ativação do cyp mcf7

MOLECULAR CARCINOGENESIS

Expression of the Aryl Hydrocarbon Receptor Is NotRequired for the Proliferation, Migration, Invasion, orEstrogen-Dependent Tumorigenesis of MCF-7 BreastCancer Cells

Barbara C. Spink,1 James A. Bennett,2 Nicole Lostritto,2 Jacquelyn R. Cole,1 and David C. Spink1,3*1Laboratory of Molecular Toxicology, Wadsworth Center, New York State Department of Health, Albany, New York2Center for Immunology and Microbial Diseases, Albany Medical College, Albany, New York3Department of Environmental Health Sciences, School of Public Health, University at Albany, State University of NewYork, Albany, New York

The AhR was initially identified as a ligand-activated transcription factor mediating effects of chlorinated dioxins andpolycyclic aromatic hydrocarbons on cytochrome P450 1 (CYP1) expression. Recently, evidence supporting involvementof the AhR in cell-cycle regulation and tumorigenesis has been presented. To further define the roles of the AhR in

cancer, we investigated the effects of AhR expression on cell proliferation, migration, invasion, and tumorigenesis ofMCF-7 human breast cancer cells. In these studies, the properties of MCF-7 cells were compared with those of twoMCF-7-derived sublines: AHR100, which express minimal AhR, and AhRexp, which overexpress AhR. Quantitative PCR,Western immunoblots, 17b-estradiol (E2) metabolism assays, and ethoxyresorufin O-deethylase assays showed the lack

of AhR expression and AhR-regulated CYP1 expression in AHR100 cells, and enhanced AhR and CYP1 expression inAhRexp cells. In the presence of 1 nM E2, rates of cell proliferation of the three cell lines showed an inverse correlationwith the levels of AhR mRNA. In comparison with MCF-7 and AhRexp cells, AHR100 cells produced more colonies in

soft agar and showed enhanced migration and invasion in chamber assays with E2 as the chemoattractant. Despitethe lack of significant AhR expression, AHR100 cells retained the ability to form tumors in severe combined immunode-ficient mice when supplemented with E2, producing mean tumor volumes comparable to those observed with MCF-7

cells. These studies indicate that, while CYP1 expression and inducibility are highly dependent on AhR expression,the proliferation, invasion, migration, anchorage-independent growth, and estrogen-stimulated tumor formation ofMCF-7 cells do not require the AhR. � 2012 Wiley Periodicals, Inc.

Key words: aryl hydrocarbon receptor; breast cancer; MCF-7 xenograft

INTRODUCTION

The aryl hydrocarbon receptor (AhR) is a tran-scription factor that is activated by a variety of envi-ronmental, dietary, and endogenous ligands [1,2].Xenobiotic AhR ligands include benzo(a)pyrene(BaP) and other polycyclic aromatic hydrocarbons(PAH), and halogenated aromatic hydrocarbons,such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).AhR agonist and antagonist activities have beenattributed to a number of dietary components,including various flavonoids [1], and a number ofendogenous compounds, notably indole derivatives[3–8]. The ligand-bound AhR, in conjunction withits heterodimerization partner, the aryl hydrocarbonnuclear transporter, facilitates transcription of a num-ber of genes including those of cytochromes P450 ofthe CYP1 family, which catalyze the metabolism ofvarious xenobiotics and endogenous substrates [9].While the concept of physiologically relevant AhRsignaling involving an endogenous ligand has yet tobe universally accepted, the rapid, AhR-mediated in-duction of CYP1A1 observed when cells were placedin suspension [10] established that the AhR can be

activated in the absence of an exogenous ligand [10–13]. The recently reported activation of the AhR bythe tryptophan-derived ligand, 6-formylindolo[3,2-b]carbazole, in response to ultraviolet irradiationsupports the existence of a physiologic mechanismthat ultimately leads to AhR-mediated activation ofgene transcription [14].

Abbreviations: ER, estrogen receptor; AhR, aryl hydrocarbonreceptor; BaP, benzo(a)pyrene; PAH, polycyclic aromatic hydrocar-bons; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; CYP, cytochromeP450; E2, 17b-estradiol; DMEM, Dulbecco’s modified Eagle’s medi-um; ICI, IC1-182,780 or Fulvestrant; DMSO, dimethylsulfoxide;EROD, ethoxyresorufin-O-deethylase; qPCR, quantitative real-timePCR; SULF, sulfatase; SCID, severe combined immunodeficient;MeOE2, methoxyestradiol; OHE2, hydroxyestradiol; TRAMP, transgenicadenocarcinoma of the mouse prostate.

*Correspondence to: Laboratory of Molecular Toxicology, Wads-worth Center, New York State Department of Health, Empire StatePlaza, P.O. Box 509, Albany, NY 12201-0509.

Received 12 July 2011; Revised 12 January 2012; Accepted 26January 2012

DOI 10.1002/mc.21889

Published online in Wiley Online Library(wileyonlinelibrary.com).

� 2012 WILEY PERIODICALS, INC.

In recent years, functions of the AhR in additionto those relating to the transcriptional regulation ofgenes encoding phase I and II enzymes have beenidentified [15–17]. Evidence supporting involvementof the AhR in immune regulation [18–21], controlof the cell-cycle and cell proliferation [22–24], cross-talk of hormone and growth-factor signaling path-ways, and physical interactions with other nuclearreceptors impacting their function and their rates ofdegradation has been presented [24–26]. Many ofthe roles of the AhR that have been characterizedare dependent on the context of specific tissue andcell type and the presence or absence of specificAhR ligands [27,28]. In MCF-7 human breast cancercells, nuclear localization of the AhR subsequent toexposure to ligands increases AhR interaction withretinoblastoma protein [29] and induces cell-cyclearrest [30].

The involvement of the AhR in cancer has tradi-tionally been viewed in terms of its role in the regu-lation of expression of the carcinogen-bioactivatingCYP1 enzymes, leading to adductive and oxidativeDNA damage and resulting in mutagenesis in theinitiation phase of the disease [9,31]. In recent stud-ies, additional roles of the AhR in the promotionand progression stages of breast cancer have beenproposed; however, they are not consistent, particu-larly with regard to the role of the AhR in develop-ment of the invasive phenotype [27,32]. In light ofthe conflicting roles of the AhR that have been pro-posed in breast cancer, this study was undertaken todetermine the involvement of the AhR in the post-initiation phases of estrogen receptor (ER)-positivebreast cancer. To further define the roles of the AhRin human breast cancer, we used MCF-7 cells, whichexpress ERa and are classified as luminal A breastcancer, and two MCF-7-derived sublines: AHR100

cells, which express minimal AhR, and AhRexp cells,which overexpress AhR. The effects of differing AhRexpression levels on CYP1 induction, metabolism of17b-estradiol (E2), cell proliferation, migration, inva-sion, anchorage-independent growth, and tumori-genesis were evaluated. Our results are consistentwith the obligatory role of the AhR in CYP1 in-ducibility, but indicate that the AhR is not requiredfor tumor formation and growth from MCF-7 cells.

MATERIALS AND METHODS

Cell Lines and Culture

MCF-7 cells were those used in our previous stud-ies [33]. AHR100 cells, which are deficient in AhR,were derived from MCF-7 cells after 6–9 mo expo-sure to BaP [34]. Stock cultures of AHR100 cells weremaintained in 0.8 mM BaP (Sigma, St. Louis, MO),which, unless otherwise indicated, was withdrawnfor at least 2 wk prior to the initiation of the experi-ments. AhRexp cells were obtained by stable transfec-tion of MCF-7 cells with the AhR-expression

construct, pcDNA3.1(þ)AhR [35]. AhRexp cells weremaintained in 0.3 mg/mL Geneticin (Invitrogen,Carlsbad, CA), although the antibiotic was removedprior to initiation of the experiments. Stock culturesof MCF-7, AHR100, and AhRexp cells were maintainedin DF5 medium, which consisted of Dulbecco’s mod-ified Eagle’s medium (DMEM) supplemented with5% (v/v) fetal bovine serum (Hyclone Laboratories,Logan, UT), 100 mM non-essential amino acids,2 mM L-glutamine, 10 mg/L insulin, 100 U/mL peni-cillin, and 100 mg/mL streptomycin. Unless other-wise indicated, all experiments were performedusing DC5 medium, which has minimal estrogencontent [35]. The formulation of DC5 differed fromthat of DF5 in that it contained 5% (v/v) bovine calfserum (cosmic calf serum; Hyclone Laboratories) inplace of the fetal bovine serum, and it was preparedwith phenol-red-free DMEM. TMX2-28, an ERa-negative subline isolated from a MCF-7 culture thathad been subjected to long-term tamoxifen expo-sure [36], was maintained in DC5 medium. As indi-cated, cultures were exposed in DC5 medium to thefollowing compounds: IC182,780 (ICI; Tocris, Ellis-ville, MO), TCDD (Cambridge Isotope Laboratories,Andover, MA), and/or E2 (Sigma) with dimethylsulf-oxide (DMSO; Sigma) as the vehicle.

Ethoxyresorufin-O-Deethylase (EROD) Assays

EROD assays were performed in 96-well plates asdescribed [37]. Confluent cultures were exposed for48 or 72 h to 10 nM TCDD, 1 nM E2, and/or100 nM ICI. After treatments, the medium wasreplaced with medium (100 mL/well) containing4 mM ethoxyresorufin and 10 mM dicumarol(Sigma), and the plates were incubated at 378C for30 min. Fluorescence measurements were made usinga Fusion Universal Microplate Analyzer (Packard In-strument Co., Meriden, CT) with 535-nm excitationand 590-nm emission filters. Data were normalizedto total protein, which was determined using theBCA Protein assay reagent (Pierce, Rockford, IL).

Reverse-Transcription and Quantitative Real-Time PCR

(qPCR)

Cultures at confluence in 6-well plates wereexposed to 10 nM TCDD with or without 1 nM E2for 48 h. Isolation of total RNA from cell lysatesand tumor homogenates, reverse-transcription of oligo-dT-primed RNA with Superscript III (Invitrogen), andqPCR using the LightCycler System with the Fast-Start DNA Master SYBR Green I kit (Roche MolecularBiochemicals, Indianapolis, IN) were performed aspreviously described [35]. Quantification of cDNAwas by comparison of cycle numbers for unknownsto those of purified cDNA standards of known con-centration. For qualitative analysis of vector-derivedAhR cDNA, the AhR-reverse primer was substitutedwith the BGH-reverse universal primer (50-TAGAA-GGCACAGTCGAGG-30).

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Molecular Carcinogenesis

Western Immunoblots

Western immunoblots of total cellular lysates forthe analysis of ERa and AhR were performed asdescribed [35]. Samples containing equal amountsof total protein were subjected to electrophoresis in10% acrylamide NuPAGE Bis–Tris denaturing gels(Invitrogen). Proteins were transferred onto Immo-bilon-P membranes (Millipore, Bethesda, MD), andblots were probed with rabbit polyclonal anti-human AhR, rabbit polyclonal anti-human ERa, orrabbit polyclonal anti-GAPDH antibodies (SantaCruz Biotechnology, Santa Cruz, CA). Immunoreac-tive proteins were detected by using the West PicoEnhanced Chemiluminescence kit (Pierce).

Estrogen Metabolism Assays

Confluent cultures in 6-well plates were exposedto 10 nM TCDD for 48 h. Cultures were then ex-posed to 1 mM E2 in 2 mL of medium for 6 h. Mediawere recovered, and 1 mL of each sample washydrolyzed at 378C with sulfatase (SULF) fromHelix pomatia (Sigma Type H-1; 24,190 units SULFand >300 units b-glucuronidase) for 6 h. Estrogenmetabolites were recovered from the hydrolyzedand non-hydrolyzed samples by solid-phase extrac-tion, converted to pyridyl-3-sulfonyl derivatives andanalyzed by liquid chromatography-tandem massspectrometry [38,39].

Cell Proliferation Assays

The Sulforhodamine B assay [40] was used for thedetermination of cell proliferation. To initiate theassay, cultures were seeded in 96-well plates at6,000 cells per well. At various times, plates werefixed with 10% trichloroacetic acid and stained fortotal protein with 0.4% Sulforhodamine B (Sigma)in 1% acetic acid. After rinsing, the dye was dis-solved by the addition of 10 mM Tris base, and theabsorbance at 490 nm was determined.

Determination of Anchorage-Independent Growth

Cells were trypsinized and suspended in DMEMwith 0.1% bovine serum albumin and diluted to3,000 cells/mL in 0.3% soft agar (Difco, BectonDickinson, Sparks, MD). Aliquots of the cell suspen-sion (500 mL) were layered over a base layer of solid-ified agar (500 mL, 0.5%) in 24-well plates [41]. Aftersolidification of the top layer, 1 mL of medium wasadded, and colonies were allowed to form for 2 wk,after which colonies of �50 mm in 6 randomly se-lected fields were counted.

Invasion and Migration Assays

Cell migration and invasion assays were per-formed using Boyden chambers with inserts having8.0 mm pores (BD Biosciences, Bedford, MA). Un-coated inserts were used for migration assays, where-as Matrigel-coated inserts were used for invasion

assays [42,43]. Cells were suspended in DMEM con-taining 0.1% bovine serum albumin, and 105 cellsper well were seeded in the upper chamber. Thelower chamber contained 1 nM E2 as the chemoat-tractant in DC10, a DMEM-based medium with 10%bovine calf serum. After 40 h, inserts were scrubbed,fixed with methanol for 1–2 min, dried, and stainedwith 0.1% crystal violet for cell counting.

Xenograft Assays for Tumorigenicity

For the xenograft studies, groups of 15 mice re-ceived inoculations of cells with or without E2 sup-plementation [44]. Each inoculation consisted of1 � 106 cells in 50 ml of culture medium injectedinto the surgically exposed mammary fat pads of6- to 8-wk-old severe combined immunodeficient(SCID) mice from Taconic Farms (Germantown,NY). E2 supplementation was accomplished by sub-cutaneous implantation of Silastic tubing capsules(2 mm in length) containing solid E2, inserted onthe day of tumor-cell implantation. Under thisprotocol, these implants produce serum E2 levels of100 pg/mL [45]. Mice were palpated daily, andemerging tumors were measured with Vernier cali-pers several times per week for 68–71 d. Tumor vol-ume was calculated using the formula V ¼ (p/6)d2D,assuming the tumor shape to be ellipsoid with D asthe long axis. The Albany Medical College AnimalCare and Use Committee approved all work withanimals.

Statistical Evaluations

Statistical evaluations of biochemical determina-tions were performed in replicates of three or moreby analysis of variance and the Bonferroni t-test formultiple comparisons. Statistical evaluations forcomparisons of tumor volumes were performed withthe Mann–Whitney rank sum test. Proliferation datawere fit to the four parameter Chapman model:y ¼ y0 þ a(1 � e�bx)c using the SigmaPlot program(SPSS). Growth rates were calculated as the firstderivative at the inflection point of the sigmoidalcurve [46].

RESULTS

Expression of AhR, ERa, and TCDD-Inducible ERODActivities in AHR100, MCF-7, and AhRexp Cells

The AhRexp clone was selected from among severalclones as having the highest level of AhR expres-sion. AhR mRNA and protein in MCF-7, AHR100, andAhRexp cells were examined by PCR and Westernimmunoblot (Figure 1). When mRNA from AhRexp

cells was isolated and analyzed by PCR, but withsubstitution of the usual reverse primer that was ho-mologous to the coding sequence with a primer thatwas homologous to the vector-derived 30 untranslat-ed RNA, the PCR product indicative of vector-derived expression was observed (Figure 1A; lane 2,

EXPRESSION OF THE ARYL HYDROCARBON RECEPTOR 3


614-bp). None of this product was observed whenRNA from MCF-7 cells was analyzed (Figure 1A; lane5). PCR product representing mRNA encoded by theendogenous gene (Figure 1A; lane 4, 377-bp) wasobtained from MCF-7 cDNA using the reverse prim-er homologous to the coding sequence. Total AhRcDNA representing endogenous plus heterologousAhR RNA from AhRexp cells was analyzed using theprimers that amplified the AhR coding sequence(Figure 1A; lane 3, 377-bp). The lack of PCR productformed in the negative-control amplification reac-tion of AhRexp RNA in which reverse transcriptase

was omitted (Figure 1A, lane 1) indicates that theheterologous cDNA was not derived from contami-nation of the RNA with vector DNA. Analysis byWestern immunoblot did not show detectableAhR protein in AHR100 cells, whereas AhRexp cellsexpressed higher levels of AhR protein than MCF-7cells (Figure 1B). ERa was expressed in the MCF-7-derived sublines, AHR100 and AhRexp. The MCF-7-de-rived cell line, TMX2-28 [36], was included as anERa-negative control.Because AHR100 cells were originally obtained from

MCF-7 cultures after continuous exposure to BaP[34], it was necessary to confirm that AhR activityremained at a minimal level after the removal ofBaP. The results presented in Figure 1C show thatAhR-mediated, TCDD-induced EROD activity inAHR100 cells was less than 1% of that of the TCDD-induced activity of MCF-7 cells. After culture for 10and 20 wk in the absence of BaP, AHR100 cells main-tained the very low response to TCDD in the ERODassay.When the three cell lines were directly compared

with regard to AhR-mediated CYP1 induction in theEROD assay (Figure 2), AhRexp cells showed approxi-mately twice the level of TCDD-induced CYP1activity as MCF-7 cells, while AHR100 cells showedapproximately 2% of the induced CYP1 activity inthe EROD assay compared to that of MCF-7 cells,which is consistent with the results of Trapani et al.[34]. Induction of EROD activity, which is primarily

Figure 1. Expression of AhR and ERa in MCF-7 and MCF-7-derivedcell lines. (A) Heterologous AhR mRNA expression in AhRexp cells.Total RNA from AhRexp (lanes 1–3) and MCF-7 cells (lanes 4–5) wasisolated, and PCR was performed with primers specific for the codingsequence of AhR mRNA representing total AhR mRNA (377-bp prod-uct) or specific for the vector-derived mRNA (614-bp product).Lane 1, reverse transcriptase-negative control using vector-specificprimers; lanes 2 and 5, PCR with vector-specific primers; lanes 3, 4,and 6, PCR with primers specific for the AhR coding sequence; lane6, minus-RNA control using primers specific for the AhR codingsequence; lane 7, 100-bp ladder. (B) AhR and ERa protein levels inAHR100, MCF-7, and AhRexp cells. Western blots of whole cell lysatesfrom MCF-7, AhRexp, and AHR100 cells were probed with anti-AhRand -ERa antibodies and detected with enhanced chemilumines-cence. Lysates from TMX2-28 cells are included as an ERa-negativecontrol. GAPDH was probed as a loading control for each blot. (C)The loss of TCDD-inducible EROD activity in AHR100 cells is persistent.BaP was withdrawn from AHR100 cultures for 10 or 20 wk, andcompared with cells without BaP withdrawal (designated as 0 wkwithout BaP), after which cells were exposed for 72 h to 10 nMTCDD (gray bars) or the vehicle, 0.1% (v/v) DMSO (black bars). CYP1activity was then measured by EROD assay. EROD activity of MCF-7cells is shown for comparison; note the difference in scale. Data arepresented as mean � SE; n ¼ 5. Significant differences for TCDD-treated cultures in comparison with the TCDD-treated control group(AHR100 cells without BaP withdrawal) are indicated (��P < 0.001).

Figure 2. Ah-responsiveness in AHR100, MCF-7, and AhRexp cells asmeasured by the EROD assay. Confluent cultures of AHR100, MCF-7,and AhRexp cells were exposed for 48 h to 10 nM TCDD (gray bars),or the vehicle, 0.16% (v/v) DMSO (black bars), with or without1 nM E2 and 100 nM ICI, as indicated. CYP1 activity was then mea-sured by the EROD assay. Data are represented as the mean � SE;n ¼ 8. Significant differences between TCDD-exposed AHR100 orAhRexp cultures and MCF-7 cultures in which all other treatmentswere identical (��P < 0.001); between groups differing only in E2exposure (þþþP < 0.001); and between groups differing only in ICIexposure (dddP < 0.001) are indicated.

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a measure of CYP1A1 activity, was modestly inhib-ited by E2. This inhibition was reversed by inclusionof the antiestrogen, ICI, indicating involvement ofthe ER in this effect.

Levels of ERa, CYP1A1, and CYP1B1 mRNAs in MCF-7-Derived Cell Lines

Cultures of MCF-7, AHR100, and AhRexp cells wereexposed to 1 nM E2 and/or 10 nM TCDD for 48 h,and RNA was isolated and was analyzed by real-timeqPCR for the following transcripts: AhR, ERa,CYP1A1, CYP1B1, and 36B4, which encodes acidicribosomal phosphoprotein PO and is commonlyused as a control mRNA that is not regulated by E2[47]. These mRNA levels are presented in Figure 3.AhR mRNA levels were 1.8-fold higher in AhRexp

cells than in MCF-7 cells, whereas AHR100 cellsexpressed approximately 2.5% of the AhR mRNAlevel of MCF-7 cells in the absence of E2 treatment.In the absence of E2 and TCDD, ERa mRNA levels inthe three cell lines were consistent with ERa proteinlevels as determined by Western blot analysis(Figure 1B). In each of the three cell lines, E2exposure caused a significant downregulation ofERa mRNA expression. Levels of the CYP1A1 andCYP1B1 mRNAs in TCDD-exposed cells correlatedwith the levels of expression of the AhR mRNA inthe three cell lines. Levels of CYP1B1 mRNA in theabsence of E2 and TCDD also correlated with AhRmRNA levels. Interestingly, E2 exposure resulted in amoderate downregulation of TCDD-induced CYP1B1mRNA levels in AhRexp cells. Consistent with itseffects on TCDD-induced EROD activity (Figure 2),E2 modestly downregulated TCDD-induced CYP1A1mRNA levels in MCF-7 and AhRexp cells.

Metabolism of E2 in MCF-7, AHR100, and AhRexp cells

Having confirmed differential expression of AhRat the protein and mRNA levels in MCF-7, AHR100,and AhRexp cells, we then determined the effect ofdifferential AhR expression on the TCDD-inducedmetabolism of E2. The rates of formation of 2-,4-, 6a, and 15a-hydroxyestradiol (OHE2), and 2- and4-methoxyestradiol (MeOE2), with and withoutexposure to 10 nM TCDD and with and without hy-drolysis of conjugates prior to analysis are shown inFigure 4. In MCF-7 and AhRexp cells, basal E2 metab-olism at the C-2 and C-4 positions was detected,and this metabolism was greatly increased by TCDDexposure. Comparison of the levels of metabolitesrecovered with and without prior SULF treatmentindicate that significant amounts of 2- and 4-OHE2,and 2- and 4-MeOE2 were present in conjugatedform, whereas 6a- and 15a-OHE2 were not conjugat-ed to an appreciable extent. In contrast to E2 metab-olism in MCF-7 and AhRexp cells, AHR100 cells didnot show appreciable E2 metabolism, with or with-out TCDD exposure.

Proliferation, Anchorage-Independent Growth, Invasion,and Migration of AHR100, MCF-7, and AhRexp Cells

We next investigated the consequences of differ-ential AhR expression on in vitro indices of tumori-genesis, with and without E2 exposure. Cellproliferation of the two MCF-7-derived lines as com-pared with that of MCF-7 cells in the presence orabsence of 1 nM E2 is shown in Figure 5A. Growthrates derived from the data in Figure 5A are pre-sented in Table 1. Proliferation without E2 supple-mentation was highest for AHR100 cells and lowestfor MCF-7 cells. MCF-7 cells, however, showed thegreatest enhancement of proliferation in responseto E2 supplementation, and AhRexp cells showed noenhancement of proliferation in response to E2.AhR100 cells showed the highest proliferation among

Figure 3. Effect of AhR expression on ERa mRNA levels and theinducibility of CYP1 mRNAs. Confluent cultures of AHR100, MCF-7,and AhRexp cells were exposed to 10 nM TCDD, 1 nM E2, or thevehicle, 0.16% (v/v) DMSO, for 48 h, and the RNA isolated. qPCRwas performed with primers specific for the transcripts as indicated.Data are represented as the average � SEM; n ¼ 3, and are normal-ized to total RNA. Significant differences between TCDD-exposedAHR100 or AhRexp cultures in comparison with MCF-7 cultures inwhich all other treatments were identical (�P < 0.05; ��P < 0.001);and between E2 exposure and the respective group differing onlyin the absence of E2 exposure (þþP < 0.01; þþþP < 0.001) areindicated.



the three cell lines with or without E2 supplementa-tion. In the presence of E2, growth rates of the threecell lines (Table 1) were inversely correlated withAhR mRNA levels (Figure 3); R2 ¼ 0.981.

The three cells lines were then evaluated for colo-ny formation in soft agar in the presence of DC5

and DC5 containing 1 nM E2 (Figure 5B). Amongthe three cell lines, anchorage-independent growthwas greatest for AHR100 cells with each medium test-ed. We then investigated migration and invasion ofthe three cell lines using Boyden chambers. The

invasion assay, which requires that the cells degradeand transverse a layer of extracellular matrix, analo-gous to the penetration of the basement membraneby cancer cells in the initial events of metastasis[42], utilized Matrigel-coated inserts, whereas un-coated chamber inserts were used to assay for migra-tion [43]. In both assays, the lower chambercontained medium supplemented with E2 as thechemoattractant. Using these assays we found thatAHR100 cells were >30-fold more invasive than MCF-7 or AhRexp cells, and that migration of AHR100 cells

Figure 4. Basal and TCDD-induced E2-metabolite formation in AHR100, MCF-7, and AhRexp cells. Confluent cultures of AHR100, MCF-7, andAhRexp cells were exposed for 48 h to 10 nM TCDD or the solvent vehicle, 0.1% (v/v) DMSO, as indicated. Metabolite formation was deter-mined with (þSULF) and without (�SULF) prior hydrolysis of conjugates as described in the Materials and Methods Section. Data are repre-sented as the mean � SE and are normalized to total cellular protein; n ¼ 3. Significant differences between TCDD-exposed AHR100 orAhRexp cultures and MCF-7 cultures, where all other treatments were identical (��P < 0.001) are indicated; ND denotes that the metaboliteswere not detected.

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was also highly elevated compared with that ofAhRexp and MCF-7 cells (Figure 5C).

Tumorigenesis of AHR100, MCF-7, and AhRexp Cells in SCID

Mice

To evaluate in vivo tumorigenicity, AHR100, MCF-7,and AhRexp cells were inoculated into the mammaryglands of SCID mice, with and without E2 supple-mentation as a Silastic implant (Figure 6). In all ofthese experiments, tumors, although palpable, werenot able to grow appreciably without E2 supplemen-tation. Included for comparison are ERa-negativeTMX2-28 cells, which, despite the fact that they arehighly proliferative and invasive in vitro [48], grewpoorly as mammary-gland xenografts with orwithout E2 supplementation (Figure 6, lower rightpanel). With E2 supplementation, tumors fromimplanted AHR100 cells (Figure 6, upper left panel)grew to an extent comparable to MCF-7 cells,indicating that the AhR is not required for tumorformation and growth in SCID mice. AhRexp-cellxenografts showed a trend toward slower tumorgrowth in comparison with those from MCF-7 cells;however, this difference was not statistically signifi-cant. Interestingly, with AhRexp cells, tumors be-came palpable but did not grow to any significantextent, which was similar to the results foundfollowing inoculation of TMX2-28 cells.

To determine whether AHR gene expression inthe xenograft tumors was comparable to that in thecells in culture, we examined the level of AhRmRNA in MCF-7 and AHR100 tumors dissected fromanimals after the final tumor-growth measurementswere taken. AhRexp tumors were of insufficient sizefor dissection. AhR cDNA and GAPDH cDNA fornormalization were amplified with human-specificprimers, which did not amplify the homologous tar-gets from murine cDNA (results not shown). Wefound that the levels of AhR mRNA in tumorsobtained from AHR100 xenografts remained low, atless than 7% the level in MCF-7 cells.

DISCUSSION

Studies have been conducted in a number of labo-ratories with the goal of deciphering the roles of theAhR in initiation, promotion, and progressionphases of cancer, and from them a variety of rolesof the receptor in cancer have been proposed[9,17,27–28,31]. Dependent on the species, tissue,and phase of carcinogenesis, evidence of both onco-genic [9,27,31,49–51] and tumor-suppressing func-tions [32,52,53] of the AhR have been reported. Inthis study, we show that, in the MCF-7 model ofluminal A breast cancer, CYP1 expression and in-ducibility by TCDD in cultured cells are highly depen-dent on AhR expression, whereas cellular proliferation,invasion, migration, anchorage-independent growth,and estrogen-stimulated formation of xenograft

Figure 5. Proliferation, migration, and invasion of AHR100, MCF-7,and AhRexp cells. (A) Proliferation: AHR100, MCF-7, and AhRexp cellswere seeded in 96-well plates at 6,000 cells per well, and the mediawere replenished every 3 or 4 d with DC5 (closed circles) or DC5 con-taining 1 nM E2 (open circles). Proliferation was measured at theindicated times using the Sulforhodamine B assay. Data are pre-sented as mean � SE; n ¼ 8; however, for all points the error barfalls within the symbol. Curves were obtained by fitting data to thefour parameter Chapman model. (B) Anchorage-independentgrowth: AHR100, MCF-7, and AhRexp cells were suspended in 500 mL0.3% soft agar in 24-well plates at 1500 cells per well. The cellswere cultured for 2 wk in DC5 or DC5 containing 1 nM E2 asindicted. Anchorage-independent growth was assessed under 10�magnification by counting colonies larger than 50 mm (black bars) orlarger than 100 mm (gray bars) in diameter. Significant differencesbetween AHR100 or AhRexp cultures in comparison with MCF-7 cul-tures when all other treatments were identical (��P < 0.01,��P < 0.001) are indicated. Data are represented as mean coloniesper field � SE; n ¼ 6. Significant differences for colonies � 50 mmare shown. (C) Migration and invasion: AHR100, MCF-7, and AhRexp

cells were suspended in DMEM containing 0.1% bovine serum albu-min, and 105 cells per well were seeded in Matrigel-coated chambersfor measurement of invasion (black bars) or in control inserts formeasurement of migration (gray bars). The lower chamber contained1 nM E2 in DC10 as a chemoattractant. After 40 h, inserts were fixedand stained with crystal violet. Data are represented as mean cellsper insert � SE; n ¼ 3. Significant differences between AHR100 orAhRexp cultures in comparison with MCF-7 cultures (��P < 0.01,��P < 0.001) are indicated.



tumors do not require the AhR. Our current studieswith MCF-7 cells suggest that the AhR may haveboth pro- and anti-carcinogenic roles within ER-pos-itive luminal breast cells during the various stages ofcarcinogenesis.

The importance of the AhR in the initiation phaseof PAH-induced carcinogenesis is well established.The AhR-mediated induction of the CYP1 enzymesthat catalyze the metabolic activation of PAH toDNA adductive forms is integral to the carcinogenicprocess [9]. AhR-null mice are refractory to PAH-induced carcinogenesis [49], and studies with CYP1single- and double-knockout animals show the roles

of these enzymes in differential organ carcinogene-sis subsequent to PAH exposure [31]. These enzymesnot only catalyze the metabolic activation ofPAHs, but also heterocyclic aromatic amines [54]and endogenous estrogens [55]. The AhR may thusbe involved in the initiation events from severalexogenous and endogenous carcinogens. In our cur-rent study, the inducibility of CYP1A1 and CYP1B1mRNAs, proteins, and CYP1 activities were consis-tent with the levels of AhR expression in AHR100,MCF-7, and AhRexp cells in culture. Increased ratesof the 2- and 4-hydroxylation pathways of E2 metab-olism in response to TCDD exposure, which reflectthe activities of CYP1A1 and CYP1B1, respectively[33], and the induction of EROD activity were notobserved in the absence of AhR expression. The ulti-mate metabolites of E2 in MCF-7 cells, the sulfateconjugates of 2- and 4-MeOE2 [56], were not pro-duced at appreciable levels in TCDD-treated AHR100

cells, nor were 6a-OHE2 and 15a-OHE2, which areproducts of CYP1A1-catalyzed metabolism [57].These results indicate that the induction of E2 me-tabolism, including the formation of the potentiallycarcinogenic 4-OHE2 [55], is reliant on AhR expres-sion in MCF-7 cells.Distinct from the roles of the AhR in CYP1 induc-

tion and the regulation of carcinogen metabolism,the AhR appears to have roles in post-initiationstages of carcinogenesis. Recently, transgenic andgene knockout animals have been used to specifical-ly determine role of the AhR in the post-initiationevents in carcinogenesis that do not rely onexogenous AhR ligands or CYP1-catalyzed carcino-gen metabolism. Fritz et al. [52] used the transgenicadenocarcinoma of the mouse prostate (TRAMP)model, in which expression of the simian virus 40large T and small t antigens are under the control ofthe androgen-dependent probasin gene promoter,to investigate the role of the AhR in tumorformation. They found that when TRAMP mice werecrossed with Ahr�/� mice, the Ahrþ/� and Ahr�/�

TRAMP progeny showed greater prostate tumor inci-dence than in Ahrþ/þ TRAMP mice, indicating aninhibitory effect of AhR expression in this modelof prostate carcinogenesis. Evidence of a tumor-suppressor function of the AhR was also reported byFan et al. [53] in their study of Ahr�/� mice exposed

Figure 6. Tumorigenicity of the MCF-7-derived cell lines with dif-ferential expression of AhR and ERa. AHR100, MCF-7, AhRexp cells, orTMX2 cells, as indicated, were implanted into the mammary glandsof SCID mice, with (closed circles) or without (open circles) E2 supple-mentation as a Silastic implant. Tumor growth was monitored bypalpation at the times indicated. The latency of tumor formation isindicated in the plots by denotation of the day at which the maximalincidence of tumor formation was observed, with the number ofmice with palpable tumors/total number of mice. Data are presentedas mean tumor volume � SE of the mice with measurable tumors.

Table 1. Proliferation Rates of AHR100, MCF-7, and AhRexp Cells

Cell line

AHR100 MCF-7 AhRexp

MediumDC5 0.382 (0.353–0.429)a 0.137 (0.129–0.150) 0.2446 (0.2443–0.2449)DC5 þ 1 nM E2 0.659 (0.623–0.744) 0.398 (0.363–0.433) 0.215 (0.167–0.275)

aRates are expressed as 105 cells/d, with 95% confidence intervals given in parenthesis.

8 SPINK ET AL.


to the direct-acting carcinogen, diethylnitrosamine.A significant increase in liver adenomas, which arethought to be the precursors of hepatocellular carci-nomas, was observed in male Ahr�/� but not femalemice in comparison with Ahrþ/þ mice after exposureto the carcinogen. Our results are consistent withthese studies of prostate tumors Ahr�/� TRAMP mice[52] and liver adenomas [53] in Ahr�/� mice, as wefound that AhR expression in MCF-7 cells is notnecessary for xenograft tumor formation in SCIDmice, and may in fact have a tumor-suppressor func-tion in these cells.A caveat that is associated with the use of AHR100

cells is that these cells were obtained after long-termexposure to BaP. Mutations and epigenetic modifica-tions in AHR100 cells in addition to those directlyrelated to the reduction of AhR expression and theirability to survive and proliferate in the presence ofBaP may have occurred. However, in this regard wenote that AHR100 cells retain ERa expression and re-main dependent on estrogen for tumor growth asxenografts. They also retain epithelial-like morphol-ogy in culture. Recently, it was reported that vector-driven re-expression of the AhR in AHR100 cellsrestored the inducibility of the CYP1A1 and CYP1B1mRNAs by TCDD [58], which supports the premisethat these cells retain many characteristics of theparental cell line. Regardless of the genetic and/orepigenetic changes that may have occurred in thedevelopment of AHR100 cells and their loss of AhRexpression, our conclusion that expression of theAhR is not required for the proliferation, migration,invasion, or estrogen-dependent tumorigenesis ofMCF-7 breast cancer cells is entirely valid.Rates of cell proliferation and alterations in cell-

cycle control are recognized as key factors impactingcarcinogenesis, and the AhR is known to affectthem, albeit in cell-specific manner [17,24,59].There are numerous studies indicating that, in theabsence of an exogenous ligand, the AhR promotesprogression through the cell cycle, whereas ligandactivation of the AhR causes cell-cycle arrest in anumber of cell types [17,22,29,30]. However, theseeffects are not universally observed; the role of theAhR in progression through the cell cycle and cellproliferation is dependent on cell type. Expressionof the AhR in AhR-defective Hepa1c1 cells enhancesthe rate of cell proliferation in the absence of anexogenous AhR ligand [11]. Exposure to the AhRligand, b-naphthoflavone, or overexpression of theAhR in A549 lung carcinoma cells caused enhancedcell proliferation [24]. The reduced rate of cell prolif-eration observed in AhR-null embryo fibroblastsappears be multi-factorial, but a significant role wasattributed to the overproduction of TGF-b [23]. Con-versely, our present study showed enhanced cell pro-liferation of AhR-deficient AHR100 cells, which isconsistent with the increased G0/G1 phase progres-sion that was observed when AhR expression was

knocked down by small inhibitory RNA in MCF-7cells [59].

An aspect of AhR activity in breast and other can-cers that remains unresolved is the role of the AhRin development of the invasive phenotype that maybe indicative of metastatic potential. Several studiessuggest that the AhR, through regulation of Slug,initiates the downregulation of E-cadherin and con-sequently the epithelial-to-mesenchymal transitionand development of the invasive phenotype[27,50,51]. In contrast, Hall et al. [32] reportedevidence for inhibitory roles of AhR expression andligand activation of the AhR in cellular invasion andprocesses related to metastasis in a panel of breastcancer cell lines representing the major breast-cancer subtypes. Exogenous AhR agonists signifi-cantly inhibited cell invasiveness and motility andinhibited colony formation in soft agar regardless ofER, progesterone receptor, or human epidermalgrowth factor receptor 2 status. Knockdown of AhRexpression using a small inhibitory RNA was alsoshown to be sufficient to increase, rather than de-crease, the invasiveness of SKBR3 and MDA-MB-231cells, and activation of the AhR by TCDD causeddecreases in colonization in soft agar of SKBR3,MDA-MB-231, ZR-75-1, and MCF-7 cells [32].

The reasons for the differences in experimentalobservations regarding the role of the AhR in cellu-lar invasiveness and anchorage-independent growthin breast cancer cells are not known, but they maybe influenced by the specific developmental lineageof the tumor. The two main differentiation path-ways in the mammary epithelium give rise to theluminal and basal/myoepithelial cell types [60]. Wesuggest that the AhR may have quite different rolesin ‘‘basal-like’’ breast tumors versus ‘‘luminal-like’’tumors. The epithelial-to-mesenchymal transitionappears to occur within the specific genetic contextof the basal phenotype [61]. If AhR-mediated regula-tion of Slug is a property of the basal-like tumorcells, then this could explain the differences in inva-sive properties of tumor cells that have beenreported for the AhR. MCF-7 cells, which are lumi-nal in phenotype, do not express significant levelsof Slug [60], and our gene expression studies [44]did not show induction of the Slug transcript byTCDD exposure in MCF-7 cells.

In numerous studies it has been suggested thatthe AhR could be a therapeutic target in cancer.However, the results of the present study, notablythose obtained with AHR100 cells, indicate that breastcancer cells can in some cases obtain a growth advan-tage when AhR expression is diminished. We foundthat cell proliferation, migration, invasion, and an-chorage-independent growth do not correlate withAhR expression in MCF-7 cells and its sublines,AHR100 and AhRexp. Over-expression of AhR inAhRexp cells did not confer increased anchorage-independent growth, invasiveness, or tumorigenicity.



As has been reported in numerous previous studies,tumor formation from MCF-7 xenografts was highlydependent on the presence of E2 in the host animal.Mice without E2 supplementation did not have mea-surable tumor growth as xenografts from MCF-7,AHR100, or AhRexp cells. Surprisingly, even the highlyinvasive and proliferative MCF-7-derived cell line,TMX2-28 [36,48], which is unresponsive to E2, wasunable to sustain appreciable tumor growth in SCIDmice.

In summary, our results show that, in the MCF-7model of luminal A breast cancer, CYP1 expressionand inducibility by TCDD are highly dependent onAhR expression, whereas cellular invasion, migra-tion, anchorage-independent growth, and estrogen-stimulated formation of tumors do not require theAhR. Our experiments with AHR100 cells show thathighly proliferative, invasive, and tumorigenic cellscan arise from AhR-deficient tumor cells. The studiesreported here indicate that the AhR and the AHRgene may represent chemoprevention targets thatare focused on reducing the cancer-initiating eventsdue to bioactivation of exogenous and endogenouscarcinogens, but they are not likely to representuniversal targets in breast cancer therapeutics aimedat inhibiting the growth and invasiveness of thedeveloping tumor.

ACKNOWLEDGMENTS

This research was supported by the National Insti-tutes of Health grant CA081243. The authors grateful-ly acknowledge the use of the Wadsworth Center’sTissue Culture Facility and the Biochemistry andMolecular Genetic Core Facilities. The AHR100 cellsline was a generous gift from Dr. Grace Chao Yeh ofthe National Cancer Institute at Frederick, NIH,Frederick, MD. The TMX2-28 cell line was kindlyprovided by Dr. John F. Gierthy of the WadsworthCenter. The authors thank Benjamin P. Leard for hisassistance in data analysis.

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