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.     

Hypoxia induces proteasome-dependent degradation of estrogen receptor alpha in ZR-75 breast cancer cells

Regulation of estrogen receptor alpha (ERalpha) plays an important role in hormone responsiveness and growth of ER-positive breast cancer cells and tumors. ZR-75 breast cancer cells were grown under conditions of normoxia (21% O(2)) or hypoxia (1% O(2) or cobaltous chloride), and hypoxia significantly increased hypoxia-inducible factor 1alpha protein within 3 h after treatment, whereas ERalpha protein levels were dramatically decreased within 6-12 h, and this response was blocked by the proteasome inhibitor MG-132. In contrast, hypoxia induced only minimal decreases in cellular Sp1 protein and did not affect ERalpha mRNA; however, hypoxic conditions decreased basal and 17beta-estradiol-induced pS2 gene expression (mRNA levels) and estrogen response element-dependent reporter gene activity in ZR-75 cells. Although 17beta-estradiol and hypoxia induce proteasome-dependent degradation of ERalpha, their effects on transactivation are different, and this may have implications for clinical treatment of mammary tumors.     

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.     

Differential expression of estrogen receptor alpha (ERalpha) protein in MCF-7 breast cancer cells chronically exposed to TCDD

Estrogens play a key role in the development and evolution of breast cancer tumors. Estrogen receptor alpha (ERalpha) mediates many of the biological activities of estrogens, and its expression is associated with low invasiveness and good prognosis. Recent epidemiological reports suggest that long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is implicated in the increased incidence of breast cancer in exposed women. TCDD interferes with the expression of some ERalpha-dependent genes and inhibits estradiol (E2)- dependent growth of breast cancer cells in vitro. However, E2-dependent xenographs of MCF-7 human breast cancer cells resumed growth after a 2-week exposure to TCDD. The mechanisms involved in the resumption of cell growth are not completely understood. In this study, we show that short term-exposure (16 days) to 1 nM TCDD results in the suppression of ERalpha protein expression, while chronic exposure for more than 1 year (LTDX cells) results in the partial re-expression of the receptor. Immunocytochemistry studies showed that re-expression of ERalpha in LTDX cells occurred in some of the cells. Analysis by Western immunoblots indicated that four out of five LTDX clones expressed ERalpha at levels comparable to those in unexposed MCF-7 cells. Removal of TCDD treatment for 16 days restored the expression of ERalpha in the ERalpha-negative clonal cells. These results suggest that MCF-7 cells chronically exposed to TCDD contain at least two cell subpopulations that may respond differently to the ERalpha-mediated effects of TCDD.     

Involvement of androgen receptor in 17beta-estradiol-induced cell proliferation in rat uterus

Although it is known that, in the uterus, estrogen receptor alpha (ERalpha) is involved in proliferation and progesterone receptor in differentiation, the role of the two other gonadal-hormone receptors expressed in the uterus, androgen receptor (AR) and estrogen receptor beta (ERbeta), remains undefined. In this study, the involvement of AR in 17beta-estradiol (E(2))-induced cellular proliferation in the immature rat uterus was investigated. AR levels were low in the untreated immature uterus, but 24 h after treatment of rats with E(2), there was an increase in the levels of AR and of two androgen-regulated genes, IGF-I and Crisp (cysteine-rich secretory protein). As expected, E(2) induced proliferation of luminal epithelial cells. These actions of E(2) were all blocked by both the antiestrogen tamoxifen and the antiandrogen flutamide. The E(2)-induced AR was found by immunohistochemistry to be localized exclusively in the stroma, mainly in the myometrium, where it colocalized with ERalpha but not with ERbeta. ERbeta, detected with two different ERbeta-specific antibodies, was expressed in both stromal and epithelial cells either alone or together with ERalpha. Treatment with E(2) caused down-regulation of ERalpha and ERbeta in the epithelium. The data suggest that, in E(2)-induced epithelial cell proliferation, ERalpha induces stromal AR and AR amplifies the ERalpha signal by induction of IGF-I. Because AR is never expressed in cells with ERbeta, it is unlikely that ERbeta signaling is involved in this pathway. These results indicate an important role for AR in proliferation of the uterus, where estrogen and androgen do not represent separate pathways but are sequential steps in one pathway.     

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.     

Aryl hydrocarbon receptor (AhR)-linked inhibition of luteal cell progesterone secretion in 2,3,7,8-tetrachlorodibenzo-p-dioxin treated cells.

In this study, we tested firstly, the hypothesis that decrease of progesterone secretion by luteal cells under the influence of 2,3,7,8-tetrachlorodibezo-p-dioxin (TCDD) is due to influence on specific enzymatic steps in the biosynthetic pathway of steroidogenesis and secondly, involvement of aryl hydrocarbon receptor (AhR) or estradiol receptor (ER) in this process. Luteal cells isolated from mature porcine corpora lutea were cultured with 25-hydroxycholesterole (25-OH) or pregnenolone (P5) as a substrate. Additionally aminoglutethimide, the inhibitor of P540scc or trilostane the inhibitor of 3 beta-HSD was added to basal and stimulated cells. The synergistic action of TCDD with aminoglutethimide in decreasing of progesterone secretion was observed. In pregnenolone treated cells 1.6 fold decrease of progesterone secretion was observed that in both TCDD alone and together with trilostane treated cells. In the second part of experiments to show the involvement of AhR and ER in TCDD action on progesterone secretion alpha- naphtophlavone, the AhR blockers and 4-hydroxytamoxifen (4-OH-TMX), the inhibitor of ER were used. alpha-naphtophlavone, the inhibitory effect of TCDD while 4-OH-TMX had no effect on TCDD-treated cells. These experiments suggest TCDD decreased progesterone secretion by luteal cells by reduction of the activity of mitochondrial enzymes, which converts cholesterol into pregnenolone. Moreover points to AhR dependent but not ER-dependent mechanisms in TCDD action in luteal cells.     

Implication of proteasome in estrogen receptor degradation

In MCF-7 breast cancer cells, estradiol (E2) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E2 and RU 58668; this selective effect was also recorded in cells prelabeled with [3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome-mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover.     

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

The aryl hydrocarbon receptor (AhR) mediates many toxic effects of environmental pollutants. AhR also interacts with multiple growth factor-driven signaling pathways. In the course of examining effects of growth factors on proliferation of human colon cancer cells, we identified cross talk between AhR and the epidermal growth factor receptor (EGFR). In the present work, we explored underlying signal transduction mechanisms and functional consequences of this interaction. With the use of two human colon cancer cell lines, H508 and SNU-C4, we examined the effects of AhR ligands including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell proliferation and activation of EGFR, ERK1/2, and Src kinases. In colon cancer cells, 5-day incubation with TCDD stimulated a twofold dose-dependent increase in cell proliferation that was detectable with 1 nM and maximal with 30 nM TCDD. TCDD induced dose- and time-dependent phosphorylation of EGFR (Tyr845) and ERK1/2; maximal phosphorylation was observed 5 to 10 min after addition of 30 nM TCDD. Both TCDD-induced ERK1/2 phosphorylation and cell proliferation were abolished by AhR small interfering RNA, AhR-specific inhibitor CH223191, Src kinase inhibitor PP2, neutralizing antibodies against matrix metalloproteinase 7, heparin-binding-EGF-like growth factor and EGFR, EGFR inhibitors (AG1478 and PD168393), and MEK1 inhibitor PD98059. Coimmunoprecipitation experiments revealed that AhR forms a protein complex with Src and regulates Src activity by phosphorylating Src (Tyr416) and dephosphorylating Src (Tyr527). These data support novel observations that, in human colon cancer cells, Src-mediated cross talk between aryl hydrocarbon and EGFR results in ERK1/2 activation, thereby stimulating cell proliferation.     

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.