Dale C. Leitman

Estrogen Receptor β Inhibits Human Breast Cancer Cell Proliferation and Tumor Formation by Causing a G2 Cell Cycle Arrest

Studies indicate that estrogen receptor (ER) α mediates breast cancer-promoting effects of estrogens. The role of ERβ in breast cancer is unknown. Elucidating the role of ERβ in the pathogenesis of breast cancer is important because many human breast tumors express both ERα and ERβ. We show that adenovirus-mediated expression of ERβ changes the phenotype of ERα-positive MCF-7 cells. Estradiol increases cell proliferation and causes tumor formation of MCF-7 cells expressing only ERα. In contrast, introducing ERβ into MCF-7 cells causes an inhibition of proliferation in vitro and prevents tumor formation in a mouse xenograft model in response to estradiol. ERβ inhibits proliferation by repressing c-myc, cyclin D1, and cyclin A gene transcription, and increasing the expression of p21Cip1 and p27Kip1, which leads to a G2 cell cycle arrest. These results demonstrate that ERα and ERβ produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ERβ-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.

Estrogen receptor beta-selective transcriptional activity and recruitment of coregulators by phytoestrogens.

Estrogens used in hormone replacement therapy regimens may increase the risk of developing breast cancer. Paradoxically, high consumption of plant-derived phytoestrogens, particularly soybean isoflavones, is associated with a low incidence of breast cancer. To explore the molecular basis for these potential different clinical outcomes, we investigated whether soybean isoflavones elicit distinct transcriptional actions from estrogens. Our results demonstrate that the estrogen 17β-estradiol effectively triggers the transcriptional activation and repression pathways with both estrogen receptors (ERs) ERα and ERβ. In contrast, soybean isoflavones (genistein, daidzein, and biochanin A) are ERβ-selective agonists of transcriptional repression and activation at physiological levels. The molecular mechanism for ERβ selectivity by isoflavones involves their capacity to create an activation function-2 surface of ERβ that has a greater affinity for coregulators than ERα. Phytoestrogens may act as natural selective estrogen receptor modulators that elicit distinct clinical effects from estrogens used for hormone replacement by selectively recruiting coregulatory proteins to ERβ that trigger transcriptional pathways.

Alternate surfaces of transcriptional coregulator GRIP1 function in different glucocorticoid receptor activation and repression contexts

Members of the mammalian p160 family, such as GRIP1, are known as glucocorticoid receptor (GR) coactivators; at certain glucocorticoid response elements (GREs), however, GRIP1 acts as a GR corepressor. We characterized functional interactions of GR and GRIP1 in a repression complex where GR tethers to DNA-bound activator protein-1 (AP-1), as at the human collagenase-3 gene, and tested whether the identified interactions were similar or different at other response elements. At the AP-1 tethering GRE, we mapped the GRIP1 corepressor activity to a domain distinct from the two known GRIP1 activation domains; it exhibited intrinsic GR-independent repression potential when recruited to DNA via Gal4 DNA-binding domain. Interestingly, neither the domain nor the activity was detected in the other two p160 family members, SRC1 and RAC3. The same GRIP1 corepression domain was required for GR-mediated repression at the nuclear factor-κB (NF-κB) tethering GRE of the human IL-8 gene. In contrast, at the osteocalcin gene GRE, where GR represses transcription by binding to a DNA site overlapping the TATA box, both GRIP1 and SRC1 corepressed, and the GRIP1-specific repression domain was dispensable. Thus, in a single cell type, GR and GRIP1 conferred one mode of activation and two modes of repression by selectively engaging distinct surfaces of GRIP1 in a response element-specific manner.

Selective Estrogen Receptor-β Agonists Repress Transcription of Proinflammatory Genes

In addition to their role in the development and function of the reproductive system, estrogens have significant anti-inflammatory properties. Although both estrogen receptors (ERs) can mediate anti-inflammatory actions, ERβ is a more desirable therapeutic target because ERα mediates the proliferative effects of estrogens on the mammary gland and uterus. In fact, selective ERβ agonists have beneficial effects in preclinical models involving inflammation without causing growth-promoting effects on the uterus or mammary gland. However, their mechanism of action is unclear. The purpose of this study was to use microarray analysis to determine whether ERβ-selective compounds produce their anti-inflammatory effects by repressing transcription of proinflammatory genes. We identified 49 genes that were activated by TNF-α in human osteosarcoma U2OS cells expressing ERβ. Estradiol treatment significantly reduced the activation by TNF-α on 18 genes via ERβ or ERα. Most repressed genes were inflammatory genes, such as TNF-α, IL-6, and CSF2. Three ERβ-selective compounds, ERB-041, WAY-202196, and WAY-214156, repressed the expression of these and other inflammatory genes. ERB-041 was the most ERβ-selective compound, whereas WAY-202196 and WAY-214156 were the most potent. The ERβ-selective compounds repressed inflammatory genes by recruiting the coactivator, SRC-2. ERB-041 also repressed cytokine genes in PBMCs, demonstrating that ERβ-selective estrogens have anti-inflammatory properties in immune cells. Our study suggests that the anti-inflammatory effects of ERB-041 and other ERβ-selective estrogens in animal models are due to transcriptional repression of proinflammatory genes. These compounds might represent a new class of drugs to treat inflammatory disorders.

Liquiritigenin is a plant-derived highly selective estrogen receptor β agonist

After the Womens Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

Estrogen Receptor β Binds to and Regulates Three Distinct Classes of Target Genes

Estrogen receptor β (ERβ) has potent antiproliferative and anti-inflammatory properties, suggesting that ERβ-selective agonists might be a new class of therapeutic and chemopreventative agents. To understand how ERβ regulates genes, we identified genes regulated by the unliganded and liganded forms of ERα and ERβ in U2OS cells. Microarray data demonstrated that virtually no gene regulation occurred with unliganded ERα, whereas many genes were regulated by estradiol (E2). These results demonstrated that ERα requires a ligand to regulate a single class of genes. In contrast, ERβ regulated three classes of genes. Class I genes were regulated primarily by unliganded ERβ. Class II genes were regulated only with E2, whereas class III genes were regulated by both unliganded ERβ and E2. There were 453 class I genes, 258 class II genes, and 83 class III genes. To explore the mechanism whereby ERβ regulates different classes of genes, chromatin immunoprecipitation-sequencing was performed to identify ERβ binding sites and adjacent transcription factor motifs in regulated genes. AP1 binding sites were more enriched in class I genes, whereas ERE, NFκB1, and SP1 sites were more enriched in class II genes. ERβ bound to all three classes of genes, demonstrating that ERβ binding is not responsible for differential regulation of genes by unliganded and liganded ERβ. The coactivator NCOA2 was differentially recruited to several target genes. Our findings indicate that the unliganded and liganded forms of ERβ regulate three classes of genes by interacting with different transcription factors and coactivators.

Regulation of specific target genes and biological responses by estrogen receptor subtype agonists

Estrogenic effects are mediated through two estrogen receptor (ER) subtypes, ERα and ERβ. Estrogens are the most commonly prescribed drugs to treat menopausal conditions, but by non-selectively triggering both ERα and ERβ pathways in different tissues they can cause serious adverse effects. The different sizes of the binding pockets and sequences of their activation function domains indicate that ERα and ERβ should have different specificities for ligands and biological responses that can be exploited for designing safer and more selective estrogens. ERα and ERβ regulate different genes by binding to different regulatory elements and recruiting different transcription and chromatin remodeling factors that are expressed in a cell-specific manner. ERα-selective and ERβ-selective agonists have been identified that demonstrate that the two ERs produce distinct biological effects. ERα and ERβ agonists are a promising new approach for treating specific conditions associated with menopause.

Drug and Cell Type-Specific Regulation of Genes with Different Classes of Estrogen Receptor β-Selective Agonists

Estrogens produce biological effects by interacting with two estrogen receptors, ERα and ERβ. Drugs that selectively target ERα or ERβ might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERβ-selective compounds have been identified. One class of ERβ-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERβ much greater than ERα. A second class of ERβ-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERβ. Diarylpropionitrile represents a third class of ERβ-selective compounds because its selectivity is due to a combination of greater binding to ERβ and transcriptional activity. However, it is unclear if these three classes of ERβ-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERβ selectivity and pattern of gene expression of these three classes of ERβ-selective compounds compared to estradiol (E2), which is a non-selective ER agonist. U2OS cells stably transfected with ERα or ERβ were treated with E2 or the ERβ-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERβ-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERβ, which is consistent with the finding that most genes regulated by the ERβ-selective compounds were similar to each other and E2. However, there were some classes of genes differentially regulated by the ERβ agonists and E2. Two ERβ-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERβ. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERβ-selective agonists and E2, there were some genes regulated that were distinct from each other and E2, suggesting that different ERβ-selective agonists might produce distinct biological and clinical effects.

Selective estrogen receptor modulators in reproductive medicine and biology.

Estrogen replacement therapy has significant potential benefits for postmenopausal women, such as improvement of menopausal symptoms and protection from osteoporosis, but it may also increase a womans risk of breast cancer. Also, some women do not take hormone replacement therapy because of such undesirable side effects as breast tenderness and uterine bleeding. Therefore, there is much interest in the development of compounds that provide the benefits of estrogen replacement therapy without the risks and side effects. The selective estrogen receptor modulators make up one class of compounds with both estrogen agonist and antagonist activity. This review discusses the clinical indications, risks, benefits, and mechanisms of action of selective estrogen receptor modulators and related compounds.

Cross Talk between Glucocorticoid and Estrogen Receptors Occurs at a Subset of Proinflammatory Genes

Glucocorticoids exert potent anti-inflammatory effects by repressing proinflammatory genes. We previously demonstrated that estrogens repress numerous proinflammatory genes in U2OS cells. The objective of this study was to determine if cross talk occurs between the glucocorticoid receptor (GR) and estrogen receptor (ER)α. The effects of dexamethasone (Dex) and estradiol on 23 proinflammatory genes were examined in human U2OS cells stably transfected with ERα or GR. Three classes of genes were regulated by ERα and/or GR. Thirteen genes were repressed by both estradiol and Dex (ER/GR-repressed genes). Five genes were repressed by ER (ER-only repressed genes), and another five genes were repressed by GR (GR-only repressed genes). To examine if cross talk occurs between ER and GR at ER/GR-repressed genes, U2OS-GR cells were infected with an adenovirus that expresses ERα. The ER antagonist, ICI 182780 (ICI), blocked Dex repression of ER/GR-repressed genes. ICI did not have any effect on the GR-only repressed genes or genes activated by Dex. These results demonstrate that ICI acts on subset of proinflammatory genes in the presence of ERα but not on GR-activated genes. ICI recruited ERα to the IL-8 promoter but did not prevent Dex recruitment of GR. ICI antagonized Dex repression of the TNF response element by blocking the recruitment of nuclear coactivator 2. These findings indicate that the ICI–ERα complex blocks Dex-mediated repression by interfering with nuclear coactivator 2 recruitment to GR. Our results suggest that it might be possible to exploit ER and GR cross talk for glucocorticoid therapies using drugs that interact with ERs.