Inhibitory Aryl Hydrocarbon Receptor−Estrogen Receptor α Cross

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JULY 2003 VOLUME 16, NUMBER 7 © Copyright 2003 by the American Chemical Society

Invited Review Inhibitory Aryl Hydrocarbon Receptor-Estrogen Receptor r Cross-Talk and Mechanisms of Action Stephen Safe* and Mark Wormke Department of Veterinary Physiology and Pharmacology, Texas A&M University, College Station, Texas 77843-4466 Received February 28, 2003

Contents 1. 2. 3. 4.

Introduction Genomic Pathways of AhR Action AhR-Mediated Responses Inhibitory AhR-ER Cross-Talk in the Female Rodent Reproductive Tract 5. Inhibitory AhR-ER Cross-Talk in Breast and Endometrial Cancer Cells 5.1. Modulation of Ah Responsiveness by ERR and Other NHRs 5.2. AhR-Mediated Inhibition of E2-Induced Gene/Protein Expression 5.3. Mechanisms of Inhibitory AhR-ERR Cross-Talk 5.3.1. Enhanced Metabolism of E2 5.3.2. Induction of Inhibitory Factors 5.3.3. Identification of Inhibitory DREs (iDREs) 5.3.4. Competition for Common Nuclear Coregulatory Proteins 5.3.5. Proteasome-Dependent Degradation of ERR 6. Therapeutic Aspects of AhR-ERR Cross-Talk 7. Summary

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1. Introduction 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally related halogenated aromatic compounds are * To whom correspondence should be addressed. Tel: 979-845-5988. Fax: 979-862-4929. E-mail: [email protected].

industrial chemicals and combustion byproducts that have been identified in fish, wildlife, humans, and throughout the environment (1, 2). Humans have also been exposed to halogenated aromatics in the workplace and as a result of numerous industrial accidents. The adverse human and environmental effects of these compounds have been extensively investigated (1-3). Early studies by Poland, Nebert, and colleagues demonstrated that for TCDD and related compounds, there were correlations between their structure-toxicity and structure-induction (CYP1A1-dependent aryl hydrocarbon hydroxylase (AHH)) relationships. Moreover, responsiveness to halogenated aromatics and polynuclear aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene also segregated among different genetically inbred strains of mice, which were deemed either aryl hydrocarbon (Ah) responsive or Ah nonresponsive (4-7). It was hypothesized that the trait of Ah responsiveness was mediated by the Ah receptor (AhR), which was subsequently identified in hepatic cytosol from Ah responsive C57BL/6 mice using [3H]TCDD as a radioligand (8). Several studies have shown that the AhR binds TCDD, other 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), and several isosteric polychlorinated biphenyls (PCBs) (2, 8-10). Research on the activation and properties of the AhR complex demonstrated that for a series of structurally related halogenated aromatics (including TCDD), there was a correlation between their structure-induction/ toxicity and structure-receptor binding relationships (2, 8-10). Although the AhR was initially linked to toxic halogenated aromatics, more recent studies show that the AhR also interacts with structurally diverse endogenous biochemicals, drugs, and chemoprotective phyto-

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Functional DREs have been identified in promoters of several genes (38); however, the overall mechanisms of AhR action are increasingly complex and dependent on cell and promoter context, phosphorylation status, and interactions with many other factors. Activation of the AhR and other nuclear hormone receptors (NHRs) can be accompanied by responses that attenuate the induced effects. For example, AhR ligands induce proteasomedependent degradation of the AhR (39-42) and also upregulate AhR repressor protein, which competitively binds Arnt and decreases AhR action (43, 44). The latter response is due to transcriptional activation pathways; however, activation of proteasomes may be nongenomic or transcriptionally independent. Figure 1. AhR ligands include toxic halogenated aromatics (TCDD; 3,3′,4,4′,5-pentachlorobiphenyl (pentaCB)) and chemoprotective phytochemicals including indole-3-cabinol (I3C), DIM, and quercetin.

Figure 2. Proposed mechanism of AhR action-mediated transactivation.

chemicals (Figure 1) (11-22). Many other ligand-activated receptors including steroid hormone receptors also bind structurally diverse ligands, which can also be toxic or chemoprotective, and this is consistent with their tissue 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 treating human disease (23-26). On the basis of the same principles of tissue selectivity, we have developed selective AhR modulators (SAhRMs) as potential therapeutic agents that target the AhR (27, 28).

2. Genomic Pathways of AhR Action Genomic pathways of ligand-dependent AhR action have been extensively investigated using CYP1A1 as a model Ah responsive gene (29-34). The classical mechanism for hepatic AhR-mediated induction of CYP1A1 is illustrated in Figure 2. The unbound cytosolic AhR is complexed with heat shock protein 90 (Hsp90) and other accessory proteins. Upon interaction with ligand, the bound AhR rapidly forms a heterodimeric nuclear complex with the AhR nuclear translocator (Arnt) protein. This complex interacts with dioxin/xenobiotic responsive elements (DREs/XREs) in the CYP1A1 gene promoter resulting in activation of gene expression. The AhR and Arnt genes from several species have been cloned and extensively analyzed revealing that both proteins are members of the basic helix-loop-helix (bHLH) family of transcription factors (33-37). Arnt is also known as hypoxia inducible factor-1β (HIF-1β), which acts as a heterodimeric partner for other members of this family of proteins. The AhR interacts primarily with Arnt and thus far is the only bHLH protein known to be activated by ligands.

3. AhR-Mediated Responses TCDD and related compounds induce or inhibit expression of multiple genes in different tissues/cells. These effects are accompanied by several highly characteristic responses including chloracne and related dermal toxicities, acute lethality, thymic atrophy, body weight loss, immune suppression, developmental and reproductive toxicity, porphyria/hepatotoxicity, and modulation of carcinogenicity (2, 9, 10). With few exceptions, TCDDinduced genes/responses are highly dependent on species, strain, age, and sex of the animal and the target tissue or organ. For example, the lethality of TCDD is generally characterized by initial weight loss and wasting prior to death of most animal species; however, the lethal potency or LD50 values for TCDD can vary by over 1000-fold among different animal species. The single dose LD50 values for TCDD in various laboratory animal species are 0.6-2.0 (guinea pig), 22-45 (rat), 25-50 (chicken), 115 (rabbit), 114-200 (mouse), 100-200 (dog), and 11575051 (hamster) µg/kg. Interspecies differences in susceptibility to TCDDinduced dermal toxicity are also striking. Chloracne is observed in humans exposed to TCDD and related compounds, and acnegenic responses are also observed in the rabbit (ear) and some strains of mice but not in most other laboratory animal species. TCDD is also a prototypical endocrine disruptor that directly or indirectly modulates multiple endocrine signaling pathways. Given that TCDD-induced toxicities resemble those observed for thyroid dysfunction, several studies have investigated the effects of TCDD on thyroid hormone (T3 and T4) levels (45-47). The results suggest that decreases in circulating T4 in animals treated with TCDD is due to induction of glucuronyl transferase activity and subsequent increased formation and excretion of T4 glucuronides (48, 49). TCDD also modulates tissue and serum distribution of retinoids in laboratory animals. For example, TCDD significantly decreases retinoid levels in the liver and this could affect retinoid signaling pathways (50). TCDD also affects steroidgenesis in vitro and in laboratory animals species (50-54) in a manner that may contribute to the profound demasculinization and feminization of rats exposed in utero to TCDD (55). Studies in laboratory animals and cells in culture show that TCDD and other AhR agonists downregulate the epidermal growth factor receptor (56-59), decrease follicle stimulating hormone and luteinizing hormone receptors mRNAs in cultured granulosa cells (60, 61), and decrease insulin-like growth factor I (IGF-I)-induced binding of the IGF-I receptor (62). TCDD also induces

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expression of transforming growth factor R (TGFR) in multiple cell lines (63-65), and mRNA levels of TGFβ3 are induced in MCF-7 breast cancer cells (63). Thus, AhR ligands modulate expression/activity of polypeptide growth factor receptors and their ligands; however, the contribution of these responses to the toxic responses induced by TCDD is unknown. Kociba and co-workers (66) reported the effects of longterm dietary exposure to TCDD (0.1, 0.01, and 0.001 µg/ kg/day) on tumor development in both male and female Sprague-Dawley rats. Dose-dependent toxicities were observed in both sexes. Increased hepatocellular carcinomas and keratinizing squamous cell carcinomas were observed in female but not male rats, whereas squamous cell carcinomas of hard palate or nasal turbinates were observed in males and females. In contrast, there were also decreases in several age-dependent spontaneous tumors in endocrine organs and the female reproductive tract of rats exposed to TCDD in the diet for up to two years. The incidence of both mammary and uterine tumors was decreased in female rats exposed to TCDD suggesting that ligand-dependent activation of the AhR inhibits formation and/or growth of two estrogen (E2)dependent tumors. These initial observations sparked research in several laboratories on the tissue specificity of inhibitory AhR-ER cross-talk, the mechanisms of this response, and the development of SAhRMs for treating E2-dependent tumors.

4. Inhibitory AhR-ER Cross-Talk in the Female Rodent Reproductive Tract Two laboratories initiated studies on the effects of TCDD in the rat or mouse uterus using immature or ovariectomized animals (67-74). 17β-Estradiol (E2) induces uterine wet weight increase, DNA synthesis, and induction of multiple uterine genes and their related proteins or enzyme activities. In animals cotreated with E2 plus TCDD, several hormone-induced uterine responses were inhibited and these include uterine wet weight 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 the observations that the AhR is expressed in the rodent uterus and the structure-dependent potencies of TCDD and related compounds as antiestrogens paralleled their corresponding AhR binding affinities. Buchanan and coworkers more recently investigated inhibitory AhR-ER cross-talk in the mouse uterus using wild-type and AhR knockout mice and tissue recombination approaches (75, 76). Their studies showed that TCDD inhibited E2induced uterine epithelial labeling index and lactoferrin expression in wild-type but not AhR knockout mice. TCDD also inhibited E2-induced cyclin A1, cyclin B1, and cyclin D2, and this was associated with increased expression of TGFβ, which may play a role in the growth inhibitory effects of TCDD. Tissue recombination experiments in wild-type and AhR knockout mice suggest that the effects of TCDD on uterine epithelial cell growth/gene expression may be dependent on stromal AhR and stromal inhibitory AhR-ER cross-talk. The decreased incidence of spontaneous mammary and uterine tumors in female Sprague-Dawley rats administered TCDD in the diet for up to two years suggests that TCDD may act to inhibit tumor formation and/or

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tumor growth. Subsequent studies (77-79) have demonstrated that administration of TCDD inhibits mammary tumor growth in carcinogen-induced rat mammary tumors and in athymic nude mice bearing human breast cancer cell xenografts. These results demonstrate that inhibitory AhR-ER cross-talk is observed in the rodent uterus and mammary tumors and this parallels the decreased incidence of mammary cancer in women accidentally exposed to high levels of TCDD in Seveso, Italy (80). Several epidemiology studies have shown that cigarette smoking protects against uterine cancers (81, 82), which may represent an example of inhibitory AhRER cross-talk since cigarette smoke condensate contains PAHs and other AhR active compounds.

5. Inhibitory AhR-ER Cross-Talk in Breast and Endometrial Cancer Cells 5.1. Modulation of Ah Responsiveness by ERr and Other NHRs The AhR is expressed in ER-positive and ER-negative breast and endometrial cancer cells; however, induction of CYP1A1 and related activities by TCDD is variable and dependent on cell context (83-88). Vickers and coworkers (83) first reported that Ah responsiveness among several breast cancer cell lines correlated with ER expression. For example, TCDD induced CYP1A1 gene expression in ER-positive T47D, ZR-75, and MCF-7 breast cancer cells, whereas in ER-negative, adriamycin resistant MCF-7 (MCF-7Adr) and MDA-MB-231 cells, no induction response (by TCDD) was observed. Moreover, transient transfection of wild-type or variant ER expression plasmids in ER-negative MDA-MB-231 or Hs578T cells restored Ah responsiveness, suggesting a possible role for ER in activating the AhR (89, 90). A recent study investigated the role of Hsp90 in mediating Ah nonresponsive in MDA-MB-231 cells. Transfection of Hsp90 expression plasmid in MCF-7 or T47D cells decreased Ah responsiveness but increased ER-mediated gene expression. MDA-MB-231 cells express relatively high levels of nuclear Hsp90, and transfection of ER appears to restore Ah responsiveness by squelching Hsp90 (91). Several studies have also reported inhibitory ER and NHR cross-talk with the AhR resulting in decreased induction of CYP1A1 by TCDD and other AhR agonists (92-95). Ricci and co-workers reported that induction of CYP1A1 gene expression by TCDD in ER-positive MCF-7 breast and ECC-1 endometrial cancer cells was significantly decreased after cotreatment with E2, whereas these interactions were not observed in ER-negative human liver cancer cells or primary keratinocytes (93). It was concluded that one possible mechanism for E2dependent inhibition of CYP1A1 induction by TCDD was due to a squelching mechanism associated with competition for limiting levels of nuclear factor 1. With one exception, our studies in ER-positive breast and endometrial cancer cells indicated minimal inhibition of induced CYP1A1-dependent activity by steroid hormones (87, 96). For example, in MCF-7 breast and mouse Hepa1c1c cancer cell lines, E2 did not inhibit induction of CYP1A1 by TCDD (96), and similar results were observed in Ishikawa endometrial cancer cells (87). In contrast, we also observed inhibition of TCDD-induced CYP1A1 by E2 in ECC1 cells as previously reported (93). We did not observe any inhibitory effects of progesterone in ECC1

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or Ishikawa endometrial cancer cells, whereas inhibitory PR-AhR cross-talk (on CYP1A1) was observed in T47D breast cancer cells (94). Differences between studies even in the same breast cancer cells is not unexpected since these cells are highly variable with respect to expression of wild-type and variant ERR and differences in cells passage number, growth conditions, and serum lots may also affect interactions between the AhR and the NHR signaling pathways.

5.2. AhR-Mediated Inhibition of E2-Induced Gene/Protein Expression Gierthy and co-workers first reported that TCDD inhibited E2-regulated excretion of extracellular plasminogen activator activity and postconfluent focus production in MCF-7 cells (97, 98) and that this was accompanied by inhibition of hormone-induced cell proliferation. Subsequent studies have reported that TCDD and other AhR agonists inhibit expression of several E2induced genes/protein and/or their related activities including cathepsin D (99-103), c-fos (104), pS2 (105, 106), Hsp27 (107), prolactin receptor (108), PR (109), and cyclin D1 (110). TCDD inhibits E2-induced cell cycle progression primarily by blocking G1 f S phase in a pathway that also decreased phosphorylation of retinoblastoma protein and cyclin-dependent kinases 2 and 4 activities (111). Other examples of inhibitory AhR-ER cross-talk include inhibition of E2-induced glucose metabolism (112) and reporter gene activity in cells transfected with constructs containing E2 responsive EREs and promoter fragments of cathepsin D, creatine kinase B, pS2, Hsp27, and c-fos genes (86, 87, 102-106, 112, 113). In addition, TCDD and other AhR agonists decrease ER protein in T47D, MCF-7, and ZR-75 breast cancer cells and the structure-dependent downregulation of ER by a series of compounds correlated with their structureAhR binding activities (28, 41, 114-116). The antiestrogenic growth inhibitory activity of TCDD and related compounds can be explained in part by the inhibition of several key pathways involved in hormoneinduced G1 f S phase progression of MCF-7 cells and inhibition of c-fos, which also plays a role in growth of breast cancer cells. Inhibitory AhR-ERR cross-talk is not confined to breast cancer cells since comparable interactions have been reported in ERR-positive ovarian and endometrial cancer cells (86, 87, 117, 118). The role of the AhR in mediating inhibition of E2-induced responses has also been investigated in transgenic MCF-7 cells, which overexpress a constitutively active form of the AhR (119). Their studies showed that overexpression of the constitutively active AhR alone inhibited E2-dependent induction of cathepsin D mRNA levels and cell proliferation; reporter gene activity was also inhibited by constitutively active AhR in cells transfected with chimeric GAL4-ERR fusion protein and a construct containing tandem GAL4 response elements linked to a luciferase reporter gene. The role of the AhR in mediating apparent antiestrogenic responses was also investigated in benzo[a]pyrene resistant MCF-7 (MCF-7BaPr) cells, which express ERR, Arnt, and only minimal levels of the AhR (120). In this cell line, the characteristic inhibitory AhRER cross-talk associated with cell proliferation and gene expression was ablated, thus confirming the requirement of AhR expression for mediating interactions between the two signaling pathways.

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

5.3. Mechanisms of Inhibitory AhR-ERr Cross-Talk Interactions between ligand-induced signaling pathways can be complex and dependent on cell or tissue context and specific genes. For example, estrogens repress prolactin-induced STAT5 activation of the β-casein promoter and this is linked to direct ER-STAT5 interactions, which are dependent on the DNA binding domain of ERR (121). In ERR knockout mice, female specific Cyp2a4 expression in the liver is repressed as compared to wild-type animals and this is related to nuclear levels of STAT5, which represses Cyp2a4 expression (122). Thus, ERR-STAT5 interactions in murine liver are associated with ERR-dependent inhibition of hepatic nuclear localization of STAT5 in female mice. Not surprisingly, there are several possible mechanisms that have 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 critical promoter regions of E2 responsive genes (no. 3), induction of inhibitory factors (no. 2), competition for common nuclear coregulatory proteins (no. 4), and proteasomedependent degradation of ERR (no. 5) (123-126). There is evidence that provides some support for most of these pathways, and these are discussed below.

5.3.1. Enhanced Metabolism of E2 Several studies have demonstrated that TCDD and other AhR agonists induce CYP1A1 and CYP1B1 in breast cancer cells resulting in enhanced metabolism of E2 and depletion of intracellular hormone levels (127129). It has been suggested that this induced oxidative metabolism of E2 results in limiting levels of E2 and decreased hormone responsiveness. Although this may contribute to the observed inhibitory crosstalk between AhR and ER signaling pathways, there is evidence that alterations in E2 metabolism are insufficient for mediating AhR-mediated antiestrogenic responses. For example, TCDD inhibits E2-induced cathepsin D mRNA levels in MCF-7 cells within 30-60 min and this precedes induction of CYP1A1 protein (102). Several SAhRMs that do not induce CYP1A1 in breast cancer cells or in rodent models in vivo maintain an ability to inhibit cell/tumor growth (27, 28). Moreover, in rodents treated with TCDD, circulating E2 levels are not altered (130). These results suggest that CYP1A1-dependent depletion of E2 through oxidative metabolism is not necessary for inhibitory AhR-ERR cross-talk.

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5.3.2. Induction of Inhibitory Factors Recent studies have demonstrated that TCDD inhibits E2-induced gene/reporter gene expression in BG-1 ovarian cancer cells, and TCDD also downregulates ERR protein and mRNA levels in BG-1 cells (118). However, inhibition of E2-induced pS2 mRNA and reporter gene activity (in cells transfected with an ERE construct) by TCDD in this cell line was blocked by cycloheximide, a protein synthesis inhibitor. These results suggest that in BG-1 cells, the inhibitory effects of TCDD may be due to induction of an inhibitory factor; however, the role of this pathway on inhibition of E2-dependent growth and induction of other genes requires further investigation.

5.3.3. Identification of Inhibitory DREs (iDREs) Studies on the rapid inhibition of E2-induced cathepsin D mRNA levels by TCDD suggested that one possible mechanism may involve direct interactions of the nuclear AhR complex with “critical” regions of E2 responsive gene promoters that repress gene activation. The proximal region (-250 to the transcription start site) of the cathepsin D gene promoter contains multiple E2 responsive elements including an upstream GC(N)19ERE halfsite (ERE1/2) motif that binds ERR/Sp1 and downstream GC-rich and E-box motifs that bind ERR/Sp1 and USF1/2 in combination with ERR, respectively (100, 102, 103). Constructs containing the upstream GC(N)19ERE1/2 motif were activated by E2 in reporter assays, and these induced responses were inhibited by TCDD (102). This region of the cathepsin D promoter also contains a GCGTG sequence at -177 to -181 that corresponds to the pentanucleotide core of a DRE required for AhRmediated transactivation. A consensus sequence for a functional DRE required for transactivation is N T/G T GCGTG A/C G/T A/T A/G N (38), and this differed significantly from the TGT GCGTG CCCGA sequence in the cathepsin D promoter, which was not Ah responsive. The requirement for the iDRE within the upstream E2 responsive GC(N)19ERE1/2 (-195 to -165) motif for mediating the ligand (TCDD)-induced inhibition of this response was confirmed in the following series of experiments (102). TCDD inhibited E2-induced transactivation of a construct (pCD) containing the GC(N)19ERE1/2 promoter, whereas this inhibition was not observed using a construct (pCDm) with a DRE mutation. Inhibitory AhR-ERR cross-talk was decreased in cells treated with AhR antagonists or with antisense AhR or Arnt, and cross-talk was not observed in AhR deficient cells. Gel mobility shift assays using the GC(N)19ERE1/2 construct showed that the core DRE was targeted by the AhR complex. This interaction blocked formation of the ERR/ Sp1 complex, resulting in inhibition of E2-induced transactivation. Subsequent studies have identified additional functional iDREs in the pS2, Hsp27, and c-fos gene promoters, with mechanisms of action that are gene promoter specific (Figure 4) (104, 106, 107). For example, the iDRE in the pS2 gene promoter is 125 bp upstream from the nonconsensus ERE at -405 to -393. However, in transactivation studies with constructs containing wild-type and mutant pS2 promoter inserts, it was apparent that hormone responsiveness of pS2 was primarily associated with an AP-1 motif (-518 to -512), which overlaps the

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

iDRE at -527 to -514. Thus, inhibitory AhR-ERR crosstalk on the pS2 gene promoter may be due to competitive interactions of the AhR and AP-1 complexes for binding to the same region of the promoter (106). This type of inhibitory interaction has also been observed for the c-fos gene promoter where both the AhR and the ERR/Sp1 complex bind the -1168 to -1159 distal region of the promoter, which contains overlapping iDRE and GC-box motifs. In contrast, the functional iDRE in the Hsp27 promoter is located at the start site, and the AhR complex may interfere with appropriate assembly of the preinitiation complex (107). A second functional iDRE in the cathepsin D promoter (-130 to -126) inhibits E2-induced transactivation from the E2 responsive adenovirus major late promoter element (MLPE), which contains an overlapping E-box and a nonconsensus ERE (103). DNA footprinting studies show that the AhR complex decreases transcription factor binding to the MLPE and this correlated with decreased transactivation.

5.3.4. Competition for Common Nuclear Coregulatory Proteins Ricci and co-workers (93) first demonstrated that induction of CYP1A1 by TCDD was inhibited by E2 and this was due to AhR and ERR competition for the transcription factor nuclear factor 1. Subsequent studies have shown that the AhR and ERR interact with several common nuclear coregulatory proteins including steroid receptor coactivators and corepressors (131-135). Moreover, subdomains of the AhR transactivation domain inhibit (or squelch) ligand-dependent activation of AhR or ERR (136). NEDD8, an ubiquitin-like protein, enhances AhR-mediated transactivation (137), whereas the activating enzyme of NEDD8 (Uba3) decreases ligand-dependent activation of ERR and ERβ using an ERE promoter in HeLa cells (138). The role of squelching mechanisms in inhibitory AhR-ERR cross-talk will depend on the relative cell context-dependent expression of the multitude of coregulatory proteins that interact with ER and AhR and modulate their transactivation function, and this requires further investigation.

5.3.5. Proteasome-Dependent Degradation of ERr Harris and co-workers (139) initially demonstrated that for a series of AhR agonists, their potency to induce

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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 Western blot analysis 3 h after treatment. The effects of proteasome inhibitors (10 µM PSI or PSII) or protease inhibitors (10 µM EST or chloroquine (chlor)) were also determined (144).

degradation of ERR in MCF-7 cells correlated with their binding affinities for the AhR. It was also reported that inhibitors of transcription (actinomycin D) and protein synthesis (cycloheximide) increase ERR levels in MCF-7 cells. However, after cotreatment with TCDD, there were significant decreases in cycloheximide/actinomycin Dinduced ERR levels. At that time, the significance of these observations was not fully appreciated. More recent studies have reported that E2 induces proteasomedependent degradation of ERR, and some selective ER modulators such as the “pure” antiestrogen ICI 182780 also induce proteasome-dependent degradation of ERR in breast cancer cells (28, 41, 140-143). Rapid degradation of ERR induced by ICI 182780 may play an important role in the antiestrogenic/antitumorigenic activity of these compounds, whereas tamoxifen, which is widely used in the treatment of breast cancer, does not induce degradation of ERR (143, 144). The ability of TCDD to enhance degradation of ERR is depicted in Figure 5. Consistent with other studies, TCDD decreased protein levels of both the AhR and the ERR in breast cancer cells in a manner that was blocked by proteasome but not protease inhibitors. Similar results have been observed in ZR-75 breast cancer cells (144). Treatment with TCDD alone results in decreased immunoreactive AhR and ERR proteins, and degradation of these receptors is blocked by proteasome but not protease inhibitors. These results are consistent with previous studies showing that TCDD induced proteasome-dependent degradation of the AhR in breast and other cell lines and degradation of ERR in breast cancer cells (28, 39, 40-42, 144). Moreover, as previously reported, E2 induces degradation of ERR but not the AhR (41). The most striking effect is observed in cells cotreated with TCDD plus E2 where 80-90% of cellular ERR levels are degraded within 3 h (41, 144), and these levels are similar to those observed after treatment with ICI 182780. These data suggest that the antiestrogenic activity of TCDD (cotreatment with E2) in breast cancer cells may be due to limiting levels of ERR and this has been extensively investigated by Wormke and co-workers (144). Their results indicate the following. (i) TCDD, E2, TCDD plus E2, and ICI 182780 enhance ubiquitination of ERR and subsequent proteasomedependent degradation of ERR. (ii) In cells cotreated with E2 plus TCDD, there was a correlation between the percentage decrease of ERR below levels observed in cells

treated with E2 alone and the corresponding decrease in levels of E2-induced transactivation and fos protein expression. (iii) TCDD (but not E2) induced interactions of the AhR with ERR in a mammalian two hybrid assay. (iv) Effects of DMSO, TCDD, E2, and TCDD plus E2 treatments on ERR levels were independent of kinase inhibitors. (v) TCDD did not affect ERR, AhR, or Arnt mRNA levels. (vi) Moreover, neither actinomycin D nor cycloheximide blocked TCDD-induced degradation of ERR. These results are consistent with a mechanism in which cotreatment of cells with TCDD plus E2 enhances proteasome-dependent degradation of ERR below critical levels required for hormone-induced transactivation. This mechanism for inhibitory AhR-ERR cross-talk represents a novel nongenomic or transcriptionally independent pathway where the degradation of ERR is due to ligand-dependent activation of AhR-ERR interactions and not transactivation through DREs.

6. Therapeutic Aspects of AhR-ERr Cross-Talk Research in this laboratory has identified two groups of SAhRMs, namely, alternate-substituted (1,3,6,8- and 2,4,6,8-) alkyl PCDFs and ring-substituted diindolylmethanes (DIMs) (27, 145, 146). These compounds interact with the AhR and exhibit partial AhR antagonist activities for many of the AhR-mediated toxic responses as well as CYP1A1. Prototypical SAhRMs such as 6methyl-1,3,8-trichlorodibenzofuran and ring-substituted DIMs are themselves relatively nontoxic; however, these compounds inhibit growth of E2-dependent mammary tumors and growth of pancreatic cancer cells (28, 147150). These results are consistent with the interaction of numerous chemoprotective phytochemicals with the AhR and their anticarcinogenic activity. For example, cruciferous vegetables and indole-3-carbinol, a major AhR phytochemical constitutent of these vegetables, inhibit mammary tumor formation in rodent mammary tumor models (151). Moreover, there is epidemiological evidence showing that cruciferous vegetable consumption is associated with prevention of several cancers including breast cancer (151) and this could be due, in part, to indole-3-carbinol and related compounds that activate the AhR (147, 148). Ongoing studies in this laboratory are

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developing new SAhRMs derived from phytochemicals as a new class of drugs for treating ER-dependent and ERnegative breast cancers and other cancers where the liganded AhR induces growth inhibition.

7. Summary Inhibitory AhR-ERR cross-talk has been observed in the rodent uterus, rodent mammary tumors, breast, ovarian, and endometrial cancer cell lines. There is evidence to support several mechanisms for interactions between these two signaling pathways, and these may be gene/response- and cell context-dependent. Recent data obtained in ER-positive breast cancer cells suggest that in cells cotreated with E2 plus TCDD, there is rapid proteasome-dependent degradation of ERR, which then becomes limiting. Similar effects have been observed in the mouse uterus (144). These data do not exclude other mechanisms, which may differentially contribute to inhibitory AhR-ERR cross-talk. These studies provide an example of the complexities of cross-talk between different biochemical/endocrine pathways and also demonstrate that the AhR is a target for development of drugs for treatment of breast and endometrial cancer.

Acknowledgment. The financial assistance of the National Institutes of Health (ES04176 and ES09106) is gratefully acknowledged.

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