2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and structurally related halogenated aromatics are environmental toxicants that bind and activate the aryl hydrocarbon receptor (AhR) complex. The effects of TCDD and other AhR agonists on mammary gland development and susceptibility to mammary tumor formation in rodent models are dependent on the rodent species/strain, dose and timing of exposure. In contrast, TCDD and selective AhR modulators (SAhRMs) inhibit mammary tumor and cell growth through a complex inhibitory AhR-estrogen receptor alpha (ER alpha) cross talk pathway involving multiple general and gene-specific mechanisms. SAhRMs are highly effective chemotherapeutic agents alone and in combination with tamoxifen as inhibitors of ER-positive mammary tumors and show promise as a new class of agents for treating ER-negative breast cancer.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the relatively non-toxic selective aryl hydrocarbon receptor (AhR) modulator 6-methyl-1,3,8-trichlorodibenzo-furan (MCDF) induced CYP1A1-dependent ethoxyresorufin O-deethylase activity and inhibited proliferation of seven estrogen receptor (ER) negative breast cancer cell lines. MCDF, TCDD and structurally related 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 3,3',4,4',5-pentachlorobiphenyl induced CYP1A1 and inhibited proliferation of BT-474 and MDA-MB-468 cells. In BT474 and MDA-MB-468 cells transfected with a small inhibitory RNA for the AhR, the antiproliferative activity of the chlorinated aromatic compounds was reversed, whereas for MCDF, only partial reversal was observed, suggesting that this compound acts through both AhR-dependent and AhR-independent pathways in these two cell lines MCDF also inhibited tumor growth in athymic nude mice in which MDA-MB-468 cells were injected directly into the mammary fat pad. These results suggest that the AhR is a potential drug target for treatment of ER-negative breast cancer.

Proc Amer Assoc Cancer Res, Volume 45, 2004 3856 A series of 1,1-bis(3’-indolyl)-1-(p-substituted phenyl)methanes activate peroxisome proliferator-activated receptor γ (PPARγ) and inhibit growth of estrogen receptor (ER)-positive breast cancer cells. The most active methylene-substituted diindolylmethane (DIM) compounds contained p-CF3 (DIM-p-pPhCF3), p-tbutyl (DIM-C-pPhtBu) and p-C6H5 (DIM-C-pPhC6H5) substituents and the effects of these compounds on proliferation of ER-negative MDA-MB-231 and MDA-MB-453 breast cancer cells were investigated. The PPARγ-active methylene substituted DIMs inhibited growth of both ER-negative cell lines and the concentrations required for 50% growth inhibition (IC50) were 1-5 μM. MDA-MB-453 cells were treated for 96 hours with DMSO, 1 or 5 uM DIM-C-pPhC6H5. FACS analysis showed that the percentage of cells in G0/G1, G2/M and S phase was 73.8, 7.54 and 18.7% respectively in DMSO (solvent) treated cells whereas these percentages were 86.3, 5.46 and 8.23% respectively after treatment with 5 μM DIM-C-pPhC6H5. No significant changes were observed in cells treated with 1 μM DIM-C-pPhC6H5, however the higher concentration significantly blocked G0/G1 to S phase progression. MDA-MB-231 cells treated for 12 hours with 1-10 μM DIM-C-pPhCF3 were also investigated in cell cycle progression studies. The percentage of cells in G0/G1, G2/M and S phase with DMSO treatment were 57.3, 14.8, 27.9% respectively and 71.5, 13.7, 14.7% with 10 μM DIM-C-pPhCF3 treatment. No substantial differences were seen with 1 or 5 μM treatment of DIM-C-pPhCF3 in MDA-MB-231 cells. The PPARγ-active compounds had minimal effects on expression of cyclin D1 and cyclin-dependent kinase inhibitors p21 and p27 in MDA-MB-453 cells whereas both p21 and p27 protein were induced in MDA-MB-231 cells. Other cell context-dependent mechanisms of growth inhibition are currently being investigated.

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.

Common risk factors for the development of endometrial and breast cancer include early menarche, late menopause, null parity, and later age at first birth, indicating that "lifetime" exposure to estrogens increases the incidence of both tumors. In contrast, smoking protects against the development of endometrial cancer, whereas the role of smoking in breast cancer incidence is equivocal and maybe dependent on the timing and duration of smoking. Constituents of cigarette smoke bind and activate the aryl hydrocarbon receptor (AhR), and research in this laboratory has focused on characterizing the inhibitory AhR-estrogen receptor (ER) alpha crosstalk in endometrial and breast cancer cell lines. Both Ishikawa and ECC1 endometrial cancer cells express the AhR and ERalpha. proteins by Western blot analysis. Moreover, AhR ligands such as benzo[a]pyrene (BaP) and/or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce CYP1Al-dependent activity or reporter gene activity in cells transfected with constructs containing dioxin-responsive elements as promoters. Estrogen responsiveness was also confirmed in these cells, as evidenced by gene/reporter gene assay and the induction of cell proliferation by 17beta-estradiol (E2). Inhibitory AhR-ERalpha crosstalk studies have shown that TCDD and/or BaP inhibit E2-induced growth of endometrial cancer cells and also block hormone-activated reporter gene/gene responses. Although there are several possible mechanisms for the interaction between AhR and ERalpha signaling pathways, the role of AhR-mediated down-regulation of ERalpha will be discussed as one possible mechanism. In addition, selective AhR modulators have been developed for the treatment of breast and endometrial cancer and the potential use of these compounds alone or in combination with tamoxifen will be outlined.