Invited ReviewInhibitory Aryl Hydrocarbon Receptor-EstrogenReceptor r Cross-Talk and Mechanisms of Action

Stephen Safe* and Mark WormkeDepartment of Veterinary Physiology and Pharmacology, Texas A&M University,

College Station, Texas 77843-4466

Received February 28, 2003

Contents1. Introduction 8072. Genomic Pathways of AhR Action 8083. AhR-Mediated Responses 8084. Inhibitory AhR-ER Cross-Talk in the

Female Rodent Reproductive Tract809

5. Inhibitory AhR-ER Cross-Talk in Breastand Endometrial Cancer Cells

809

5.1. Modulation of Ah Responsiveness byERR and Other NHRs

809

5.2. AhR-Mediated Inhibition ofE2-Induced Gene/Protein Expression

810

5.3. Mechanisms of Inhibitory AhR-ERRCross-Talk

810

5.3.1. Enhanced Metabolism of E2 8105.3.2. Induction of Inhibitory Factors 8115.3.3. Identification of Inhibitory DREs

(iDREs)811

5.3.4. Competition for Common NuclearCoregulatory Proteins

811

5.3.5. Proteasome-DependentDegradation of ERR

811

6. Therapeutic Aspects of AhR-ERRCross-Talk

812

7. Summary 813

1. Introduction2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and struc-

turally related halogenated aromatic compounds are

industrial chemicals and combustion byproducts thathave been identified in fish, wildlife, humans, andthroughout the environment (1, 2). Humans have alsobeen exposed to halogenated aromatics in the workplaceand as a result of numerous industrial accidents. Theadverse human and environmental effects of these com-pounds have been extensively investigated (1-3). Earlystudies by Poland, Nebert, and colleagues demonstratedthat for TCDD and related compounds, there werecorrelations between their structure-toxicity and struc-ture-induction (CYP1A1-dependent aryl hydrocarbonhydroxylase (AHH)) relationships. Moreover, responsive-ness to halogenated aromatics and polynuclear aromatichydrocarbons (PAHs) such as 3-methylcholanthrene alsosegregated among different genetically inbred strains ofmice, which were deemed either aryl hydrocarbon (Ah)responsive or Ah nonresponsive (4-7). It was hypoth-esized that the trait of Ah responsiveness was mediatedby the Ah receptor (AhR), which was subsequentlyidentified in hepatic cytosol from Ah responsive C57BL/6mice using [3H]TCDD as a radioligand (8). Severalstudies have shown that the AhR binds TCDD, other2,3,7,8-substituted polychlorinated dibenzo-p-dioxins(PCDDs) and dibenzofurans (PCDFs), and several iso-steric polychlorinated biphenyls (PCBs) (2, 8-10). Re-search on the activation and properties of the AhRcomplex demonstrated that for a series of structurallyrelated halogenated aromatics (including TCDD), therewas a correlation between their structure-induction/toxicity and structure-receptor binding relationships (2,8-10). Although the AhR was initially linked to toxichalogenated aromatics, more recent studies show thatthe AhR also interacts with structurally diverse endog-enous biochemicals, drugs, and chemoprotective phyto-* To whom correspondence should be addressed. Tel: 979-845-5988.

Fax: 979-862-4929. E-mail: ssafe@cvm.tamu.edu.

JULY 2003

VOLUME 16, NUMBER 7

© Copyright 2003 by the American Chemical Society

10.1021/tx034036r CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 05/23/2003

chemicals (Figure 1) (11-22). Many other ligand-acti-vated receptors including steroid hormone receptors alsobind structurally diverse ligands, which can also be toxicor chemoprotective, and this is consistent with theirtissue specific action, which is also observed for the AhR.Selective steroid hormone receptor agonists/antagonists,such as tamoxifen and raloxifene (estrogen receptor (ER)ligands), are being developed and used for treatinghuman disease (23-26). On the basis of the sameprinciples of tissue selectivity, we have developed selec-tive AhR modulators (SAhRMs) as potential therapeuticagents that target the AhR (27, 28).

2. Genomic Pathways of AhR Action

Genomic pathways of ligand-dependent AhR actionhave been extensively investigated using CYP1A1 as amodel Ah responsive gene (29-34). The classical mech-anism for hepatic AhR-mediated induction of CYP1A1is illustrated in Figure 2. The unbound cytosolic AhR iscomplexed with heat shock protein 90 (Hsp90) and otheraccessory proteins. Upon interaction with ligand, thebound AhR rapidly forms a heterodimeric nuclear com-plex with the AhR nuclear translocator (Arnt) protein.This complex interacts with dioxin/xenobiotic responsiveelements (DREs/XREs) in the CYP1A1 gene promoterresulting in activation of gene expression. The AhR andArnt genes from several species have been cloned andextensively analyzed revealing that both proteins aremembers of the basic helix-loop-helix (bHLH) familyof transcription factors (33-37). Arnt is also known ashypoxia inducible factor-1â (HIF-1â), which acts as aheterodimeric partner for other members of this familyof proteins. The AhR interacts primarily with Arnt andthus far is the only bHLH protein known to be activatedby ligands.

Functional DREs have been identified in promoters ofseveral genes (38); however, the overall mechanisms ofAhR action are increasingly complex and dependent oncell and promoter context, phosphorylation status, andinteractions with many other factors. Activation of theAhR and other nuclear hormone receptors (NHRs) canbe accompanied by responses that attenuate the inducedeffects. For example, AhR ligands induce proteasome-dependent degradation of the AhR (39-42) and alsoupregulate AhR repressor protein, which competitivelybinds Arnt and decreases AhR action (43, 44). The latterresponse is due to transcriptional activation pathways;however, activation of proteasomes may be nongenomicor transcriptionally independent.

3. AhR-Mediated Responses

TCDD and related compounds induce or inhibit ex-pression of multiple genes in different tissues/cells. Theseeffects are accompanied by several highly characteristicresponses including chloracne and related dermal tox-icities, acute lethality, thymic atrophy, body weight loss,immune suppression, developmental and reproductivetoxicity, porphyria/hepatotoxicity, and modulation ofcarcinogenicity (2, 9, 10). With few exceptions, TCDD-induced genes/responses are highly dependent on species,strain, age, and sex of the animal and the target tissueor organ. For example, the lethality of TCDD is generallycharacterized by initial weight loss and wasting prior todeath of most animal species; however, the lethal potencyor LD50 values for TCDD can vary by over 1000-foldamong different animal species. The single dose LD50

values for TCDD in various laboratory animal species are0.6-2.0 (guinea pig), 22-45 (rat), 25-50 (chicken), 115(rabbit), 114-200 (mouse), 100-200 (dog), and 1157-5051 (hamster) µg/kg.

Interspecies differences in susceptibility to TCDD-induced dermal toxicity are also striking. Chloracne isobserved in humans exposed to TCDD and relatedcompounds, and acnegenic responses are also observedin the rabbit (ear) and some strains of mice but not inmost other laboratory animal species. TCDD is also aprototypical endocrine disruptor that directly or indirectlymodulates multiple endocrine signaling pathways. Giventhat TCDD-induced toxicities resemble those observed forthyroid dysfunction, several studies have investigated theeffects of TCDD on thyroid hormone (T3 and T4) levels(45-47). The results suggest that decreases in circulatingT4 in animals treated with TCDD is due to induction ofglucuronyl transferase activity and subsequent increasedformation and excretion of T4 glucuronides (48, 49).TCDD also modulates tissue and serum distribution ofretinoids in laboratory animals. For example, TCDDsignificantly decreases retinoid levels in the liver and thiscould affect retinoid signaling pathways (50). TCDD alsoaffects steroidgenesis in vitro and in laboratory animalsspecies (50-54) in a manner that may contribute to theprofound demasculinization and feminization of ratsexposed in utero to TCDD (55).

Studies in laboratory animals and cells in culture showthat TCDD and other AhR agonists downregulate theepidermal growth factor receptor (56-59), decrease fol-licle stimulating hormone and luteinizing hormone re-ceptors mRNAs in cultured granulosa cells (60, 61), anddecrease insulin-like growth factor I (IGF-I)-inducedbinding of the IGF-I receptor (62). TCDD also induces

Figure 1. AhR ligands include toxic halogenated aromatics(TCDD; 3,3′,4,4′,5-pentachlorobiphenyl (pentaCB)) and chemo-protective phytochemicals including indole-3-cabinol (I3C), DIM,and quercetin.

Figure 2. Proposed mechanism of AhR action-mediated trans-activation.

808 Chem. Res. Toxicol., Vol. 16, No. 7, 2003 Safe and Wormke

expression of transforming growth factor R (TGFR) inmultiple cell lines (63-65), and mRNA levels of TGFâ3are induced in MCF-7 breast cancer cells (63). Thus, AhRligands modulate expression/activity of polypeptide growthfactor receptors and their ligands; however, the contribu-tion of these responses to the toxic responses induced byTCDD is unknown.

Kociba and co-workers (66) reported the effects of long-term dietary exposure to TCDD (0.1, 0.01, and 0.001 µg/kg/day) on tumor development in both male and femaleSprague-Dawley rats. Dose-dependent toxicities wereobserved in both sexes. Increased hepatocellular carci-nomas and keratinizing squamous cell carcinomas wereobserved in female but not male rats, whereas squamouscell carcinomas of hard palate or nasal turbinates wereobserved in males and females. In contrast, there werealso decreases in several age-dependent spontaneoustumors in endocrine organs and the female reproductivetract of rats exposed to TCDD in the diet for up to twoyears. The incidence of both mammary and uterinetumors was decreased in female rats exposed to TCDDsuggesting that ligand-dependent activation of the AhRinhibits formation and/or growth of two estrogen (E2)-dependent tumors. These initial observations sparkedresearch in several laboratories on the tissue specificityof inhibitory AhR-ER cross-talk, the mechanisms of thisresponse, and the development of SAhRMs for treatingE2-dependent tumors.

4. Inhibitory AhR-ER Cross-Talk in theFemale Rodent Reproductive Tract

Two laboratories initiated studies on the effects ofTCDD in the rat or mouse uterus using immature orovariectomized animals (67-74). 17â-Estradiol (E2) in-duces uterine wet weight increase, DNA synthesis, andinduction of multiple uterine genes and their relatedproteins or enzyme activities. In animals cotreated withE2 plus TCDD, several hormone-induced uterine re-sponses were inhibited and these include uterine wetweight increase, DNA synthesis, progesterone receptor(PR) binding, peroxidase activity, EGF receptor binding,EGF receptor mRNA, and c-fos mRNA levels (67-74).Evidence supporting a role for the AhR include theobservations that the AhR is expressed in the rodentuterus and the structure-dependent potencies of TCDDand related compounds as antiestrogens paralleled theircorresponding AhR binding affinities. Buchanan and co-workers more recently investigated inhibitory AhR-ERcross-talk in the mouse uterus using wild-type and AhRknockout mice and tissue recombination approaches (75,76). Their studies showed that TCDD inhibited E2-induced uterine epithelial labeling index and lactoferrinexpression in wild-type but not AhR knockout mice.TCDD also inhibited E2-induced cyclin A1, cyclin B1, andcyclin D2, and this was associated with increased expres-sion of TGFâ, which may play a role in the growthinhibitory effects of TCDD. Tissue recombination experi-ments in wild-type and AhR knockout mice suggest thatthe effects of TCDD on uterine epithelial cell growth/geneexpression may be dependent on stromal AhR andstromal inhibitory AhR-ER cross-talk.

The decreased incidence of spontaneous mammary anduterine tumors in female Sprague-Dawley rats admin-istered TCDD in the diet for up to two years suggeststhat TCDD may act to inhibit tumor formation and/or

tumor growth. Subsequent studies (77-79) have dem-onstrated that administration of TCDD inhibits mam-mary tumor growth in carcinogen-induced rat mammarytumors and in athymic nude mice bearing human breastcancer cell xenografts. These results demonstrate thatinhibitory AhR-ER cross-talk is observed in the rodentuterus and mammary tumors and this parallels thedecreased incidence of mammary cancer in women ac-cidentally exposed to high levels of TCDD in Seveso, Italy(80). Several epidemiology studies have shown thatcigarette smoking protects against uterine cancers (81,82), which may represent an example of inhibitory AhR-ER cross-talk since cigarette smoke condensate containsPAHs and other AhR active compounds.

5. Inhibitory AhR-ER Cross-Talk in Breastand Endometrial Cancer Cells

5.1. Modulation of Ah Responsiveness by ERrand Other NHRs

The AhR is expressed in ER-positive and ER-negativebreast and endometrial cancer cells; however, inductionof CYP1A1 and related activities by TCDD is variableand dependent on cell context (83-88). Vickers and co-workers (83) first reported that Ah responsiveness amongseveral breast cancer cell lines correlated with ERexpression. For example, TCDD induced CYP1A1 geneexpression in ER-positive T47D, ZR-75, and MCF-7breast cancer cells, whereas in ER-negative, adriamycinresistant MCF-7 (MCF-7Adr) and MDA-MB-231 cells, noinduction response (by TCDD) was observed. Moreover,transient transfection of wild-type or variant ER expres-sion plasmids in ER-negative MDA-MB-231 or Hs578Tcells restored Ah responsiveness, suggesting a possiblerole for ER in activating the AhR (89, 90). A recent studyinvestigated the role of Hsp90 in mediating Ah non-responsive in MDA-MB-231 cells. Transfection of Hsp90expression plasmid in MCF-7 or T47D cells decreased Ahresponsiveness but increased ER-mediated gene expres-sion. MDA-MB-231 cells express relatively high levels ofnuclear Hsp90, and transfection of ER appears to restoreAh responsiveness by squelching Hsp90 (91).

Several studies have also reported inhibitory ER andNHR cross-talk with the AhR resulting in decreasedinduction of CYP1A1 by TCDD and other AhR agonists(92-95). Ricci and co-workers reported that induction ofCYP1A1 gene expression by TCDD in ER-positive MCF-7breast and ECC-1 endometrial cancer cells was signifi-cantly decreased after cotreatment with E2, whereasthese interactions were not observed in ER-negativehuman liver cancer cells or primary keratinocytes (93).It was concluded that one possible mechanism for E2-dependent inhibition of CYP1A1 induction by TCDD wasdue to a squelching mechanism associated with competi-tion for limiting levels of nuclear factor 1. With oneexception, our studies in ER-positive breast and endome-trial cancer cells indicated minimal inhibition of inducedCYP1A1-dependent activity by steroid hormones (87, 96).For example, in MCF-7 breast and mouse Hepa1c1ccancer cell lines, E2 did not inhibit induction of CYP1A1by TCDD (96), and similar results were observed inIshikawa endometrial cancer cells (87). In contrast, wealso observed inhibition of TCDD-induced CYP1A1 by E2in ECC1 cells as previously reported (93). We did notobserve any inhibitory effects of progesterone in ECC1

Invited Review Chem. Res. Toxicol., Vol. 16, No. 7, 2003 809

or Ishikawa endometrial cancer cells, whereas inhibitoryPR-AhR cross-talk (on CYP1A1) was observed in T47Dbreast cancer cells (94). Differences between studies evenin the same breast cancer cells is not unexpected sincethese cells are highly variable with respect to expressionof wild-type and variant ERR and differences in cellspassage number, growth conditions, and serum lots mayalso affect interactions between the AhR and the NHRsignaling pathways.

5.2. AhR-Mediated Inhibition of E2-InducedGene/Protein Expression

Gierthy and co-workers first reported that TCDDinhibited E2-regulated excretion of extracellular plas-minogen activator activity and postconfluent focus pro-duction in MCF-7 cells (97, 98) and that this wasaccompanied by inhibition of hormone-induced cell pro-liferation. Subsequent studies have reported that TCDDand other AhR agonists inhibit expression of several E2-induced genes/protein and/or their related activitiesincluding cathepsin D (99-103), c-fos (104), pS2 (105,106), Hsp27 (107), prolactin receptor (108), PR (109), andcyclin D1 (110). TCDD inhibits E2-induced cell cycleprogression primarily by blocking G1 f S phase in apathway that also decreased phosphorylation of retino-blastoma protein and cyclin-dependent kinases 2 and 4activities (111). Other examples of inhibitory AhR-ERcross-talk include inhibition of E2-induced glucose me-tabolism (112) and reporter gene activity in cells trans-fected with constructs containing E2 responsive EREsand promoter fragments of cathepsin D, creatine kinaseB, pS2, Hsp27, and c-fos genes (86, 87, 102-106, 112,113). In addition, TCDD and other AhR agonists decreaseER protein in T47D, MCF-7, and ZR-75 breast cancercells and the structure-dependent downregulation of ERby a series of compounds correlated with their structure-AhR binding activities (28, 41, 114-116).

The antiestrogenic growth inhibitory activity of TCDDand related compounds can be explained in part by theinhibition of several key pathways involved in hormone-induced G1 f S phase progression of MCF-7 cells andinhibition of c-fos, which also plays a role in growth ofbreast cancer cells. Inhibitory AhR-ERR cross-talk is notconfined to breast cancer cells since comparable interac-tions have been reported in ERR-positive ovarian andendometrial cancer cells (86, 87, 117, 118). The role ofthe AhR in mediating inhibition of E2-induced responseshas also been investigated in transgenic MCF-7 cells,which overexpress a constitutively active form of the AhR(119). Their studies showed that overexpression of theconstitutively active AhR alone inhibited E2-dependentinduction of cathepsin D mRNA levels and cell prolifera-tion; reporter gene activity was also inhibited by consti-tutively active AhR in cells transfected with chimericGAL4-ERR fusion protein and a construct containingtandem GAL4 response elements linked to a luciferasereporter gene. The role of the AhR in mediating apparentantiestrogenic responses was also investigated in benzo-[a]pyrene resistant MCF-7 (MCF-7BaPr) cells, which ex-press ERR, Arnt, and only minimal levels of the AhR(120). In this cell line, the characteristic inhibitory AhR-ER cross-talk associated with cell proliferation and geneexpression was ablated, thus confirming the requirementof AhR expression for mediating interactions between thetwo signaling pathways.

5.3. Mechanisms of Inhibitory AhR-ERrCross-Talk

Interactions between ligand-induced signaling path-ways can be complex and dependent on cell or tissuecontext and specific genes. For example, estrogens re-press prolactin-induced STAT5 activation of the â-caseinpromoter and this is linked to direct ER-STAT5 interac-tions, which are dependent on the DNA binding domainof ERR (121). In ERR knockout mice, female specificCyp2a4 expression in the liver is repressed as comparedto wild-type animals and this is related to nuclear levelsof STAT5, which represses Cyp2a4 expression (122).Thus, ERR-STAT5 interactions in murine liver areassociated with ERR-dependent inhibition of hepaticnuclear localization of STAT5 in female mice. Not sur-prisingly, there are several possible mechanisms thathave been proposed for inhibitory AhR-ERR cross-talk(Figure 3), and these include increased metabolism of E2(no. 1), direct interactions of the AhR with criticalpromoter regions of E2 responsive genes (no. 3), inductionof inhibitory factors (no. 2), competition for commonnuclear coregulatory proteins (no. 4), and proteasome-dependent degradation of ERR (no. 5) (123-126). Thereis evidence that provides some support for most of thesepathways, and these are discussed below.

5.3.1. Enhanced Metabolism of E2Several studies have demonstrated that TCDD and

other AhR agonists induce CYP1A1 and CYP1B1 inbreast cancer cells resulting in enhanced metabolism ofE2 and depletion of intracellular hormone levels (127-129). It has been suggested that this induced oxidativemetabolism of E2 results in limiting levels of E2 anddecreased hormone responsiveness. Although this maycontribute to the observed inhibitory crosstalk betweenAhR and ER signaling pathways, there is evidence thatalterations in E2 metabolism are insufficient for mediat-ing AhR-mediated antiestrogenic responses. For example,TCDD inhibits E2-induced cathepsin D mRNA levels inMCF-7 cells within 30-60 min and this precedes induc-tion of CYP1A1 protein (102). Several SAhRMs that donot induce CYP1A1 in breast cancer cells or in rodentmodels in vivo maintain an ability to inhibit cell/tumorgrowth (27, 28). Moreover, in rodents treated with TCDD,circulating E2 levels are not altered (130). These resultssuggest that CYP1A1-dependent depletion of E2 throughoxidative metabolism is not necessary for inhibitoryAhR-ERR cross-talk.

Figure 3. Proposed mechanisms of inhibitory AhR-ERR cross-talk (123-126).

810 Chem. Res. Toxicol., Vol. 16, No. 7, 2003 Safe and Wormke

5.3.2. Induction of Inhibitory Factors

Recent studies have demonstrated that TCDD inhib-its E2-induced gene/reporter gene expression in BG-1ovarian cancer cells, and TCDD also downregulates ERRprotein and mRNA levels in BG-1 cells (118). However,inhibition of E2-induced pS2 mRNA and reporter geneactivity (in cells transfected with an ERE construct) byTCDD in this cell line was blocked by cycloheximide, aprotein synthesis inhibitor. These results suggest thatin BG-1 cells, the inhibitory effects of TCDD may be dueto induction of an inhibitory factor; however, the role ofthis pathway on inhibition of E2-dependent growth andinduction of other genes requires further investigation.

5.3.3. Identification of Inhibitory DREs(iDREs)

Studies on the rapid inhibition of E2-induced cathepsinD mRNA levels by TCDD suggested that one possiblemechanism may involve direct interactions of the nuclearAhR complex with “critical” regions of E2 responsive genepromoters that repress gene activation. The proximalregion (-250 to the transcription start site) of thecathepsin D gene promoter contains multiple E2 respon-sive elements including an upstream GC(N)19ERE half-site (ERE1/2) motif that binds ERR/Sp1 and downstreamGC-rich and E-box motifs that bind ERR/Sp1 and USF1/2in combination with ERR, respectively (100, 102, 103).Constructs containing the upstream GC(N)19ERE1/2motif were activated by E2 in reporter assays, and theseinduced responses were inhibited by TCDD (102). Thisregion of the cathepsin D promoter also contains aGCGTG sequence at -177 to -181 that corresponds tothe pentanucleotide core of a DRE required for AhR-mediated transactivation. A consensus sequence for afunctional DRE required for transactivation is N T/G TGCGTG A/C G/T A/T A/G N (38), and this differedsignificantly from the TGT GCGTG CCCGA sequence inthe cathepsin D promoter, which was not Ah responsive.The requirement for the iDRE within the upstream E2responsive GC(N)19ERE1/2 (-195 to -165) motif formediating the ligand (TCDD)-induced inhibition of thisresponse was confirmed in the following series of experi-ments (102). TCDD inhibited E2-induced transactivationof a construct (pCD) containing the GC(N)19ERE1/2promoter, whereas this inhibition was not observed usinga construct (pCDm) with a DRE mutation. InhibitoryAhR-ERR cross-talk was decreased in cells treated withAhR antagonists or with antisense AhR or Arnt, andcross-talk was not observed in AhR deficient cells. Gelmobility shift assays using the GC(N)19ERE1/2 constructshowed that the core DRE was targeted by the AhRcomplex. This interaction blocked formation of the ERR/Sp1 complex, resulting in inhibition of E2-induced trans-activation.

Subsequent studies have identified additional func-tional iDREs in the pS2, Hsp27, and c-fos gene promoters,with mechanisms of action that are gene promoterspecific (Figure 4) (104, 106, 107). For example, the iDREin the pS2 gene promoter is 125 bp upstream from thenonconsensus ERE at -405 to -393. However, in trans-activation studies with constructs containing wild-typeand mutant pS2 promoter inserts, it was apparent thathormone responsiveness of pS2 was primarily associatedwith an AP-1 motif (-518 to -512), which overlaps the

iDRE at -527 to -514. Thus, inhibitory AhR-ERR cross-talk on the pS2 gene promoter may be due to competitiveinteractions of the AhR and AP-1 complexes for bindingto the same region of the promoter (106). This type ofinhibitory interaction has also been observed for the c-fosgene promoter where both the AhR and the ERR/Sp1complex bind the -1168 to -1159 distal region of thepromoter, which contains overlapping iDRE and GC-boxmotifs. In contrast, the functional iDRE in the Hsp27promoter is located at the start site, and the AhR complexmay interfere with appropriate assembly of the pre-initiation complex (107). A second functional iDRE in thecathepsin D promoter (-130 to -126) inhibits E2-inducedtransactivation from the E2 responsive adenovirus majorlate promoter element (MLPE), which contains an over-lapping E-box and a nonconsensus ERE (103). DNAfootprinting studies show that the AhR complex de-creases transcription factor binding to the MLPE and thiscorrelated with decreased transactivation.

5.3.4. Competition for Common NuclearCoregulatory Proteins

Ricci and co-workers (93) first demonstrated thatinduction of CYP1A1 by TCDD was inhibited by E2 andthis was due to AhR and ERR competition for thetranscription factor nuclear factor 1. Subsequent studieshave shown that the AhR and ERR interact with sev-eral common nuclear coregulatory proteins includingsteroid receptor coactivators and corepressors (131-135).Moreover, subdomains of the AhR transactivation do-main inhibit (or squelch) ligand-dependent activation ofAhR or ERR (136). NEDD8, an ubiquitin-like protein,enhances AhR-mediated transactivation (137), whereasthe activating enzyme of NEDD8 (Uba3) decreasesligand-dependent activation of ERR and ERâ using anERE promoter in HeLa cells (138). The role of squelchingmechanisms in inhibitory AhR-ERR cross-talk willdepend on the relative cell context-dependent expres-sion of the multitude of coregulatory proteins thatinteract with ER and AhR and modulate their trans-activation function, and this requires further investiga-tion.

5.3.5. Proteasome-Dependent Degradation ofERr

Harris and co-workers (139) initially demonstratedthat for a series of AhR agonists, their potency to induce

Figure 4. Functional iDREs in promoters of the cathepsin D,c-fos, heat shock protein 27, and pS2 genes (102-107).

Invited Review Chem. Res. Toxicol., Vol. 16, No. 7, 2003 811

degradation of ERR in MCF-7 cells correlated with theirbinding affinities for the AhR. It was also reported thatinhibitors of transcription (actinomycin D) and proteinsynthesis (cycloheximide) increase ERR levels in MCF-7cells. However, after cotreatment with TCDD, there weresignificant decreases in cycloheximide/actinomycin D-induced ERR levels. At that time, the significance of theseobservations was not fully appreciated. More recentstudies have reported that E2 induces proteasome-dependent degradation of ERR, and some selective ERmodulators such as the “pure” antiestrogen ICI 182780also induce proteasome-dependent degradation of ERRin breast cancer cells (28, 41, 140-143). Rapid degrada-tion of ERR induced by ICI 182780 may play an impor-tant role in the antiestrogenic/antitumorigenic activityof these compounds, whereas tamoxifen, which is widelyused in the treatment of breast cancer, does not inducedegradation of ERR (143, 144). The ability of TCDD toenhance degradation of ERR is depicted in Figure 5.Consistent with other studies, TCDD decreased proteinlevels of both the AhR and the ERR in breast cancer cellsin a manner that was blocked by proteasome but notprotease inhibitors. Similar results have been observedin ZR-75 breast cancer cells (144). Treatment with TCDDalone results in decreased immunoreactive AhR and ERRproteins, and degradation of these receptors is blockedby proteasome but not protease inhibitors. These resultsare consistent with previous studies showing that TCDDinduced proteasome-dependent degradation of the AhRin breast and other cell lines and degradation of ERR inbreast cancer cells (28, 39, 40-42, 144). Moreover, aspreviously reported, E2 induces degradation of ERR butnot the AhR (41). The most striking effect is observed incells cotreated with TCDD plus E2 where 80-90% ofcellular ERR levels are degraded within 3 h (41, 144),and these levels are similar to those observed aftertreatment with ICI 182780. These data suggest that theantiestrogenic activity of TCDD (cotreatment with E2)in breast cancer cells may be due to limiting levels of ERRand this has been extensively investigated by Wormkeand co-workers (144). Their results indicate the following.(i) TCDD, E2, TCDD plus E2, and ICI 182780 enhanceubiquitination of ERR and subsequent proteasome-dependent degradation of ERR. (ii) In cells cotreated withE2 plus TCDD, there was a correlation between thepercentage decrease of ERR below levels observed in cells

treated with E2 alone and the corresponding decreasein levels of E2-induced transactivation and fos proteinexpression. (iii) TCDD (but not E2) induced interactionsof the AhR with ERR in a mammalian two hybrid assay.(iv) Effects of DMSO, TCDD, E2, and TCDD plus E2treatments on ERR levels were independent of kinaseinhibitors. (v) TCDD did not affect ERR, AhR, or ArntmRNA levels. (vi) Moreover, neither actinomycin D norcycloheximide blocked TCDD-induced degradation ofERR.

These results are consistent with a mechanism inwhich cotreatment of cells with TCDD plus E2 enhancesproteasome-dependent degradation of ERR below criticallevels required for hormone-induced transactivation. Thismechanism for inhibitory AhR-ERR cross-talk repre-sents a novel nongenomic or transcriptionally independ-ent pathway where the degradation of ERR is due toligand-dependent activation of AhR-ERR interactionsand not transactivation through DREs.

6. Therapeutic Aspects of AhR-ERrCross-Talk

Research in this laboratory has identified two groupsof SAhRMs, namely, alternate-substituted (1,3,6,8- and2,4,6,8-) alkyl PCDFs and ring-substituted diindolyl-methanes (DIMs) (27, 145, 146). These compounds in-teract with the AhR and exhibit partial AhR antagonistactivities for many of the AhR-mediated toxic responsesas well as CYP1A1. Prototypical SAhRMs such as 6-methyl-1,3,8-trichlorodibenzofuran and ring-substitutedDIMs are themselves relatively nontoxic; however, thesecompounds inhibit growth of E2-dependent mammarytumors and growth of pancreatic cancer cells (28, 147-150). These results are consistent with the interactionof numerous chemoprotective phytochemicals with theAhR and their anticarcinogenic activity. For example,cruciferous vegetables and indole-3-carbinol, a major AhRphytochemical constitutent of these vegetables, inhibitmammary tumor formation in rodent mammary tumormodels (151). Moreover, there is epidemiological evidenceshowing that cruciferous vegetable consumption is as-sociated with prevention of several cancers includingbreast cancer (151) and this could be due, in part, toindole-3-carbinol and related compounds that activate theAhR (147, 148). Ongoing studies in this laboratory are

Figure 5. Inhibitory AhR-ERR cross-talk and the role of proteasomes. MCF-7 (A) or T47D (B) cells were treated with DMSO (D),10 nM E2 (E), 10 nM TCDD (T), or E2 plus TCDD (TE), and ERR, AhR, or Sp1 (loading control) proteins were determined by Westernblot analysis 3 h after treatment. The effects of proteasome inhibitors (10 µM PSI or PSII) or protease inhibitors (10 µM EST orchloroquine (chlor)) were also determined (144).

812 Chem. Res. Toxicol., Vol. 16, No. 7, 2003 Safe and Wormke

developing new SAhRMs derived from phytochemicals asa new class of drugs for treating ER-dependent and ER-negative breast cancers and other cancers where theliganded AhR induces growth inhibition.

7. Summary

Inhibitory AhR-ERR cross-talk has been observed inthe rodent uterus, rodent mammary tumors, breast,ovarian, and endometrial cancer cell lines. There isevidence to support several mechanisms for interactionsbetween these two signaling pathways, and these maybe gene/response- and cell context-dependent. Recentdata obtained in ER-positive breast cancer cells suggestthat in cells cotreated with E2 plus TCDD, there is rapidproteasome-dependent degradation of ERR, which thenbecomes limiting. Similar effects have been observed inthe mouse uterus (144). These data do not exclude othermechanisms, which may differentially contribute toinhibitory AhR-ERR cross-talk. These studies providean example of the complexities of cross-talk betweendifferent biochemical/endocrine pathways and also dem-onstrate that the AhR is a target for development ofdrugs for treatment of breast and endometrial cancer.

Acknowledgment. The financial assistance of theNational Institutes of Health (ES04176 and ES09106) isgratefully acknowledged.

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