Identification of estrogen-induced genes downregulated by AhR agonists in MCF-7 breast cancer cells using suppression subtractive hybridization

Aryl hydrocarbon receptor (AhR) agonists inhibit 17beta-estradiol (E2) induced growth of MCF-7 human breast cancer cells in vitro and rodent mammary tumor growth in vivo. Genes associated with inhibitory AhR-estrogen receptor (ER) crosstalk were investigated in MCF-7 human breast cancer cells using poly(A)(+)RNA from cells treated with either 1 nM E2 (target) or E2 plus 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (reference) or 25 microM diindolylmethane (DIM) as AhR agonists in MCF-7 cells. Suppression subtractive hybridization (SSH) was subsequently used to identify 33 genes with sequence homology to known human genes that are induced by E2 and inhibited by AhR agonists in MCF-7 cells; two unknown genes were also identified. Many of these genes are involved in cell proliferation and these include cell cycle regulators (cdc28/cdc2-associated protein), nucleotide synthases (thymidylate synthase), early intermediate genes (early growth response alpha, EGRalpha) and other proteins involved in signaling pathways (calmodulin, ATP synthase alpha subunit). Thus SSH has identified a diverse spectrum of new genes that are affected by inhibitory AhR-ER crosstalk and among this group are a subset of genes that may be critical for the in vivo antitumorigenic effects of AhR agonists.     

DNA sequence determinants for binding of transformed Ah receptor to a dioxin-responsive enhancer.

We have utilized gel retardation analysis and DNA mutagenesis to examine the specific interaction of transformed guinea pig hepatic cytosolic TCDD.AhR complex with a dioxin-responsive element (DRE). Sequence alignment of the mouse CYPIA1 upstream DREs has identified a common invariant "core" consensus sequence of TNGCGTG flanked by several variable nucleotides. Competitive gel retardation analysis using a series of DRE oligonucleotides containing single or multiple base substitutions has allowed identification of those nucleotides important for TCDD.AhR.DRE complex formation. A putative TCDD.AhR DNA-binding consensus sequence of GCGTGNNA/TNNNC/G has been derived. The four core nucleotides, CGTG, appear to be critical for TCDD-inducible protein-DNA complex formation since their substitution decreased AhR binding affinity by 100-800-fold; the remaining conserved bases are also important, albeit to a lesser degree (3-5-fold). The 5'-ward thymine, present in the invariant core sequence of all the DREs identified to date, appears not to be involved in DNA binding of the AhR. The results obtained here indicate that although the primary interaction of the TCDD.AhR complex with the DRE occurs with the conserved "core" sequence, nucleotides flanking the core also contribute to the specificity of DRE binding.     

The aryl hydrocarbon receptor mediates degradation of estrogen receptor alpha through activation of proteasomes

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other aryl hydrocarbon receptor (AhR) ligands suppress 17beta-estradiol (E)-induced responses in the rodent uterus and mammary tumors and in human breast cancer cells. Treatment of ZR-75, T47D, and MCF-7 human breast cancer cells with TCDD induces proteasome-dependent degradation of endogenous estrogen receptor alpha (ERalpha). The proteasome inhibitors MG132, PSI, and PSII inhibit the proteasome-dependent effects induced by TCDD, whereas the protease inhibitors EST, calpain inhibitor II, and chloroquine do not affect this response. ERalpha levels in the mouse uterus and breast cancer cells were significantly lower after cotreatment with E plus TCDD than after treatment with E or TCDD alone, and our results indicate that AhR-mediated inhibition of E-induced transactivation is mainly due to limiting levels of ERalpha in cells cotreated with E plus TCDD. TCDD alone or in combination with E increases formation of ubiquitinated forms of ERalpha, and both coimmunoprecipitation and mammalian two-hybrid assays demonstrate that TCDD induces interaction of the AhR with ERalpha in the presence or absence of E. In contrast, E does not induce AhR-ERalpha interactions. Thus, inhibitory AhR-ERalpha cross talk is linked to a novel pathway for degradation of ERalpha in which TCDD initially induces formation of a nuclear AhR complex which coordinately recruits ERalpha and the proteasome complex, resulting in degradation of both receptors.     

Crosstalk between estrogen receptor alpha and the aryl hydrocarbon receptor in breast cancer cells involves unidirectional activation of proteasomes

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxin that activates the aryl hydrocarbon receptor (AhR) and disrupts multiple endocrine signaling pathways. T47D human breast cancer cells express a functional estrogen receptor alpha (ERalpha) and AhR, and treatment of these cells with 17beta-estradiol (E2) or TCDD resulted in a rapid proteasome-dependent decrease in immunoreactive ERalpha and AhR proteins (>60-80%), respectively. E2 did not affect the AhR, whereas TCDD induced proteasome-dependent degradation of both the AhR and ERalpha in T47D and MCF-7 human breast cancer cells, and these responses were specifically blocked by proteasome inhibitors. Thus, TCDD-induced degradation of ERalpha may contribute to the antiestrogenic activity of AhR agonists and this pathway may be involved in AhR-mediated disruption of other endocrine responses.     

The aryl hydrocarbon receptor (AhR) tyrosine 9, a residue that is essential for AhR DNA binding activity, is not a phosphoresidue but augments AhR phosphorylation

We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1-23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR.     

A potential endogenous ligand for the aryl hydrocarbon receptor has potent agonist activity in vitro and in vivo

The aryl hydrocarbon receptor (AhR) is best known as a mediator of toxicity of a diverse family of xenobiotic chemicals such as dioxins and PCBs. However, many naturally occurring compounds also activate AhR. One such compound, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), was isolated from tissue and found to be potent in preliminary tests [J. Song, M. Clagett-Dame, R.E. Peterson, M.E. Hahn, W.M. Westler, R.R. Sicinski, H.F. DeLuca, Proc. Natl. Acad. Sci. USA 99 (2002) 14694-14699]. We have synthesized ITE and [(3)H]ITE and further evaluated its AhR activity in several in vitro and in vivo assays in comparison with the toxic ligand, TCDD. AhR in Hepa1c1c7 cell cytosol bound [(3)H]ITE with high affinity and the AhR.ITE complex formed in vitro bound dioxin response element (DRE) oligonucleotide as potently as TCDD.AhR. In cells treated with ITE, nuclear translocation of AhR, and induction of CYP1A1 protein and of a DRE-dependent luciferase reporter gene were observed. ITE administered to pregnant DRE-LacZ transgenic mice activated fetal AhR, observed as X-gal staining in the same sites as in TCDD-treated mice. However, unlike TCDD, ITE did not induce cleft palate or hydronephrosis. TCDD but not ITE induced thymic atrophy in young adult mice, but both ITE and TCDD caused similar loss of cells and alterations of cell profiles in cultured fetal thymi. These data demonstrate that ITE is a potent AhR agonist in cell extracts, cultured cells, and intact animals, but does not cause the toxicity associated with the more stable xenobiotic ligand, TCDD.     

Aryl hydrocarbon receptor-mediated inhibition of LNCaP prostate cancer cell growth and hormone-induced transactivation

LNCaP prostate cancer cells express the aryl hydrocarbon receptor (AhR), and treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and an Ah-responsive reporter gene. Similar results were obtained with the selective AhR modulator 6-methyl-1,3,8-trichlorodibenzofuran (6-MCDF); however, TCDD but not 6-MCDF induced degradation of the AhR protein. TCDD and 6-MCDF inhibited growth of LNCaP cells, and inhibitory AhR-androgen receptor (AR) crosstalk was investigated in cells transfected with constructs containing the androgen-responsive probasin promoter (-288 to +28) (pPB) or three copies of the -244 to -96 region of this promoter (pARR(3)). Ten nanomolar dihydrotestosterone (DHT) and 17 beta-estradiol (E2) induced transactivation in LNCaP cells transfected with pPB or pARR(3); however, inhibitory AhR-AR crosstalk was observed only with the latter construct. 6-MCDF and TCDD did not inhibit DHT- or E2-induced transactivation in ZR-75 human breast cancer cells, indicating that these interactions were promoter and cell context-dependent. Both E2 and DHT stabilized AR protein in LNCaP cells; however, cotreatment with TCDD or 6-MCDF decreased AR protein levels. These results indicate that inhibitory AhR-AR crosstalk in prostate cancer cells is complex and for some responses, AR protein stability may play a role.     

Interactions of nuclear receptor coactivator/corepressor proteins with the aryl hydrocarbon receptor complex.

MCF-7 human breast cancer cells express the aryl hydrocarbon receptor (AhR), and treatment with AhR agonists such as 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) inhibits estrogen receptor (ER)-mediated responses. This study investigates physical and functional interactions of the AhR complex with a prototypical coactivator (estrogen receptor associating protein 140, ERAP 140) and corepressor (silencing mediator for retinoic acid and thyroid hormone receptor, SMRT) for ER and other members of the nuclear receptor superfamily. The AhR, AhR nuclear translocator (Arnt), and AhR/Arnt proteins were coimmunoprecipitated with 35S-ERAP 140 and 35S-SMRT and, in gel mobility shift assays, AhR/Arnt binding to 32P-dioxin response element (DRE) was enhanced by ERAP-140 and inhibited by SMRT; supershifted bands were not observed. In transactivation assays, coactivator and corepressor proteins enhanced or inhibited AhR-mediated gene expression; however, these responses varied with the amount of coactivator/corepressor expression. These results confirmed functional and physical interactions of AhR/Arnt with ERAP 140 and SMRT in breast cancer cells.     

Cyclophilin-40 has a cellular role in the aryl hydrocarbon receptor signaling

Cyclophilin-40 (CyP40) promotes the formation of the gel shift complex that contains the aryl hydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and dioxin response element (DRE) using baculovirus expressed proteins. Here we reported that CyP40 plays a role in the AhR signaling. When the CyP40 content in MCF-7 cells is reduced, up-regulation of cyp1a1 and cyp1b1 by 3-methylchloranthrene (3MC) is also reduced, suggesting that CyP40 is essential for maximal AhR function. The CyP40 region containing amino acids 186-215, but not the peptidyl-prolyl cis-trans isomerase and tetratricopeptide repeat domains, is essential for forming the AhR/Arnt/DRE complex. CyP40 is found in the cell nucleus after 3MC treatment and appears to promote the DRE binding form of the AhR/Arnt heterodimer.     

Egr-1 is activated by 17beta-estradiol in MCF-7 cells by mitogen-activated protein kinase-dependent phosphorylation of ELK-1

Early growth response-1 (Egr-1) is an immediate-early gene induced by E2 in the rodent uterus and breast cancer cells. E2 induces Egr-1 mRNA and protein levels in MCF-7 human breast cancer cells and reporter gene activity in cells transfected with pEgr-1A, a construct containing the -600 to +12 region of the Egr-1 promoter linked to the firefly luciferase gene. Deletion analysis of the Egr-1 promoter identified a minimal E2-responsive region of the promoter that contained serum response element (SRE)3 (-376 to -350) which bound Elk-1 and serum response factor (SRF) in gel mobility shift assays. Hormone-responsiveness of Egr-1 in MCF-7 cells was specifically inhibited by PD98059, a mitogen-activated protein kinase kinase inhibitor, but not by LY294002, an inhibitor of phosphatidylinositol-3-kinase (PI3-K). These results contrasted with hormone-dependent activation of the SRE in the c-fos promoter, which was inhibited by both PD98059 and LY294002. Differences in activation of the SREs in Egr-1 and c-fos were related to promoter sequence, which defines the affinities of Elk-1 and SRF to their respective binding sites. Thus, Egr-1, like c-fos, is activated through non-genomic (extranuclear) pathways of estrogen action in breast cancer cells.     

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β-estradiol (E₂) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERα, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E₂ metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998. © 1998 Wiley-Liss, Inc.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin elicits aryl hydrocarbon receptor-mediated apoptosis in the avian DT40 pre-B-cell line through activation of caspases 9 and 3

The halogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce immunotoxicity, but relatively little is known regarding its effects on B-lymphocytes, and on avian B-cells in particular. In this study, the avian bursal pre-B-cell line DT40 was exposed to TCDD ranging from 1 to 500 nM for 1 and 6 h. At 100 nM, TCDD caused a significant increase in the number of apoptotic cells, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay, and induced the expression of the chicken cytochrome P450 1A4 (CYP1A4) mRNA, a hallmark of TCDD exposure. TCDD induced transient upregulation of aryl hydrocarbon receptor (AhR) mRNA. At 100 nM, both caspase 3 and caspase 9 were transiently upregulated after 1 h, but returned to normal levels after 6 h of exposure. Challenge with TCDD after AhR blockade with resveratrol, a competitive AhR antagonist, prevented changes in caspases 3 and 9 and in the AhR message itself, suggesting that the effects of TCDD were mediated via the AhR. TCDD did not cause significant changes in the relative gene expression of caspase 8, Bcl-2 and Bcl-xL. We conclude that avian DT40 pre-B-cells exposed to TCDD are susceptible to apoptosis, likely through activation of executioner caspase 3.     

The aryl hydrocarbon receptor activates the retinoic acid receptoralpha through SMRT antagonism

Aryl hydrocarbon receptor (AhR) ligands such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo(a)pyrene interfere with hormonal regulatory pathways, leading to endocrine disruption. Notably, the activated AhR exerts complex effects on estrogens and retinoids at both levels of their metabolism and regulation of cognate genes. Our current investigation of these AhR effects revealed the TCDD-dependent activation of a subset of retinoid-dependent genes (tissue-transglutaminase, IGF binding protein-3, AhR) in MCF-7 breast cancer cells. A collection of in vitro hormone-dependent reporter gene models showed that AhR activation by TCDD stimulated transactivation by several class I heteromeric receptors (retinoic and thyroid hormone receptors) while it antagonized homodimeric nuclear receptors (estrogen and progesterone receptors, ER and PR). TCDD exerted a dose-dependent effect on a retinoic acid-dependent reporter gene expressed in MCF-7 cells. AhR was shown to be involved in a mutual antagonism with RARalpha corepressor SMRT (silencing mediator of retinoid and thyroid receptors). This, and the documented physical interaction between AhR and SMRT suggested that SMRT sequestration by AhR might activate RARalpha in the absence of ligand. Immunocytochemical studies of AhR and SMRT strongly suggested they colocalized in nuclear bodies during this sequestration. Concurring with this interpretation, we observed an interaction in vitro between AhR and the PML protein, the core component of nuclear bodies. This ability of AhR to elicit spurious activation of retinoid receptors expands the scope of AhR ligands influence beyond ER antagonism and specific Dioxin-responsive genes. Unknown AhR endogenous ligands may also elicit gene transactivation by class I receptors, while being inactive on classic xenobiotic-responsive genes.