patterns ofAHR and FOXA1 using ChIP-sequencing and RNAi-medi-ated knockdown approaches will be helpful in determiningwhether FOXA1 is a general modulator of AHR signaling ora gene-specific regulator.In summary, we report that the AHR-dependent reg-

ulation of CCNG2 requires FOXA1 to stabilize thebinding of activated AHR and aiding in the recruitmentof other co-regulatory proteins, such as NCoA3. Ourfindings also show that activated AHR is able to overcomethe repressive actions of ERa at CCNG2, providingfurther evidence supporting the antiestrogenic effects ofAHR. Although additional studies are needed to fullycharacterize the depth and role of FOXA1 in AHRsignaling, the data presented here provide new insight

into the antiproliferative action of AHR in breast cancercells and further support AHR as a therapeutic target forbreast cancer treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

AcknowledgmentsThe authors thank Dr. Chun Peng for providing materials for this study, all

members of the Matthews' laboratory for their assistance and helpful discussionsduring the course of this work, andMeghan Larin andNishani Rajakulendran for theirhelp with the cell preparation and analysis of the FACS samples.

Grant SupportThe study was supported by Canadian Institute of Health Research operating

grant (MOP-82715) and Canadian Breast Cancer Foundation-Ontario funded thisresearch. S. Ahmed is supported by the Canadian Institute of Health ResearchDoctoral Research Award. J. Matthews is the recipient of the Canadian Institute ofHealth Research New Investigator Award and Early Researcher Award from theOntario Ministry of Innovation.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received October 24, 2011; revised February 2, 2012; accepted March 13, 2012;published online May 17, 2012.

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2012;10:636-648. Mol Cancer Res Shaimaa Ahmed, Sarra Al-Saigh and Jason Matthews Regulation of Cyclin G2

Dependent−FOXA1 Is Essential for Aryl Hydrocarbon Receptor

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