Estrogen Responsiveness of IBEP-2, A New Human Cell Line Derived from Breast Carcinoma

IBEP-2, an established cell line recently derived from breast carcinoma, was characterized with regard to estrogen receptor (ER) expression, cell mitogenic response to estrogenic stimulation and sensitivity to antiestrogens. In addition, we examined ER modulation following binding of agonist and antagonists, and the ER-mediated induction of progesterone receptor (PgR). ER level in IBEP-2 cells, determined by enzyme-linked immunoassay (EIA), was slightly higher than that measured in MCF-7 cells (662 v.s. 595 fmol/mg protein). When tested on IBEP-2 and MCF-7, various agonists stimulated cell growth with EC50's reflecting different estrogenic potencies (E(2) approximately diethylstilbestrol > E(1) > genistein). IBEP-2 appeared slightly more sensitive than MCF-7, especially to E(2) (at least 4-fold difference between EC50 values). By contrast, IBEP-2 and MCF-7 were equally sensitive to the growth inhibitory effect of antiestrogens 4-hydroxy-tamoxifen (OH-Tam) and ICI 182,780. As revealed by immunoblotting and immunofluorescence using anti-ER alpha antibodies, ER expression in IBEP-2 cells was modulated by E(2) and estrogen antagonists like it has been shown in other ER-positive cell lines, that is, E(2) and ICI 182,780 caused ER downregulation, whereas OH-Tam induced ER accumulation. Ligand-induced downregulation of ER involved degradation in proteasomes, since it was suppressed by the proteasome inhibitor MG-132. Exposure of IBEP-2 cells to E(2) resulted in a marked (at least 25-fold) induction of PgR, documented by EIA, immunoblotting and immunofluorescence. PgR induction due to E(2) was not modified by MG-132. Interestingly, MG-132 alone produced an ER-independent increase of PgR expression. IBEP-2 might prove to be valuable to study ER-mediated induction of PgR.     

Distinct effects of gonadotropin-releasing hormone analogs and 4-hydroxytamoxifen on pS2 mRNA expression with respect to cell proliferation in MCF-7 breast cancer cells.

Gonadotropin-releasing hormone (GnRH) analogs and antiestrogens display direct antiestrogenic effects on the proliferation of hormone-sensitive breast cancer cells. This study aimed to determine whether growth inhibition of 17 beta-estradiol-stimulated MCF-7 breast cancer cells by the agonist D-Trp6 GnRH, the GnRH antagonist BIM 21009C and 4-hydroxy-tamoxifen (OHT) respectively occurred through alterations of the estrogen receptor (ER)-mediated intracellular pathway. The pS2 mRNA expression is primarily dependent on activated ER in MCF-7 cells, and pS2 protein could act as a growth factor. Drug effect on pS2 mRNAs were qualitatively compared to those on the cell cycle. Unlike OHT, GnRH analogs did not suppress the 17 beta-estradiol-induced pS2 mRNA expression whilst the cell cycle was blocked. The pS2 mRNA expression was induced by D-Trp6 GnRH alone without effect on the cell cycle. The outcome of our study is double. Firstly, GnRH analogs are distinct from OHT as regards their effects on the ER-mediated intracellular pathway. Secondly, pS2 mRNA expression is not strictly related to MCF-7 cell proliferation, suggesting that pS2 protein has a function other than that of critical growth regulator.     

In vitro regulation of vascular endothelial growth factor by estrogens and antiestrogens in estrogen-receptor positive breast cancer

BACKGROUND: The effects of antiestrogens on angiogenesis in breast cancer are not fully defined. In this study we investigated the in vitro effects of antiestrogens at different concentrations on vascular endothelial growth factor (VEGF) production in estrogen receptor (ER)-positive breast cancer cells. METHODS: The dose-dependent effects of 17beta-estradiol (E2), 4-hydroxytamoxifen (4OHT), and ICI182,780 were analyzed both with reference to growth rates and VEGF protein production using enzyme-linked immunosorbent assay (ELISA) in MCF-7 cells. RESULTS: E2 stimulated both the growth rates and VEGF production of MCF-7 cells in the same manner. Although 4OHT stimulated the growth rates as an agonistic effect in an estrogen-free media at levels ranging from 1 nM to 1 micro M, it did not stimulate VEGF expression at the same levels except for at 1 micro M. Although 4OHT had a weak agonistic effect on VEGF production at 1 micro M in an estrogen-free media, it significantly inhibited E2-stimulated VEGF production at the same level. A cytotoxic effect was observed with 10 micro M 4OHT that paradoxically caused a prominent increase in VEGF production. ICI182,780 had no significant effects on the growth rates or VEGF production in this cell line. CONCLUSIONS: These results support the hypothesis that tamoxifen could inhibit angiogenesis induced by estrogens in ER-positive breast cancer cells.     

Distinct promoter usage of mdm2 gene in human breast cancer

Human breast cancers, especially estrogen receptor alpha (ER(alpha))-positive ones, often overexpress the oncoprotein MDM2 without mdm2 gene amplification. The mdm2 gene is transcribed into two different mRNAs, namely L-mdm2 and S-mdm2, which are generated from promoters P1 (constitutive) and P2 (regulated by tumor suppressor p53), respectively. To cast light on the mechanisms of MDM2 overexpression, we measured the expression levels of these mdm2 mRNAs using RT-PCR analysis in three human breast cancer cell lines and 15 breast cancer samples obtained from surgery. ER(alpha)-positive MCF-7 cells, which possess wild-type p53, displayed dominant expression of S-mdm2. In contrast, two other cell lines with mutant p53, T47-D (ER(alpha)-positive) and MDA-MB-231 (ER(alpha)-negative), showed almost equivalent expression of L-mdm2 and S-mdm2. Treatment of 17beta-estradiol (E2) significantly enhanced the expression of S-mdm2 but not that of L-mdm2 in MCF-7. Among 6 breast cancer samples regarded as ER(alpha)-positive with wild-type p53, 5 samples showed increased expression of S-mdm2. Expression of S-mdm2 was stimulated in 2 ER(alpha)-positive samples with mutant p53. In contrast, 4 of 5 samples which express mutant p53 without ER(alpha) showed poor expression of S-mdm2. There is a tendency that ER(alpha)-positive breast cancers with wild-type p53 preferably use P2 promoter for the expression of mdm2, possibly through E2-induced accumulation of p53. However, wild-type p53 and ER(alpha) are not necessarily enough for the utilization of S-mdm2. Tumors with mutant p53 also showed expression of S-mdm2 in some cases. These results strongly suggest that other factor(s) is also implicated in the promoter usage of mdm2 gene in human breast cancer tissues.     

Antiestrogens are pro-apoptotic in normal human breast epithelial cells

Estrogens promote cell proliferation in normal and transformed mammary epithelial cells by inducing expression of hormone-responsive genes involved in the cell cycle. The action of antiestrogens is therefore central in regard to their potent inhibitory effects on estrogen-induced cell growth. We used normal human epithelial breast cells from primary cultures (HBE cells) to study hormonal (estrogen and antiestrogen) regulation on 3 key proteins involved in the apoptotic process: Bcl-2, p53 and caspase-3. The mammary adenocarcinoma cell line, MCF-7, was also used to study the molecular regulation of Bcl-2. In both HBE and MCF-7 cells, we found that estradiol (E2) induced an increase in Bcl-2 mRNA levels. This effect was counteracted in the presence of a pure antiestrogen, ICI 182780 (ICI). Alone, ICI did not modify either the Bcl-2 protein or mRNA levels in HBE cells, whereas in MCF-7, a strong downregulation of Bcl-2 mRNA was observed. In parallel, in HBE cells, we observed that E2 caused a decrease in p53 and caspase-3 protein levels, whereas ICI alone increased p53 and caspase-3 protein levels. The ICI effects on p53 and caspase-3 were partially counteracted by E2. Under the same experimental conditions, ICI exerts a potent pro-apoptotic effect, which was not counteracted by E2. In contrast, 4-hydroxytamoxifen was slightly weaker as a pro-apoptotic agent in HBE cells and its effects were reversed by E2. We demonstrate that in HBE cells, ICI reverses the anti-apoptotic action of E2 and alone acts as a highly potent pro-apoptotic molecule. These results provide new insight into treatment for breast cancer prevention.     

Mechanisms of growth arrest by c-myc antisense oligonucleotides in MCF-7 breast cancer cells: implications for the antiproliferative effects of antiestrogens

The proto-oncogene c-myc is up-regulated by estrogen stimulation of hormone-dependent breast cancer cells and is frequently overexpressed in breast and other cancers. Therapeutic interventions that inhibit c-Myc expression have been extensively investigated, including antisense oligonucleotides that have high specificity and potential clinical application. This investigation compared antiestrogen-mediated growth arrest with the molecular events after repression of c-Myc expression in MCF-7 breast cancer cells using an antisense oligonucleotide. We show that the decreased cellular proliferation of MCF-7 cells after direct inhibition of c-Myc is a consequence of inhibition of cyclin D1 expression, subsequent redistribution of p21(WAF1/CIP1) from cyclin D1-Cdk4 to cyclin E-Cdk2 complexes, and a decline in cyclin E-Cdk2 enzymatic activity. Simultaneous repression of p21(WAF1/CIP1) can attenuate the growth-inhibitory effects of reduced c-Myc expression emphasizing the importance of this cyclin-dependent kinase (CDK) inhibitor in growth arrest. These molecular events are similar to the initial changes in cyclin gene expression, CDK complex formation and CDK activity seen after antiestrogen (ICI 182780)-mediated growth inhibition of MCF-7 cells, which suggests that the down-regulation of c-Myc by ICI 182780 is a primary event that culminates in cell cycle arrest.     

Treatment with the pure antiestrogen faslodex (ICI 182780) induces tumor necrosis factor receptor 1 (TNFR1) expression in MCF-7 breast cancer cells

Apoptosis induction by the pure antiestrogen faslodex, also known as ICI 182780 (ICI), is associated with an effective down-regulation of Bcl-2 expression in the human breast cancer cell line MCF-7. Recent observations point out that beside members of the Bcl-2 family also the TNFR1 signaling pathway may be involved in apoptosis induction by antiestrogens. In this report we have analyzed the expression of members of the TNFR1 signaling pathway during the apoptotic process induced by the pure antiestrogen faslodex and by tamoxifen (Tam) in MCF-7 breast cancer cells. Treatment with 10^–7M ICI or 10^–7M Tam leads to a time dependent increase of TNFR1 and TRADD steady-state mRNA levels in MCF-7 cells. In contrast, Bcl-2 expression was strongly decreased following administration of ICI but only weakly after administration of Tam. Western blot analysis and studies by the use of fluorescence microscopy and flow cytometry revealed a time dependent induction of TNFR1 protein and cell surface expression in MCF-7 cells in response to treatment with ICI. To investigate if TNFR1 is functionally involved in apoptosis induction by antiestrogens, we tested whether TNFR1 blocking antibodies can counteract the growth inhibitory action of Tam and ICI. Coincubation of MCF-7 cells with antiestrogens (ICI or Tam) and blocking TNFR1 antibodies lead to an increase in cell viability. Our results provide evidence for a cross talk between the TNFR1 signaling pathway and antiestrogens during the process of apoptosis induction in MCF-7 breast cancer cells. The superiority of the pure antiestrogen ICI to induce apoptosis in MCF-7 cells may result from its capability to modulate the induction of apoptosis via Bcl-2 as well as TNF-associated signal transduction pathways.     

Farnesol, a mevalonate pathway intermediate, stimulates MCF-7 breast cancer cell growth through farnesoid-X-receptor-mediated estrogen receptor activation

Farnesoid X receptor (FXR) is a metabolic nuclear receptor expressed in the liver and traditionally considered as a bile acid sensor. Yet, FXR has been recently demonstrated in other tissues and cells, such as the kidneys, the adrenals, and arterial smooth muscle cells. Immunohistochemical data reported in this study point to the expression of FXR in human breast cancer. In addition, FXR expression was also found by Western blotting and immunofluorescence microscopy in breast-cancer-derived cell lines MCF-7 (estrogen receptor [ER]-positive) and MDA-MB-231 (ER-negative). The FXR activator farnesol, a mevalonate pathway intermediate, exerts a mitogenic effect on MCF-7 cells. The growth stimulation is completely suppressed by antiestrogens. In contrast, MDA-MB-231 cells appear farnesol-insensitive, suggesting an involvement of ER in farnesol mitogenicity. In accordance with this interpretation, farnesol induces in MCF-7 cells a decrease of ER level, consistent with a phenomenon of receptor downregulation. Farnesol also increases progesterone receptor (PgR) expression in MCF-7 cells and stimulates ER-mediated gene transactivation in MVLN cells (MCF-7 cells stably transfected with an ER reporter gene). Of note, both effects of farnesol on ER expression and activity are completely suppressed by antiestrogens. In addition, farnesol-induced PgR is markedly reduced by FXR gene silencing (siRNA), demonstrating the involvement of FXR in the estrogenic effects of farnesol. Finally, coimmunoprecipitation experiments (FXR immunoprecipitation followed by Western blot analysis of ER in the immunoprecipitate) produced definite evidence that FXR interacts with ER. Altogether, these observations reveal the hitherto unreported presence of FXR in breast cancer and show that the latter receptor functionally interacts with ER. The occurrence of such a crosstalk calls for some caution regarding the pharmacological use of FXR agonists. Fabrice Journe and Guy Laurent contributed equally to this work and should be considered as joint first authors.     

Modulation of the folate receptor alpha gene by the estrogen receptor: mechanism and implications in tumor targeting

The folate receptor (FR) type alpha is a promising target for diagnostic imaging agents and therapeutic intervention in major subtypes of gynecological malignancies; however, the receptor levels in the tumors are variable and are generally relatively low in estrogen receptor (ER)-positive tumors. Here we report that the FR-alpha gene promoter is repressed in the presence of 17beta-estradiol and derepressed by the antiestrogens tamoxifen and ICI 182780 in a promoter-specific and ER-alpha-dependent manner in carcinoma cell lines including HeLa (cervical carcinoma), BG-1 (ovarian carcinoma), and IGROV-1 (ovarian carcinoma). The ligand and ER dose response of the FR-alpha promoter and its time course paralleled those of a classical estrogen response element-mediated effect. Antiestrogens produced an ER-dependent increase of up to 36-fold in the expression of the endogenous FR-alpha gene. Deletion analysis and FR-alpha/SV40 promoter chimeras showed that the ER effect is mediated exclusively within the G/C-rich region in the TATA-less P4 promoter of FR-alpha; electrophoretic mobility shift analysis demonstrated interaction of ER at only one of three G/C-rich elements. ER-beta only modestly affected FR-alpha promoter activity but did not diminish the ER-alpha-mediated effects. The ER corepressor, SMRT, enhanced the repression by 17beta-estradiol/ER, but ER coactivators, including SRC family members, did not appreciably impact the ER ligand response. The results suggest that in ER+ tumors, FR-alpha expression is directly and actively suppressed and predict that a brief treatment with antiestrogens will boost FR-alpha expression by passive derepression, enhancing the efficacy of FR-targeted diagnostic and therapeutic applications. They also reveal novel aspects of gene repression by ER.     

Resveratrol acts as an estrogen receptor (ER) agonist in breast cancer cells stably transfected with ER alpha

Resveratrol (Res) is a phytoestrogen found in grapes and present in red wine. Res has been shown to function as an estrogen receptor (ER) agonist, but it remains unclear whether it may also exert antagonist activity. Our aim was to study the effects of Res at both the molecular (TGFalpha gene activation) and the cellular (cell growth) levels in breast cancer cells stably transfected with wild-type (wt) ER(D351) and mutant (mut) ER (D351Y). TGFalpha mRNA induction was used as a specific marker of estradiol (E(2)) responsiveness. Res caused a concentration-dependent (10(-8)-10(-4) M) stimulation of TGFalpha mRNA, indicating that it acts as an estrogen agonist in these cell lines. The pure antiestrogen ICI 182,780 (ICI) blocked Res-induced activation of TGFalpha, consistent with action through an ER-mediated pathway. Further studies that combined treatments with E(2) and Res showed that Res does not act as an antagonist in the presence of various (10(-11)-10(-8) M) concentrations of E(2). To determine whether Res can be classified as a type I or type II estrogen (Jordan et al., Cancer Res 2001;61:6619-23,), we examined Res with the D351G ER in the TGFalpha assay and found that Res belongs to the type I estrogens. Both Res and E(2) had concentration-dependent growth inhibitory effects in cells expressing wtER and D351Y ER. Although the pure antiestrogen ICI blocked the growth inhibitory effects of E(2), it did not block the inhibitory effects of Res, suggesting that the antiproliferative effects of Res also involve ER-independent pathways. Interestingly, Res differentially affected the levels of ER protein in these 2 cell lines: Res down-regulated wtER levels while significantly up-regulating the amount of mutD351Y ER. Co-treatment with ICI resulted in strongly reduced ER levels in both cell lines. Gene array studies revealed Res-induced up-regulation of more than 80 genes, among them a profound activation of p21(CIP1)/WAF1, a gene associated with growth arrest. The p21(CIP1)/WAF1 protein levels measured by Western blotting confirmed Res-induced significant up-regulation of this protein in both cell lines. In summary, Res acts as an ER agonist at low doses but also activates ER-independent pathways, some of which inhibit cell growth.     

The interaction of raloxifene and the active metabolite of the antiestrogen EM-800 (SC 5705) with the human estrogen receptor.

A naturally occurring mutation at amino acid 351 (D351Y) in the human estrogen receptor (ER) can change the pharmacology of antiestrogens. Raloxifene is converted from an antiestrogen to an estrogen, whereas the biological properties of the steroidal pure antiestrogen ICI 182,780 are not affected by the D351Y ER (Levenson, A. S., and Jordan, V. C. Cancer Res., 58: 1872-1875, 1998). We propose an assay system that can be used to classify antiestrogens by determining their ability to up-regulate transforming growth factor alpha (TGF-alpha) mRNA in MDA-MB-231 cells stably transfected with either wild-type or D351Y ER. The novel compound EM-800 and its active metabolite, EM-652, have been reported to be p.o. active nonsteroidal pure antiestrogens. Using the D351Y cell line, EM-652 is able to up-regulate TGF-alpha mRNA in a dose-dependent manner and to a similar extent as estradiol, whereas in the wild-type cell line, it acts as an antiestrogen. In addition, the pure antiestrogen ICI 182,780 is capable of inhibiting EM-652-induced TGF-alpha mRNA expression at the D351Y ER. In MCF-7 cells expressing wild-type ER, it has previously been shown that ICI 182,780 decreases ER only at the protein level. EM-652 treatment does not decrease ER protein levels to a similar extent as ICI 182,780 treatment, and, in addition, EM-652 has no effect on ER mRNA levels. In proliferation assays, EM-652 is as effective as raloxifene in inhibiting cell growth. From these studies, we conclude that the reason the pharmacology of EM-652 is similar to that of raloxifene is because they both fit the ER in the same manner, and their biology depends on an interaction of the antiestrogenic side chain with amino acid 351.     

Antiestrogens modulate MT1 melatonin receptor expression in breast and ovarian cancer cell lines

An interaction between cellular estrogen response and melatonin signaling mediated by G-protein coupled receptors is present in breast cancer cells. In this study, the effect of antiestrogens on basal and melatonin-modulated expression of MT1 melatonin receptor in breast and ovarian cancer cells was examined. For this purpose, the effects of the selective estrogen receptor modulator tamoxifen and pure antiestrogen ICI 182,780 on MT1 expression in estrogen receptor (ER) alpha-positive and -negative breast and ovarian cancer cell lines cultured in medium supplemented with 1 nM 17-beta estradiol were assessed by Western blot analysis. We were able to detect expression of the MT1 receptor in SK-OV-3 and OVCAR-3 cells and report its up-regulation by melatonin in both ovarian cancer cell lines. MT1 expression was observed to be significantly weaker in ERalpha-positive MCF-7 and OVCAR-3 cells than in ERalpha-negative MDA-MB-231 and SK-OV-3 cells. Treatment with the pure antiestrogen ICI 182,780 increased MT1 receptor expression in OVCAR-3 ovarian cancer cells, but decreased MT1 expression in MCF-7 breast cancer cells. No effect of ICI 182,780 on MT1 expression was observed in the ERalpha-negative cell lines SK-OV-3 and MDA-MB-231. After treatment with 4-OH tamoxifen, down-regulation of basal MT1 receptor expression in ERalpha-positive MCF-7 cells and inhibition of melatonin-induced up-regulation of MT1 in OVCAR-3 ovarian cancer cells were observed. In contrast, treatment with 4-OH tamoxifen increased the MT1 receptor level in ERalpha-negative SK-OV-3 ovarian cancer cells. Our findings support the existence of close interaction between estrogen and melatonin signaling. Moreover, our data suggest that melatonin signaling is modulated by antiestrogens in breast and ovarian cancer cells.