Estrogen In Breast Cancer Cells Research Articles

The rearranged during transfection/papillary thyroid carcinoma tyrosine kinase is an estrogen-dependent gene required for the growth of estrogen receptor positive breast cancer cells

The rearranged during transfection/papillary thyroid carcinoma (RET/PTC) tyrosine kinase is an oncogene implicated in the tumorigenesis of thyroid cancer. Recent studies by us and others have shown that RET/PTC kinase expression is induced by estrogen in breast cancer cells. Due to the critical involvement of estrogen-regulated genes in the pathogenesis of breast cancer, we investigated the expression, regulation, and function of RET/PTC kinase in breast cancer cells. We found that RET/PTC kinase expression correlates with estrogen receptor (ER) expression in breast cancer cells and tumor specimens, and that RET/PTC kinase expression is associated with a poor prognosis in ER-positive breast cancer patients. We found that estrogen rapidly induces RET/PTC kinase expression in an ER-dependent manner in breast cancer cells and that this induction is through a transcriptional regulatory mechanism. Using reporter assays, small interfering RNA (siRNA) assays, and chromatin immunoprecipitation (ChIP) assays, we demonstrated the necessity of crosstalk between ER and the forkhead box A1 (FOXA1) transcription factor in regulating RET/PTC kinase expression. In functional studies, increased expression of RET/PTC kinase induced by estrogen stimulation resulted in elevated phosphorylation of multiple downstream kinase signaling pathways. Conversely, knockdown of RET/PTC expression was associated with the inhibition of these same kinase signaling pathways, and, in fact, decreased the stimulatory effect of estrogen on the proliferation of ER-positive breast cancer cells. These results demonstrate a novel pathway of ER and FOXA1 transcription factor crosstalk in regulating RET/PTC kinase expression, and demonstrate that RET/PTC kinase is a critical regulator for the proliferation of ER-positive breast cancer cells. Taken together, our study suggests that RET/PTC kinase may serve as a novel prognostic biomarker and therapeutic target for prevention and treatment of ER-positive breast cancer.

Differential Estrogen-Regulation of CXCL12 Chemokine Receptors, CXCR4 and CXCR7, Contributes to the Growth Effect of Estrogens in Breast Cancer Cells

CXCR4 and CXCR7 are the two receptors for the chemokine CXCL12, a key mediator of the growth effect of estrogens (E2) in estrogen receptor (ER)-positive breast cancers. In this study we examined E2-regulation of the CXCL12 axis components and their involvement in the growth of breast cancer cells. CXCR4 and CXCR7 were differentially regulated by E2 which enhanced the expression of both CXCL12 and CXCR4 but repressed the expression of CXCR7. Formaldehyde-associated isolation of regulatory elements (FAIRE) revealed that E2-mediated transcriptional regulation of these genes is linked to the control of the compaction state of chromatin at their promoters. This effect could be accomplished via several distal ER-binding sites in the regions surrounding these genes, all of which are located 20–250 kb from the transcription start site. Furthermore, individual down-regulation of CXCL12, CXCR4 or CXCR7 expression as well as the inhibition of their activity significantly decreases the rate of basal cell growth. In contrast, E2-induced cell growth was differentially affected. Unlike CXCR7, the inhibition of the expression or activity of either CXCL12 or CXCR4 significantly blunted the E2-mediated stimulation of cellular growth. Besides, CXCR7 over-expression increased the basal MCF-7 cell growth rate and decreased the growth effect of E2. These findings indicate that E2 regulation of the CXCL12 signaling axis is important for the E2-mediated growth effect of breast cancer cells. These data also provide support for distinct biological functions of CXCR4 and CXCR7 and suggest that targeting CXCR4 and/or CXCR7 would have distinct molecular effects on ER-positive breast tumors.

FOXM1 is a transcriptional target of ERα and has a critical role in breast cancer endocrine sensitivity and resistance

Previous data have shown that FOXM1 expression is elevated in breast cancer tissues and is strongly correlated with the expression pattern of ERα in breast cancer cells. The expression of ERα is a good prognostic factor in breast cancer, as about two-thirds of these ERα-positive patients respond to treatment with antiestrogens. However, approximately one-half of the patients that initially respond to hormonal therapy develop resistance. Since FOXM1 is critical for the progression of the cell cycle, we investigated the regulation of FOXM1 by ERα and its role in endocrine sensitivity and resistance in breast cancer cells. We firstly observed by quantitative RT-PCR a strong and significant positive correlation between ERα and FOXM1 mRNA expression in breast cancer patient samples. We showed that FOXM1 protein and mRNA expression was regulated by ER ligands. We also demonstrated that ectopic conditional expression of ERα, in the presence of estrogens, leads to induction of FOXM1 expression in ER-negative U2OS cells. Using reporter gene assays, we demonstrated that ERα activates FOXM1 transcription through an estrogen-response element site. The direct binding of ERα to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation analysis. Importantly, silencing of FOXM1 by RNA interference abolishes the estrogen-mediated MCF-7 cell proliferation. Conversely, ectopic expression of a constitutively active FOXM1 can abrogate the cell cycle arrest mediated by the antiestrogen tamoxifen. Taken together, the results clearly demonstrate FOXM1 as a key mediator of the mitogenic functions of ERα and estrogen in breast cancer cells. Our findings that antiestrogens repress FOXM1 expression in endocrine-sensitive but not endocrine-resistant breast carcinoma cell lines and that ectopic expression of an active FOXM1 can abrogate the anti-proliferative effects of tamoxifen also suggest that deregulation of FOXM1 may also contribute to antiestrogen insensitivity.

Abstract LB-203: Aromatase expression promotes breast cancer tumorigenesis and bone metastasis

Abstract Aromatase P450 (CYP19) is the rate-limiting enzyme catalyzing the final step in estrogen biosynthesis, and an important target in breast cancer therapy. In post-menopausal women, ovaries cease to make estrogen, yet the incidence of breast cancer increases with age as aromatase present in breast adipose stroma and epithelium catalyzes local estrogen formation. The circulating estrogen level in postmenopausal women is very low and it reflects the sum of local estrogen biosynthesis in various sites. Although breast cancer cells have been shown to express aromatase prompting the hypothesis that intracrine action of estrogen in breast cancer cells may drive tumor progression, there has been little evidence in addressing the hypothesis. We stably transfected the ER positive (ER+) breast cancer ZR-75-1 cells with an human aromatase expression vector and obtained a few clones expressing a moderately higher level of aromatase in comparison with the control vector-transfected cells. One of the clones called ZR-75-1/Aro/Clone10 (Cl10) was selected for further study due to its increased intracellular estradiol (E2) concentration compared to other clones. The Cl10 cells formed tumors after being xenografted into the inguinal mammary fat pad of female athymic mice while the control cells were incapable of forming tumors without estrogen supplementation. Implantation of testosterone pellet further stimulated the growth of the tumor and a cell line, ZR-75-1/Aro/Clone10/TT1 (TT1) was established from the tumor. Both Cl10 and TT1 cells expressed similar levels of aromatase. Interestingly, the aromatase expression increased when the cells were cultured in suspension suggesting circulating ER+ breast cancer cells may up-regulate intracrine estrogen activity for survival after leaving the estrogen-rich adipose stroma at primary site. To investigate the importance of intracrine estrogen in promoting tumorigenesis and metastasis, we implanted the Cl10 and TT1 cells orthotopically and intracardiacally (I.C.) into female athymic mice. Notably, both cell lines generated orthotopic tumors with no estrogen supplementation after three weeks of inoculation. More interestingly, the mice with I.C. inoculated aromatase-expressing cells presented distant metastasis to bones in mandible and legs after two weeks of inoculation detected by whole mouse fluorescence and bioluminescence imaging as both cells were stably transfected with a luciferase and GFP expression vector. We will determine whether growth of the orthotopic tumors and bone metastases can be inhibited with systemic administration of an aromatase inhibitor and whether bone metastasis is osteoclastic or osteoblastic. Our studies show that a moderate expression of aromatase in breast cancer cells is sufficient to generate intracrine estrogenic action and promote tumorigenesis and skeletal metastasis in ER+ breast cancer cells. These aromatase-expressing models should be useful for future investigation on how ER+ breast cancer cells up-regulate aromatase expression in suspension and induces bone remodeling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-203.

FOXM1 is a transcriptional target of ERα and has a critical role in breast cancer endocrine sensitivity and resistance

In this study we investigated the regulation of FOXM1 expression by estrogen receptor α (ERα) and its role in hormonal therapy and endocrine resistance. FOXM1 protein and mRNA expression was regulated by ER-ligands, including estrogen, tamoxifen (OHT), and fulvestrant (ICI182780; ICI) in breast carcinoma cell lines. Depletion of ERα by RNA interference (RNAi) in MCF-7 cells down-regulated FOXM1 expression. Reporter gene assays demonstrated that ERα activates FOXM1 transcription through an estrogen-response element (ERE) located within the proximal promoter region. The direct binding of ERα to the FOXM1 promoter was confirmed in vitro by mobility shift and DNA pull-down assays and in vivo by chromatin immunoprecipitation (ChIP) analysis. Our data also revealed that upon OHT treatment ERα recruits histone deacetylases (HDACs) to the ERE site of the FOXM1 promoter, which is associated with a decrease in histone acetylation and transcription activity. Importantly, silencing of FOXM1 by RNAi abolished estrogen-induced MCF-7 cell proliferation and overcame acquired tamoxifen resistance. Conversely, ectopic expression of FOXM1 abrogated the cell cycle arrest mediated by the anti-estrogen OHT. OHT repressed FOXM1 expression in endocrine sensitive but not resistant breast carcinoma cell lines. Further, qRT-PCR analysis of breast cancer patient samples revealed there was a strong and significant positive correlation between ERα and FOXM1 mRNA expression. Collectively, these results demonstrate FOXM1 to be a key mediator of the mitogenic functions of ERα and estrogen in breast cancer cells, and also suggest that the deregulation of FOXM1 may contribute to anti-estrogen insensitivity.

Estrogen Receptor-Mediated Repression of Target Genes: Mechanism of Action, and Biological Significance.

Abstract Estrogen-bound estrogen receptor (ER) can regulate the transcription of a large number of genes. Although estrogen represses as many genes as it induces, induction has been studied in detail while the repression mechanisms remain largely unknown. In this study we characterized several estrogen-repressed ERα target genes that are directly repressed by estrogen in breast cancer cells. Interestingly, many repressed genes are tumor suppressor genes, playing critical roles in cell cycle inhibition and/or apoptosis. One gene that particularly piqued our interest is Reprimo (RPRM) because of its robust repression in a number of cell lines, and its role as a cell cycle inhibitor. Additionally, it is highly methylated in a variety of cancers including breast cancer and maps to a locus that displays loss of heterozygosity, suggesting that it may be a tumor suppressor gene. As a result, E2-mediated repression of this gene may be a crucial step in the progression of breast cancer. We did show that RPRM decrease (by siRNA) enhances estrogen-mediated S-phase entry, strongly suggesting a biological role of its repression. RPRM was actively and strongly repressed by estrogen and this repression was not due to a quicker turnover of the RPRM mRNA by estrogen. E2-mediated repression of RPRM levels did not require new protein synthesis since it is also repressed in the presence of the translation inhibitor, cycloheximide. We find that estrogen repression of RPRM requires a tripartite interplay between ERα, FoxA1, and HDAC7 as knockdown of these proteins abrogates its repression. Remarkably, silencing of HDAC7 significantly relieved repression of the majority of eleven estrogen-repressed genes tested. Further examination of the interplay between HDAC7 and ERα revealed that HDAC7 can interact with ERα and repress its transcriptional activity in a deacetylase-independent manner. Chromatin immunoprecipitation assays demonstrate that ERα, FoxA1, and HDAC7 are all recruited to a cis-regulatory enhancer (-4.8 kb) in the RPRM gene in the presence of estrogen, which was associated with a release of RNA Pol II from the proximal promoter. Repression requires FoxA1 to be recruited to an epigenetic signature characterized by H3K4 mono- and di-methylation. In summary, we have uncovered a unique requirement for a deacetylase-independent function of HDAC7 in estrogen repression of genes such as RPRM. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4136.

The Impact of Estrogen Receptor-beta on the Actions of Endoxifen in Breast Cancer Cells.

Abstract Background: We have previously demonstrated that endoxifen is the most potent tamoxifen metabolite responsible for eliciting the anti-estrogenic effects of this drug in breast cancer cells expressing ERα (Wu, Hawse et al. Cancer Res 2009;69:1722). These studies support the clinical findings which demonstrate that women with genetically impaired CYP2D6 metabolism have significantly reduced endoxifen levels and a higher risk of breast cancer recurrence. More recently, data has emerged from several laboratories suggesting that expression of ERβ in breast tumors leads to increased benefits from tamoxifen therapy. However, the role of ERβ in mediating endoxifen action has yet to be explored.Methods: Using U2OS osteosarcoma cells, as well as Hs578T and MCF7 breast cancer cells, stably expressing ERβ, we examined the effects of endoxifen on ERβ protein levels by western blotting, ERβ transcriptional activity by real-time RT-PCR, and on ERβ inhibition of breast cancer cell growth through the use of proliferation assays.Results: We have discovered that endoxifen stabilizes ERβ protein, unlike its targeted degradation of ERα, and induces ERα/ERβ heterodimerization in a concentration dependent manner. We have also found that endoxifen is a more potent inhibitor of estrogen induced gene expression in MCF7 cells stably expressing ERβ. Concentrations of endoxifen that mimic patients with impaired CYP2D6 activity (20-40 nM) do not significantly alter E2-induced cell proliferation in MCF7 cells expressing only ERα. However, these concentrations of endoxifen significantly reduce the estrogen induced proliferation rate of MCF7 cells expressing ERβ.Conclusions: Our data demonstrate that endoxifen stabilizes ERβ protein, induces ER heterodimerization and more potently inhibits the actions of estrogen in breast cancer cells expressing ERβ. The presence of ERβ sensitizes cells to the anti-estrogenic effects of endoxifen and growth inhibition occurs at substantially lower endoxifen concentrations (20-40 nM) than in cells that express only ERα (100 nm). Our data suggest that tamoxifen therapy may be beneficial even in patients with reduced CYP2D6 metabolism when ERβ is expressed. These studies also suggest that the increased effectiveness of tamoxifen therapy in patients whose tumors express ERβ may be due to the effects of endoxifen. Finally, these findings underscore the need to further elucidate the role of ERβ in the biology and treatment of breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 405.

A Paradox of Cadmium: A Carcinogen That Impairs the Capability of Human Breast Cancer Cells To Induce Angiogenesis

Cadmium, a highly persistent heavy metal, has been categorized as a human carcinogen. Even though it is known that cadmium acts as estrogens in breast cancer cells, several studies failed to demonstrate whether cadmium is a causal factor for breast cancer. The lack of a strong association between cadmium and breast cancer could be found in the antiangiogenic properties of this heavy metal, which might counteract its carcinogenic properties in the progression of breast cancer. In this study, we exposed estrogen-responsive breast cancer cells to subtoxic levels of cadmium, and we evaluated their angiogenic potential using the chick embryo chorioallantoic membrane assay. Exposure of breast cancer cells to subtoxic levels of cadmium significantly inhibited the angiogenic potential of the breast cancer cell line, suggesting the possibility that cadmium might negatively regulate the production of proangiogenic factors in breast cancer cells. Our results suggest that cadmium might exert a paradoxical effect in breast cancer: on the one hand, it could promote carcinogenesis, and, on the other hand, it could delay the onset of tumors by inhibiting breast cancer cell-induced angiogenesis.

Serum polyamines in pre- and post-operative patients with breast cancer corrected by menopausal status

Polyamines are essential for cell growth and differentiation of cells and its increased production is observed in many cancers. Due to one of key enzymes in polyamine synthesis ornithine decarboxylase is stimulated by estrogens in breast cancer cells, the polyamine levels in serum samples obtained from patients with breast cancer (BCa; pre-menopause: 45.6 ± 0.8 years old, n = 58; post-menopause: 55.2 ± 1.1 years old; n = 11), who were also classified as pre- and post-surgery, and from normal controls (pre-menopause: ages 43.6 ± 0.9 years, n = 45, post-menopause: ages 55.2 ± 1.0 years, n = 18) were determined by liquid chromatography–mass spectrometry. In pre-menopausal patients, the concentrations of 1,3-diaminopropane, N-acetyl putrescine ( N-actPut), N-acetyl spermidine and spermidine (Sp) were higher ( p < 0.05) in pre-surgery breast cancer patients than in normal controls, with N-actPut ( p < 0.0005) and Sp ( p < 0.005) levels markedly increased. All polyamine levels were slightly decreased post-surgery, and were comparable to levels in normal subjects. Based on menopausal-dependent data, the results imply that serum polyamines levels may correlate with estrogen levels in association with estrogen-induced cell growth and ornithine decarboxylase activity.

Increased wound-response revealed by proteomics of estrogen receptor-negative breast cancers

Besides the mitogenic action of estrogens in breast cancer cells there is also evidence that estrogen receptors mediate protective, anti-invasive effects. The aim of this project was to identify downstream ER-dependent proteins differentially expressed in fresh-frozen ER-positive and –negative breast cancer. Tissue sections from eight ER-positive and eight ER-negative cryo-preserved breast cancer specimen were analysed by differential multiplex ProteoTope imaging of 2D-PAGE gels. This method is based on labeling proteins with chemically identical iodine radio-isotopes (I–125, I–131) and provides a direct quantitative and multi color differential proteome display at the low-attomole level. Spots were matched across gels, and their intensities were analysed relative to ER status. The statistically most significant differential protein spots were identified by mass spectrometry using picked co-migrating non-radioactive tumor proteins. In total, proteins from 325 spots were identified by MALDI-TOF PMF with MASCOT scores greater than 70. 72 spots represented 16 proteins that were identified in more than one protein spot. Proteins significantly differentially abundant across all pools at the 0.1% level were consistent with previously published literature. We observed elevated levels of keratins 19, 18 and 8 in ER-positive tumors suggesting a luminal phenotype for ER-positive tumors. Additionally, proteins associated with increased inflammation and wound response were observed in ER-negative tumors. Our data are consistent with previously published literature, suggesting an altered keratin pool associated with increased inflammation and wound response in ER-negative tumors.

Anti-estrogenic effect of unliganded progesterone receptor is estrogen-selective in breast cancer cells MCF-7

We have reported that the ectopic expression of progesterone receptor (PR) in MCF-7 cells disrupted the effects of estradiol-17β (E2), and this anti-estrogenic effect of PR was associated with heightened E2 metabolism and inhibition of estrogen receptor (ER) binding to estrogen response element (ERE). This study determined if the transfected PR was also able to abolish the effect of other estrogens including estrone (E1), estriol (E3) and estradiol-17α. The study revealed that the transfected PR in MCF-7 cells abolished the growth-stimulatory effects of E1 and E2, but had minimal effect on the effects of E3 and estradiol-17α. This observation is associated with a faster metabolic inactivation of E1 and E2 than that of E3 and estradiol-17α. The conditioned media collected after 72 h of E3 treatment from PR-transfected MCF-7 cells retained full estrogenic potential whereas E2-treated conditioned media after 72 h have lost all of the estrogenic activity. On the other hand, PR was able to inhibit ER–ERE binding induced by all the estrogens. This suggests that the estrogen-selective anti-estrogenic effect of transfected PR was due to the difference in rate of metabolic inactivation of different estrogens in MCF-7 cells. It is plausible that inhibition of ER–ERE interaction by PR resulted in faster estrogen-ER dissociation, and the dissociated E1 and E2 were metabolically inactivated but enzymes for E3 and estradiol-17α inactivation were lacking in MCF-7 cells. The findings suggest an interesting mechanism by which the disruption of ER–ERE interaction heightens the inactivation of estrogens in breast cancer cells.

ERα negative breast cancer cells restore response to endocrine therapy by combination treatment with both HDAC inhibitor and DNMT inhibitor

Estrogen receptor alpha (ER alpha) mediates the growth stimulation of estrogen in breast cancer cells and is a useful predictive factor for response to endocrine therapy. It is reported that ER alpha was induced in ER alpha negative breast cancer cells by both DNA methyltransferase-1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (AZA) and histone deacetylase (HDAC) inhibitor trichostatin A (TSA). However, whether the breast cancer cells with induced ER alpha restore response to endocrine therapy requires to be further researched. Reverse transcriptase-polymerase chain reaction (RT-PCR) method was used to explore the change in the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 treated with two chemicals (AZA + TSA). Water-soluble tetrazolium salt-8 (WST-8) method was used to study the proliferation rate of the breast cancer cells. Flow cytometer (FCW) was used to analyze the distribution of cell cycle of these breast cancer cells. Some xenograft models in nude mice were used to further study the results we found in vitro. In this study we observed that the mRNA of ER alpha, PR and pS2 in the ER alpha negative breast cancer cells MDA-MB-435 was re-expressed by treatment with AZA + TSA. The proliferation assay analysis showed AZA + TSA suppressed the proliferation of MDA-MB-435 cells, which were further suppressed by addition of 4-OH Tamoxifen (4-OHT). On the contrary, the proliferation of cells treated with 4-OHT alone showed no difference compared with the vehicle control. Cell cycle analysis showed AZA + TSA treated cells showed S phase arrest, which was partially attenuated by addition of estradiol (E2); furthermore, the effect of E2 on stimulation of cell cycle could be reversed by 4-OHT in the treated cells with induced ER alpha. In vivo experiment xenograft volume of MDA-MB-435 cells treated with AZA + TSA was smaller than that of the control (P < 0.01), and the xenograft of AZA + TSA treated cells was further suppressed by ovariectomy (P < 0.01). Our data indicate that DNMT1 inhibitor AZA and HDAC inhibitor TSA play important roles in restoring sensitivity of the ER alpha negative breast cancer cells to endocrine therapy in vitro and in vivo.

Identification of transcriptional regulatory elements in the human somatostatin receptor sst2 promoter and regions including estrogen response element half-site for estrogen activation

The somatostatin receptor subtype 2 (sst2) mediates inhibition of hormone secretion and cell proliferation, and modulates neurotransmission. Its expression is widespread in various normal tissues and many malignant cells, and is up-regulated by estrogen in breast cancer cells. This study was undertaken to investigate molecular mechanism of transcriptional regulation of the human sst2 gene, for which an additional exon (exon 1) in the 5'-untranslated region was recently found. Transient transfection and mutational analysis showed that the immediate 5'-upstream region containing two Sp1 (-54/-45 and -88/-79) and an ATF/CRE (-69/-62) sites provided full promoter activity. An EMSA together with transfection analysis in Sp1-deficient Drosophila Schneider line (SL2) cells showed that Sp1 acted on the proximal Sp1 site, whereas Sp3, Sp1, and Sp2 did on the distal Sp1 site. Activating transcription factor-2 (ATF)-2, c-Jun, and cyclic AMP response element-binding protein (CREB) interacted with the ATF/CRE site. Transcriptional activation by estrogen occurred through two different regions; one included these proximal elements and the other existed in the upstream region containing estrogen response element (ERE) half-site (-348/-344) and GC-rich sequence (-447/-414). This upstream estrogen responsiveness was observed in a human breast cancer T47D cell, but not in GH(3) or estrogen receptor alpha (ERalpha) -expressing HeLa cells, and was potentiated by overexpression of ERalpha or ERbeta, whose binding to the ERE half-site was verified by EMSAs. A chromatin immunoprecipitation assay suggested that ERalpha was recruited to the ERE half-site after estrogen treatment in T47D cells. The present results should provide a molecular basis for transcriptional regulation in a variety of physiological and pathological contexts of sst2-expressing tissues.

Mechanisms of primary and secondary estrogen target gene regulation in breast cancer cells

Estrogen receptors (ERs), which mediate the proliferative action of estrogens in breast cancer cells, are ligand-dependent transcription factors that regulate expression of their primary target genes through several mechanisms. In addition to direct binding to cognate DNA sequences, ERs can be recruited to DNA through other transcription factors (tethering), or affect gene transcription through modulation of signaling cascades by non-genomic mechanisms of action. To better characterize the mechanisms of gene regulation by estrogens, we have identified more than 700 putative primary and about 1300 putative secondary target genes of estradiol in MCF-7 cells through microarray analysis performed in the presence or absence of the translation inhibitor cycloheximide. Although siRNA-mediated inhibition of ERα expression antagonized the effects of estradiol on up- and down-regulated primary target genes, estrogen response elements (EREs) were enriched only in the vicinity of up-regulated genes. Binding sites for several other transcription factors, including proteins known to tether ERα, were enriched in up- and/or down-regulated primary targets. Secondary estrogen targets were particularly enriched in sites for E2F family members, several of which were transcriptionally regulated by estradiol, consistent with a major role of these factors in mediating the effects of estrogens on gene expression and cellular growth.

High tumoral levels of Kiss1 and G-protein-coupled receptor 54 expression are correlated with poor prognosis of estrogen receptor-positive breast tumors

KiSS1 is a putative metastasis suppressor gene in melanoma and breast cancer-encoding kisspeptins, which are also described as neuroendocrine regulators of the gonadotropic axis. Negative as well as positive regulation of KiSS1 gene expression by estradiol (E(2)) has been reported in the hypothalamus. Estrogen receptor alpha (ERalpha level is recognized as a marker of breast cancer, raising the question of whether expression of KiSS1 and its G-protein-coupled receptor (GPR54) is down- or upregulated by estrogens in breast cancer cells. KiSS1 was found to be expressed in MDA-MB-231, MCF7, and T47D cell lines, but not in ZR75-1, L56Br, and MDA-MB-435 cells. KiSS1 mRNA levels decreased significantly in ERalpha-negative MDA-MB-231 cells expressing recombinant ERalpha. In contrast, tamoxifen (TAM) treatment of ERalpha-positive MCF7 and T47D cells increased KiSS1 and GPR54 levels. The clinical relevance of this negative regulation of KiSS1 and GPR54 by E(2) was then studied in postmenopausal breast cancers. KiSS1 mRNA increased with the grade of the breast tumors. ERalpha-positive invasive primary tumors expressed sevenfold lower KiSS1 levels than ERalpha-negative tumors. Among ERalpha-positive breast tumors from postmenopausal women treated with TAM, high KiSS1 combined with high GPR54 mRNA tumoral levels was unexpectedly associated with shorter relapse-free survival (RFS) relative to tumors expressing low tumoral mRNA levels of both genes. The contradictory observation of putative metastasis inhibitor role of kisspeptins and RFS to TAM treatment suggests that evaluation of KiSS1 and its receptor tumoral mRNA levels could be new interesting markers of the tumoral resistance to anti-estrogen treatment.

Influence of estrogens and antiestrogens on the expression of selected hormone-responsive genes

Estrogen exerts a primary regulatory role on a wide variety of physiological processes in different tissues and organs. Agonistic ad antagonistic compounds are widely used in human health and, therefore, a deep understanding of their mechanisms of action at the molecular level is mandatory. The effect of 17β-estradiol and three antiestrogenic drugs, comprising two selective estrogen receptor modulator (SERM, 4-OH-tamoxifen, Raloxifene) and the pure antiestrogen ICI 182,780, on genome-wide gene expression levels was evaluated in breast carcinoma cell lines by DNA microarray analysis. Different clusters of genes, showing specific coregulation patterns, were found. First, several groups of genes displaying temporal-specific up- or down-regulation were characterized. Second, clusters of genes responding to different antiestrogenic drugs in either antagonstic or agonistic fashion, were found. Genes responding specifically to antiestrogens, but not to estrogen, were also identified. In addition, each individual compound exhibited a very specific gene regulation. Bioinformatic analysis was applied to the regulatory sequences of different groups of genes and confirmed that specific pathways and secondary responses are activated at each temporal point and in response to different compounds. Our results underline the complexity of genomic responses to estrogen in breast cancer cells and strongly suggest that the molecular characterization of estrogen agonists and antagonists used in human therapy should be carefully studied.

Estrogen Receptor β Inhibits Angiogenesis and Growth of T47D Breast Cancer Xenografts

Estrogens, which are stimulators of growth of both the normal breast and malignant breast, mediate their effects through two estrogen receptors (ER), namely ERalpha and ERbeta. ERalpha mediates the proliferative effect of estrogen in breast cancer cells, whereas ERbeta seems to be antiproliferative. We engineered ERalpha-positive T47D breast cancer cells to express ERbeta in a Tet-Off-regulated manner. These cells were then injected orthotopically into severe combined immunodeficient mice, and the growth of the resulting tumors was compared with tumors resulting from injecting the parental T47D cells that do not express ERbeta. The presence of ERbeta resulted in a reduction in tumor growth. Comparison of the ERbeta-expressing and non-ERbeta-expressing tumors revealed that the expression of ERbeta caused a reduction in the number of intratumoral blood vessels and a decrease in expression of the proangiogenic factors vascular endothelial growth factor (VEGF) and platelet-derived growth factor beta (PDGFbeta). In cell culture, with the Tet-Off-regulated ERbeta-expressing cells, expression of ERbeta decreased expression of VEGF and PDGFbeta mRNA under normoxic as well as hypoxic conditions and reduced secreted VEGF and PDGFbeta proteins in cell culture medium. Transient transfection assays with 1,026 bp VEGF and 1,006 bp PDGFbeta promoter constructs revealed a repressive effect of ERbeta at the promoter level of these genes. Taken together, these data show that introduction of ERbeta into malignant cells inhibits their growth and prevents tumor expansion by inhibiting angiogenesis.

Cadmium induces mitogenic signaling in breast cancer cell by an ERα-dependent mechanism

Breast cancer (BC) is linked to estrogen exposure. Estradiol (E 2) stimulates BC cells proliferation by binding the estrogen receptor (ER). Hormone-related cancers have been linked to estrogenic environmental contaminants. Cadmium (Cd) a toxic pollutant, acts as estrogens in BC cells. Purpose of our study was to evaluate whether Cd regulates MCF-7 cell proliferation by activating ERK1/2, Akt and PDGFRα kinases. Cd increased cell proliferation and the ER-antagonist ICI 182,780 blunted it. To characterize an ER-dependent mechanism, ERα/β expression was evaluated. Cd decreased ERα expression, but not ERβ. Cd also increased ERK1/2, Akt and PDGFRα phosphorylation while ICI blocked it. Since stimulation of phosphorylation was slower than expected, c- fos and c- jun proto-oncogenes, and PDGFA were analyzed. Cd rapidly increased c- jun, c- fos and PDGFA expression. Cells were also co-incubated with the Cd and specific kinases inhibitors, which blocked the Cd-stimulated proliferation. In conclusion, our results indicate that Cd increases BC cell proliferation in vitro by stimulating Akt, ERK1/2 and PDGFRα kinases activity likely by activating c- fos, c- jun and PDGFA by an ERα-dependent mechanism.

Estrogen receptor β represses breast tumor growth and angiogenesis in vivo

10101 Background: Estrogens are well known stimulators of breast cancer cell growth in vitro as well as in vivo. Two different estrogen receptors exist, namely estrogen receptor (ER) α and β. ERα mediates the proliferative effect of estrogen in breast cancer cells and we have earlier shown that ERβ inhibits cell-cycle progression in vitro. Estrogens are well known stimulators of in vivo breast cancer cell growth as well as angiogenesis, and the effect is mediated through ERα. The function of ERβ in this context is not well understood. Methods: We have used ERα-positive T47D breast cancer cells stably transfected with a Tet/Off regulated ERβ expression vector system. The ERβ-inducible tumor cells are studied in vitro as well as in vivo. Results: By transplanting ERβ-inducible breast cancer cells into SCID-mice, we show that ERβ inhibits tumor growth and reduces the volume of established tumors. Furthermore, we show by immunohistochemistry, that the number of blood microvessels in the tumor periphery is decreased by ERβ expression, counteracting the well-known pro-angiogenic effect of ERα. By Western blot analysis on tumor extracts, we show that the concentration of the important pro-angiogenic growth factors VEGF and bFGF, normally expressed by breast tumor cells, is decreased in the ERβ-expressing tumors compared to the normal tumors. To exclude that the observed anti-angiogenic effect is just a result of reduced tumor growth, we incubated Tet/Off regulated ERβ expressing cells in vitro, during non-hypoxic conditions. We found that the expression of ERβ leads to decreased expression of VEGF and PDGFβ at the mRNA and protein-levels. In transient transfection assays, we found estrogen-ERα mediated up regulation of VEGF, PDGFβ and bFGF-promoter activities in T47D cells, and these activities were all suppressed following co-transfection with an ERβ-expression vector. Conclusions: We conclude that ERβ inhibits growth factor expression at transcriptional level in breast cancer cells; taken together, our data indicates that ERβ inhibits growth and angiogenesis of tumors formed by T47D breast cancer cells. This makes ERβ an interesting therapeutic target in breast cancer and perhaps treatment with the newly designed ERβ-selective ligands might work as a new anti-proliferative and anti-angiogenic therapy. No significant financial relationships to disclose.

Comparative gene expression profiling reveals partially overlapping but distinct genomic actions of different antiestrogens in human breast cancer cells

Antiestrogens used for breast cancer (BC) treatment differ among each other for the ability to affect estrogen receptor (ER) activity and thereby inhibit hormone-responsive cell functions and viability. We used high-density cDNA microarrays for a comprehensive definition of the gene pathways affected by 17beta-estradiol (E2), ICI 182,780 (ICI), 4OH-tamoxifen (Tamoxifen), and raloxifene (RAL) in ER-positive ZR-75.1 cells, a suitable model to investigate estrogen and antiestrogen actions in hormone-responsive BC. The expression of 601 genes was significantly affected by E2 in these cells; in silico analysis reveals that 86 among them include one or more potential ER binding site within or near the promoter and that the binding site signatures for E2F-1, NF-Y, and NRF-1 transcription factors are significantly enriched in the promoters of genes induced by estrogen treatment, while those for CAC-binding protein and LF-A1 in those repressed by the hormone, pointing to novel transcriptional effectors of secondary responses to estrogen in BC cells. Interestingly, expression of 176 E2-regulated mRNAs was unaffected by any of the antiestrogens tested, despite the fact that under the same conditions the transcriptional and cell cycle stimulatory activities of ER were inhibited. On the other hand, of 373 antiestrogen-responsive genes identified here, 52 were unresponsive to estrogen and 25% responded specifically to only one of the compounds tested, revealing non-overlapping and clearly distinguishable effects of the different antiestrogens in BC cells. As some of these differences reflect specificities of the mechanism of action of the antiestrogens tested, we propose to exploit this gene set for characterization of novel hormonal antagonists and selective estrogen receptor modulators (SERMs) and as a tool for testing new associations of antiestrogens, more effective against BC.