Estrogen-responsive Breast Cancer Cell Line Research Articles

Nuclear accumulation of MKL1 in luminal breast cancer cells impairs genomic activity of ERα and is associated with endocrine resistance

Estrogen receptor (ERα) is central in driving the development of hormone-dependent breast cancers. A major challenge in treating these cancers is to understand and overcome endocrine resistance. The Megakaryoblastic Leukemia 1 (MKL1, MRTFA) protein is a master regulator of actin dynamic and cellular motile functions, whose nuclear translocation favors epithelial-mesenchymal transition. We previously demonstrated that nuclear accumulation of MKL1 in estrogen-responsive breast cancer cell lines promotes hormonal escape. In the present study, we confirm through tissue microarray analysis that nuclear immunostaining of MKL1 is associated with endocrine resistance in a cohort of breast cancers and we decipher the underlining mechanisms using cell line models. We show through gene expression microarray analysis that the nuclear accumulation of MKL1 induces dedifferentiation leading to a mixed luminal/basal phenotype and suppresses estrogen-mediated control of gene expression. Chromatin immunoprecipitation of DNA coupled to high-throughput sequencing (ChIP-Seq) shows a profound reprogramming in ERα cistrome associated with a massive loss of ERα binding sites (ERBSs) generally associated with lower ERα-binding levels. Novel ERBSs appear to be associated with EGF and RAS signaling pathways. Collectively, these results highlight a major role of MKL1 in the loss of ERα transcriptional activity observed in certain cases of endocrine resistances, thereby contributing to breast tumor cells malignancy.

Cycloartane and Stigmastane Type Triterpenoids from Pothos scandens Inhibit Estradiol (E2) Induced Proliferations in Breast Cancer Cells

Four cycloartane type triterpenoids and three stigmastane type steroids were isolated from the methanolic extract of stem and root part of Pothos scandens L. (Araceae), a Bangladeshi medicinal plant by high performance liquid chromatographic technique. The compounds were characterized as 24-methylenecycloartanol (1), 24-methylenecycloartenone (2), 24-en-cycloartenone (3), 24-methylenecycloartanyl ferulate (4), stigmast-4-en-3-one (5), stigmast-4,22-diene-3-one (6) and β-sitosterol glucoside (7) through extensive 1D and 2D NMR spectroscopic studies. All the isolates were evaluated for their estrogenic/antiestrogenic activity using the estrogen-responsive breast cancer cell lines, MCF-7 and T47D. The results showed that all the compounds possess mild to strong antiestrogenic activity in both cell lines which was compared to positive control tamoxifen. 24- Methylenecycloartanol (1), which contains a hydroxyl group in its C-3 position, inhibited 90% of estradiol (E2)- induced cell proliferation in MCF-7 and T47D cell lines at a concentration of 0.01 μM only. 24- Methylenecycloartanyl ferulate (4) and stigmast-4,22-diene-3-one (6) showed 90% of estradiol (E2)-induced cell proliferation in T47D cell only at a concentration of 0.01 μM whereas 10.0 μM was required for 24- methylenecycloartanyl ferulate (4) for the same activity in MCF-7 cells. This is the first report of isolation of these compounds from the plant along with their antiestrogenic property.
 Dhaka Univ. J. Pharm. Sci. 18(1): 93-102, 2019 (June)

Estrogen-mediated inactivation of FOXO3a by the G protein-coupled estrogen receptor GPER

BackgroundEstrogen (17β-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERβ as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear.MethodsMCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation.ResultsIn the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions.ConclusionsOur results suggest that non-genomic signaling by GPER contributes, at least in part, to the survival of breast cancer cells, particularly in the presence of ER-targeted therapies involving SERMs and SERDs. Our results further suggest that GPER expression and FOXO3a localization could be utilized as prognostic markers in breast cancer therapy and that GPER antagonists could promote apoptosis in GPER-positive breast cancers, particularly in combination with chemotherapeutic and ER-targeted drugs, by antagonizing estrogen-mediated FOXO3a inactivation.

Abstract P2-03-16: Examining 3-dimensional genome architecture and transcriptional profiles using an optimized HiC-seq protocol in breast cancer cells

Abstract The post-genomic era ushered in a cadre of techniques to study epigenetic mechanisms involved in disease progression and cancer. One understudied mechanism of gene expression/transcriptional control is 3-dimensional architecture of the genome. Several high-throughput technologies have been adapted to examine 3D genome architecture; in particular those derived from chromatin conformation capture (3C) sequencing technologies. Of 3C-derived techniques, HiC-seq, is the most extensive, and examines all chromatin-chromatin contact sites within a genome, elucidating the entire 3D genome architecture. Although, HiC-seq technique was published 2009, it has been under-utilized in research laboratories due to its technical complexity, expense, and requirement for large volumes of starting material (50 million cells/500 μg of DNA). Other barriers to use of HiC-seq include the requirement for specialized equipment and laborious extraction techniques. However, the benefits of HiC-seq are high for cancer researchers because a genome-wide "snap-shot" of the entire 3D genome of the cancer is displayed. This allows for analysis of higher-order epigenetic control of gene expression. Furthermore, understanding the chromatin conformation of the genome provides new insights into the cellular disease state and potentially novel therapeutic approaches to drug-resistant cancers. Our study presents significant technical innovations, permitting HiC-seq technique to be adopted in laboratories without extensive experience in 3C-seq technologies, and with the use of standard equipment and reagents, as well as smaller volumes of starting materials. Our modified technique utilizes up to 100 fold lower amounts of starting material (500,000 cells/5-10 μg of DNA) per reaction. This adaptation of HiC-seq preparation offers the potential for simultaneous extraction of total RNA and Hi-C processed chromatin for sequencing. To optimize our technique we utilized the estrogen-responsive breast cancer cell line, MCF7. This cell line is typically utilized to study potential drug therapies in the context of estrogen-sensitive cancers. We stream lined HiC-seq by optimizing nuclei extraction and allowing for collection of a cytoplasmic fraction for mRNA extraction and sequencing analysis. We also optimized enzymatic digestion, ligation and DNA extraction methods by utilizing newer enzymatic chemistries, commercially available silica column extraction methods and reagents in place of multiple phenol-based DNA extraction methods. Our quality control results suggest that sample processing for reduced amounts of material is similar if not superior to conventional HiC-seq. We leverage this powerful genomic high-throughput technique in order to simultaneously examine the 3D architecture and transcriptional profiles of MCF7 breast cancer cells. Our computational workflow includes further quality controls using HiC-User Pipeline software from the Bahraham Institute and the R Bioconductor package HiTC package. Overall our optimizations in MCF7 breast cancer cells, and post-acquisition work-flow, make HiC-seq an accessible technology. These innovations permit simultaneous analysis of architectural and transcriptional profiles in cancer cells. Citation Format: Jennifer D Davis, Christian Ross, Hu Li, Amy Brock. Examining 3-dimensional genome architecture and transcriptional profiles using an optimized HiC-seq protocol in breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-16.

Activation of the MKL1/actin signaling pathway induces hormonal escape in estrogen-responsive breast cancer cell lines

Estrogen receptor alpha (ERα) is generally considered to be a good prognostic marker because almost 70% of ERα-positive tumors respond to anti-hormone therapies. Unfortunately, during cancer progression, mammary tumors can escape from estrogen control, resulting in resistance to treatment. In this study, we demonstrate that activation of the actin/megakaryoblastic leukemia 1 (MKL1) signaling pathway promotes the hormonal escape of estrogen-sensitive breast cancer cell lines. The actin/MKL1 signaling pathway is silenced in differentiated ERα-positive breast cancer MCF-7 and T47D cell lines and active in ERα-negative HMT-3522 T4-2 and MDA-MB-231 breast cancer cells, which have undergone epithelial-mesenchymal transition. We showed that MKL1 activation in MCF-7 cells, either by modulating actin dynamics or using MKL1 mutants, down-regulates ERα expression and abolishes E2-dependent cell growth. Interestingly, the constitutively active form of MKL1 represses PR and HER2 expression in these cells and increases the expression of HB-EGF, TGFβ, and amphiregulin growth factors in an E2-independent manner. The resulting expression profile (ER-, PR-, HER2-) typically corresponds to the triple-negative breast cancer expression profile.

Tamoxifen-resistant, ER-positive MAC 51 cell line with a high metastatic potential developed from a spontaneous breast cancer mouse model

We developed and characterized an estrogen-responsive and ER-positive murine breast cancer cell line (MAC51) from a spontaneous breast cancer animal model. These cells are overexpressed with K8, K18 and K19 proteins in an immunofluoresence assay. Upregulation of ER alpha was observed in the immunofluoresence assay, real-time PCR analysis and western blot assay. A colocalization experiment in MAC 51 showed cytoplasmic colocalization of K18 and K19 proteins with ER α. Real-time analysis of tumor samples from engrafted animals, MAC 51, metastatic liver and metastatic ovary revealed overexpression of K8 and K18 compared to the respective controls. A hormone responsive experiment in immunodeficient mice showed highly significant decreases in estrogen and tumor volume after 14 days ovariectomization. The tumorogenicity assay showed higher (3 × 10 (5)) and lower (3 × 10(4)) concentrations of MAC 51 cells that developed tumors within 2weeks post-transplantation. Tumor morphology and histology resembled a sarcoma pattern but our spontaneous model appeared in an adenocarcinoma pattern. Metastasis to different organs occurred through hematogenous and lymphatic routes. We assessed the potency of the anticancer effect in MAC 51 cells by treating various anticancer drugs with E2, followed by studying apoptotic gene expression profiles. E2 and E2+ tamoxifen-treated cells showed upregulation of apoptotic genes caspase 1, 3, 9, P53 and Bcl-xl but the tamoxifen- and paclitaxel-treated cells did not upregulate the apoptotic genes. Tamoxifen-resistant, ER-positive and high metastatic potential cell lines from murine origin are very rare. Also, estrogen greatly induced apoptosis in this cell line, hence MAC 51 has a greater application potential to evaluate low doses of estrogen with other targeted therapeutic drugs to treat breast cancer.

Estrogenic Activities of Ten Medicinal Herbs from the Middle East

Traditional medicinal plants have long been recognized as remedies and important sources of treatment for developing countries. In the present study, we report on a detailed study to quantify the presence of five known phytoestrogens in 10 widely used herbs used in the Middle East. Surprisingly some of these plants were almost devoid of tested phytoestrogens, whereas others were very rich in known phytoestrogens. For example, Hibiscus sabdariffa was found to be the richest in quercetin and daidzein, whereas Cyperus conglomeratus had the highest concentrations of kaempferol and genistein. On the other hand, Salvadora persica was almost devoid of the screened phytoestrogens. Ethanolic extracts were further tested for their proliferative activities in cell-culture using estrogen-responsive breast cancer cell lines (MCF-7) and were found to fall into three distinct groups based on their estrogenic activities. The most potent herbal extract (O. vulgare) was further fractionated and the fractions were analyzed again for phytoestrogenic content (using high-performance liquid chromatography) and proliferative activity. Our results indicate that the proliferative activities of some of the extracts and fractions are not completely attributable to the phytoestrogens screened, thus it is likely that some of these plants may have other (perhaps yet unknown) phytoestrogens.

Environmental factors and breast cancer

This review summarizes the results of studies on the effects of environment on breast cancer risk. As known risk factors such as reproductive life, inheritance, and socioeconomic status are estimated to explain only about half of the breast cancer cases, it has been thought that environmental factors could also be related to the risk of this disease. It is known that ionizing radiation is an environmental risk factor increasing the risk of breast cancer. The data of experimental studies show that some organochlorines could be associated with breast cancer risk although the data from epidemiological studies are not consistent due to the difficulties to assess exposure and other risk factors. Recent experimental studies show that cadmium is an environmental factor that mimics the effects of estradiol in estrogen-responsive breast cancer cell lines while solar radiation possibly decreases the risk due to protective effect of vitamin D. The data on the effect of electromagnetic fields are not consistent. Although evidence about the effect of environmental factors on the risk of breast cancer is not convincing, some of these factors together with inheritance, reproductive life, and age at exposure could be associated with an increased risk of the disease.

PRIMA-1 inhibits growth of breast cancer cells by re-activating mutant p53 protein

Mutation of the p53 tumor suppressor gene is a common event in many types of tumors, including breast cancers. Mutant p53 (mtp53) protein is thought to promote tumor cell survival and resistance to chemotherapeutic drugs. Therefore, restoring p53 function by converting existing mtp53 to the wild-type p53 (wtp53) conformation is being pursued as one strategy to promote apoptosis of tumor cells. PRIMA-1 (p53 re-activation and induction of massive apoptosis) is a non-toxic small molecule that converts mtp53 to the active conformation and induces apoptosis in tumor cells. Here we examined whether PRIMA-1 activates mtp53 and induces cell death in vitro and in vivo in estrogen-responsive breast cancer cell lines that express mtp53 (BT-474, HCC-1428, and T47-D). Fluorescent staining with conformation-specific p53 antibodies demonstrated that PRIMA-1 converted mtp53 into the wtp53 conformation. In vitro treatment of tumor cells with PRIMA-1 (0-50 microM) led to a dose-dependent loss of cell viability and induced cell death markers. In contrast, PRIMA-1 had no effect on the viability of MCF-7 cells, normal breast cells, and endothelial cells, all of which express wtp53 protein. PRIMA-1 treatment of mice inhibited the growth of tumors from xenografts of BT-474, HCC-1428, and T47-D cells but did not influence xenografts obtained from MCF-7 cells. Mechanistic studies showed that PRIMA-1 induced the mitochondrial-dependent apoptotic pathway in mtp53-expressing breast cancer cells. Our findings suggest that PRIMA-1 renews the susceptibility of mtp53-expressing breast tumors to apoptosis and should be investigated for use in breast cancer therapy.

Nitrophenols isolated from diesel exhaust particles promote the growth of MCF-7 breast adenocarcinoma cells

Diesel exhaust particles (DEPs) cause many adverse health problems, and reports indicate increased risk of breast cancer in men and women through exposure to gasoline and vehicle exhaust. However, DEPs include vast numbers of compounds, and the specific compound(s) responsible for these actions are not clear. We recently isolated two nitrophenols from DEPs-3-methyl-4-nitrophenol (4-nitro- m-cresol; PNMC) and 4-nitro-3-phenylphenol (PNMPP)—and showed that they had estrogenic and anti-androgenic activities. Here, we tried to clarify the involvement of these two nitrophenols in promoting the growth of the MCF-7 breast cancer cell line. First, comet assay was used to detect the genotoxicity of PNMC and PNMPP in a CHO cell line. At all doses tested, PNMC and PNMPP showed negative genotoxicity, indicating that they had no tumor initiating activity. Next, the estrogen-responsive breast cancer cell line MCF-7 was used to assess cell proliferation. Proliferation of MCF-7 cells was stimulated by PNMC, PNMPP, and estradiol-17β and the anti-estrogens 4-hydroxytamoxifen and ICI 182,780 inhibited the proliferation. To further investigate transcriptional activity through the estrogen receptor, MCF-7 cells were transfected with a receptor gene that allowed expression of luciferase enzyme under the control of the estrogen regulatory element. PNMC and PNMPP induced luciferase activity in a dose-dependent manner at submicromolar concentrations. ICI 182,780 inhibited the luciferase activity induced by PNMC and PNMPP. These results clearly indicate that PNMC and PNMPP do not show genotoxicity but act as tumor promoters in an estrogen receptor α-predominant breast cancer cell line.

Determining the estrogen‐sensitivity of the ER‐alpha mRNA half‐life in human breast cell lines

In two normal estrogen-responsive tissues mammalian endometrium and fish liver, the ER-alpha mRNA half-life increases in response to estrogen treatment. However, Mary Beth Martin and colleagues (1998) found estrogen treatment decreased the ER-alpha mRNA stability in the MCF7 breast cancer cell line. The goal of this research was to test the estrogen-sensitivity of the half-life for the ER-alpha mRNA in other breast cancer cell lines and in a normal breast epithelial cell line. Thus in addition to MCF7 cells, we tested two estrogen-responsive breast cancer cell lines, BT474 and T47D, and the normal estrogen-responsive breast epithelial cell line, HCC1500. 17beta-estradiol treated (6hrs) cells were harvested after exposure to actinomycin D for various times (0.5 to 6 hours) and the total RNA was isolated using a Qiagen RNeasy kit. The isolated RNA was used in quantitative real time RT-PCR in order to determine the half-life of the ER-alpha mRNA. Preliminary data indicates that ER-alpha mRNA half-life decreases similarly in the tested breast cancer cell lines but increases in the normal breast epithelial cell line.

Expression of Insulin-Like Growth Factor Binding Protein-2 by MCF-7 Breast Cancer Cells Is Regulated through the Phosphatidylinositol 3-Kinase/AKT/Mammalian Target of Rapamycin Pathway

IGF binding protein-2 (IGFBP-2) has been implicated in the development and spread of a number of tumor types, and its abrogation in experimental models of cancer is associated with decreased tumor growth. This suggests that targeted inhibition of IGFBP-2 expression in some cancers may have therapeutic benefit. In this study, we investigated signaling pathways involved in extracellular IGFBP-2 expression in an IGF- and estrogen-responsive breast cancer cell line, MCF-7. IGFBP-2 was present at approximately 150 ng per 10(6) cells in serum-free MCF-7-conditioned medium and constituted the predominant IGFBP. Inhibition of the phosphatidylinositol 3-kinase signaling pathway using LY294002, or the downstream signaling intermediate mammalian target of rapamycin using rapamycin, markedly reduced IGFBP-2 in conditioned medium to approximately 25% of untreated levels (P < 0.001); there was no effect of inhibition of p38 MAPK, and an inhibitor of p44/42 MAPK activation, PD98059, caused only a slight reduction in extracellular IGFBP-2. IGFBP-2 levels were increased 25-30% by estradiol, whereas IGF-I (100 ng/ml) increased IGFBP-2 levels 2-fold (P < 0.001) by a type 1 IGF receptor (IGFR1)-dependent mechanism. Estradiol enhanced the effect of IGF-I on IGFBP-2 levels, and this was associated with increased phosphorylation of IGFR1. Basal, IGF-, or estradiol-stimulated IGFBP-2 was abrogated by LY294002 and rapamycin and an inhibitor of IGFR1 tyrosine kinase activity, AG1024. Modulation of intracellular hypoxia-inducible factor-1alpha had no effect on IGFBP-2 expression. These findings indicate that IGFBP-2 is regulated predominantly through the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin pathway, the target of a number of anticancer agents currently in clinical trial and use.

SET-mediated Promoter Hypoacetylation Is a Prerequisite for Coactivation of the Estrogen-responsive pS2 Gene by PRMT1

Induction of transcription requires an ordered recruitment of coregulators and specific combinations of histone modifications at the promoter. Occurrence of histone H4 arginine (Arg) 3 methylation by protein arginine methyltransferase 1 (PRMT1) represents an early promoter event in ER (estrogen receptor)-regulated gene activation. However, its in vivo significance in ER signaling and the prerequisites for PRMT1 recruitment to promoters have not been established yet. We show here that endogenous PRMT1 is a crucial and non-redundant coactivator of ER-mediated pS2 gene induction in MCF7 cells. By investigating promoter requirements for PRMT1 recruitment we find that the patient SE translocation (SET) protein, which was reported to protect histone tails from acetylation, associates with the uninduced pS2 gene promoter and dissociates early upon estrogen treatment. Knockdown of SET or trichostatin A (TSA) treatment causes premature acetylation of H4 and abrogation of H4 Arg3 methylation at the pS2 gene promoter resulting in diminished transcriptional induction. Thus, SET prevents promoter acetylation and is a prerequisite for the initial acetylation-sensitive steps of pS2 gene activation, namely PRMT1 function. Similar to pS2 we identify lactoferrin as a PRMT1-dependent and TSA-sensitive ER target gene. In contrast, we find that the C3 gene, another ER target, is activated in a PRMT1-independent manner and that SET is involved in C3 gene repression. These findings establish the existence of PRMT1-dependent and -independent ER target genes and show that proteins guarding promoter hypoacetylation, like SET, execute a key function in the coactivation process by PRMT1.

Melatonin inhibits both ERα activation and breast cancer cell proliferation induced by a metalloestrogen, cadmium

Cadmium (Cd) is a heavy metal affecting human health both through environmental and occupational exposure. There is evidence that Cd accumulates in several organs and is carcinogenic to humans. In vivo, Cd mimics the effect of estrogens in the uterus and mammary gland. In estrogen-responsive breast cancer cell lines, Cd stimulates proliferation and can also activate the estrogen receptor independent of estradiol. The ability of this metalloestrogen to increase gene expression in MCF7 cells is blocked by anti-estrogens suggesting that the activity of these compounds is mediated by ER alpha. The aims of this work were to test whether melatonin inhibits Cd-induced proliferation in MCF7 cells, and also to study whether melatonin specifically inhibits Cd-induced ER alpha transactivation. We show that melatonin prevents the Cd-induced growth of synchronized MCF7 breast cancer cells. In transient transfection experiments, we prove that both ER alpha- and ER beta-mediated transcription are stimulated by Cd. Melatonin is a specific inhibitor of Cd-induced ER alpha-mediated transcription in both estrogen response elements (ERE)- and AP1-containing promoters, whereas ER beta-mediated transcription is not inhibited by the pineal indole. Moreover, the mutant ER alpha-(K302G, K303G), unable to bind calmodulin, is activated by Cd but becomes insensitive to melatonin treatment. These results proved that melatonin inhibits MCF7 cell growth induced by Cd and abolishes the stimulatory effect of the heavy metal in cells expressing ER alpha at both ERE-luc and AP1-luc sites. We can infer from these experiments that melatonin regulates Cd-induced transcription in both ERE- and AP1 pathways. These results also reinforce the hypothesis of the anti-estrogenic properties of melatonin as a valuable tool in breast cancer therapies.

Expression of Wnt genes and frizzled 1 and 2 receptors in normal breast epithelium and infiltrating breast carcinoma

The Wnt genes encode a family of related, secreted proteins which initiate a signal cascade upon binding to cell surface receptor molecules. The signaling pathway has been shown to be critical for normal growth and development in model organisms and is implicated in the genesis of numerous human cancers. Wnt proteins regulate mammary development in the mouse but their precise role in normal breast development and malignant transformation in humans remains poorly defined. In this study, we have examined the expression of several Wnt ligands by in situ anti-sense RNA hybridization in normal and malignant human breast tissue, as well as in several estrogen-responsive and estrogen-independent human breast cancer cell lines. The specific Wnt genes tested included Wnt1, Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7b and Wnt10b. We have also studied the expression of frizzled receptors 1 and 2 by immunohistochemistry in these tissues. Our results indicate that several of the Wnt ligands, especially Wnt1 and Wnt6, are strongly expressed in both normal and malignant breast tissue and that Wnt7b is down-regulated in breast cancer, compared to normal breast epithelium. The expression of frizzled 1 and 2 receptors was found to be up-regulated in breast cancer. These studies provide additional support to the hypothesis that the Wnt signaling pathway is involved in human breast cancer.

Evaluation of Cell Death Caused by Triterpene Glycosides and Phenolic Substances from Cimicifuga racemosa Extract in Human MCF-7 Breast Cancer Cells

We previously reported that the antiproliferative effect of an isopropanolic-aqueous extract of black cohosh (iCR) on MCF-7 estrogen-responsive breast cancer cell line was due to the induction of apoptosis. Here we address the question to what extent apoptosis induction can be ascribed to one of the two major fractions of iCR, the triterpene glycosides (TTG) or the cinnamic acid esters (CAE). Furthermore, as black cohosh is routinely administered orally, we studied whether its pharmacological effects would withstand simulated liver metabolism. The antiproliferative activity of TTG and CAE as well as of rat liver microsomal S9 fraction-pretreated iCR on MCF-7 cells were investigated by WST-1 assay. The features of cell death induced were tested for apoptosis by flow cytometry (light scatter characteristics, Annexin V binding). Irrespective of S9-pretreatment, 72 h iCR treatment induced a dose-dependent down regulation of cell proliferation with the same IC50 of 55.3 microg/ml dry residue which corresponds to 19.3 microg/ml TTG and 2.7 microg/ml CAE. The degree of apoptotic MCF-7 cells was also comparable. Both, isolated TTG and CAE fractions inhibited cell growth, the IC50 being 59.3 microg/ml and 26.1 microg/ml, respectively. Interestingly, whereas IC50 and apoptosis induction correspond well for the whole extract, TTG and CAE fractions induced apoptosis at concentrations (25 and 5 microg/ml) well below those required for significant growth inhibition. Observation of this study firstly showed that the cell death induced by iCR withstood a metabolic activation system. In addition, TTG and CAE compounds significantly contributed to its apoptotic effect, CAE being the more potent inhibitor of proliferation and apoptosis inducer.

The estrogen-regulated protein, TFF1, stimulates migration of human breast cancer cells.

The human trefoil protein TFF1 is a small cysteine-rich secreted protein that is frequently expressed in breast tumors under the control of estrogen. The function of TFF1 in breast cancer is unknown. To test the hypothesis that it promotes tumor dissemination, we produced recombinant TFF1 and assessed its ability to stimulate the movement of breast cancer cells by using in vitro wounding and migration assays. Recombinant TFF1 stimulated migration at concentrations of TFF1 found in culture medium. Migration of MCF-7 breast cancer cells, which secrete TFF1, was stimulated by lower concentrations of TFF1 than MDA MB231 cells that do not produce TFF1. Dimeric TFF1, linked by a disulfide bond, and monomeric TFF1 are produced by estrogen-responsive breast cancer cell lines. Recombinant TFF1 dimer was eightfold more potent than TFF1 monomer, implying that the interaction of TFF1 with its receptor is facilitated by dimerization. The majority of TFF1-stimulated migration resulted from chemotaxis, but dimeric TFF1 stimulated some chemokinesis. These results show that estrogens can stimulate the motility of breast cancer cells via the induction of TFF1 and suggest that one reason for the efficacy of hormonal therapies is their ability to reduce expression of TFF1 and, hence, the migration of breast tumor cells.

Estrogen suppression of EGFR expression in breast cancer cells: a possible mechanism to modulate growth.

Epidermal growth factor receptor (EGFR) is a transmembrane receptor whose overexpression in breast cancer predicts for poor prognosis and is inversely correlated with expression of estrogen receptor (ER). This study was designed to investigate whether estrogen plays an active role in suppression of EGFR expression in estrogen-responsive breast cancer cell lines expressing low levels of EGFR. Upon withdrawal of estrogen, EGFR mRNA and protein increased 3-6 fold in MCF-7, T47D, and BT474 ER+ breast cancer cells. This was reversible upon addition of estradiol back to the culture media, but only after prolonged treatment. Nuclear run-on assays and studies with the transcription inhibitor actinomycin D demonstrated that regulation is at the transcriptional level. These results indicate that in the presence of estrogen, ER+ breast cancer cells possess active mechanisms to suppress EGFR expression. Up-regulation of EGFR in response to estrogen depletion and growth inhibition could represent an attempt to rescue cell growth by utilizing an alternative pathway. Indeed, we found that estrogen-depleted breast cancer cells are more sensitive to the mitogenic effects of EGF and TGF-alpha, and simultaneous blockade of both estrogen and EGFR signaling pathways induced cell death. J. Cell. Biochem. Suppl. 36: 232-246, 2001.

Activation of estrogen receptor-alpha by the heavy metal cadmium.

Previous studies from this laboratory have shown that the heavy metal cadmium (Cd) mimics the effects of estradiol in estrogen-responsive breast cancer cell lines. To understand the mechanism by which cadmium activates estrogen receptor-alpha (ER-alpha), the ability of cadmium to bind to and activate wild-type and various mutants of ER-alpha was examined. When tested in transient cotransfection assays in COS-1 cells, cadmium concentrations as low as 10(-11) M activated ER-alpha. Scatchard analysis employing either purified human recombinant ER-alpha or extracts from ER-containing MCF-7 cells demonstrated that l09Cd binds to the ER with an equilibrium dissociation constant of approximately 4 to 5 x 10(-10) M. Cadmium also blocks the binding of estradiol to ER-alpha in a noncompetitive manner (K(i) = 2.96 x 10(-10) M), suggesting that the heavy metal interacts with the hormone-binding domain of the receptor. To study the role of the hormone-binding domain in cadmium activation, COS-1 cells were transiently cotransfected with GAL-ER, a chimeric receptor containing the DNA-binding domain of the transcription factor GAL4 and the hormone-binding domain of ER-alpha, and a GAL4-responsive reporter gene. Treatment of the transfected cells with either 10(-6) M cadmium or 10(-9) M estradiol resulted in a 4-fold increase in reporter gene activity. The effect of cadmium on the chimeric receptor was blocked by the antiestrogen, ICI-164,384, suggesting that cadmium activates ER-alpha through an interaction with the hormone-binding domain of the receptor. Transfection and binding assays with ER-alpha mutants identified C381, C447, E523, H524, and D538 as possible interaction sites of cadmium with the hormone-binding domain of ER-alpha.

Selective promoter usage of the human estrogen receptor-alpha gene and its regulation by estrogen.

Three promoters have been identified for the human estrogen receptor-alpha gene. The positions of promoters A and B are known whereas that of the recently identified promoter C is not. Cloning and hybridization experiments demonstrated that promoter C is located more than 21 kb upstream of promoter A. The use of the three promoters was examined in estrogen receptor-positive breast cancer cell lines, cell lines derived from other malignancies, and some normal tissues by RT-PCR and transient transfection. All estrogen-responsive breast cancer cell lines used all three promoters, apart from ZR-75 cells, which did not use promoter B in one of two sublines examined. Cell lines derived from other malignancies and other normal tissues that express lower levels of estrogen receptor-alpha showed more selective promoter usage. This suggests that the level of expression of estrogen receptor-alpha is determined by the number of promoters used, rather than the selective use of specific promoters. We also show that promoter C is used more widely than suggested by others. Analysis of a series of estrogen receptor-positive primary breast tumors showed that all three promoters were used in all the tumors. All three promoters were modulated by estrogen in estrogen-responsive breast cancer cell lines: all three promoters were down-regulated by estrogen in MCF-7 cells in which estrogens reduce receptor expression whereas all promoters used were upregulated in T47D, ZR-75, and EFM-19 cells in which estrogens increase receptor expression. This suggests that it is the repertoire of transcription factors present within a cell rather than the selective use of a specific promoter that determines whether estrogen receptor-alpha expression is increased or decreased by estrogen.