Inhibition Of Estrogen Signaling Research Articles

MiR-206 inhibits estrogen signaling and ovarian cancer cell migration without affecting GPER

AimsEstrogen-regulated pathways are involved in the etiology and progression of epithelial ovarian cancer (EOC), but the relative contribution of estrogen receptor isoforms is unclear. Only a subset of patients responds to antiestrogens including tamoxifen. Based on our previous evidence that miR-206 behaves as an oncosuppressor in EOC, we hypothesized that miR-206 would interfere with G protein-coupled estrogen receptor (GPER)-mediated signaling and cell motility. Main methodsPFKFB3 and FAK proteins from OC cells challenged with selective estrogen receptor agonist and antagonist were measured by Western blotting. Cell proliferation and motility were analyzed by MTT and Boyden chamber, respectively. Estrogen-dependent cells were transfected with miR-206 mimic or control using Lipofectamine. Key findingsThe migration of SKOV3 and OVCAR5 cells significantly increased following treatment with 17β-estradiol (E2) and the selective GPER agonist G1. However, tamoxifen failed to inhibit E2 effect and even promoted SKOV3 cell migration. Estrogen receptor ligands did not affect SKOV3 proliferation. The GPER antagonist G15 significantly prevented E2-mediated upregulation of PFKFB3 expression, while G1 concentration-dependently upregulated PFKFB3 levels. Consistent with the functional link between PFKFB3 and FAK activation, E2 and G1 increased FAK phosphorylation at Tyr397. Transfection with miR-206 abolished estrogen-induced EOC migration and down-regulated PFKFB3 protein levels. Notably, miR-206 transfection reduced ERα protein abundance, whereas GPER amount was unchanged. SignificanceBy blocking estrogen signaling and G1-induced EOC cell invasiveness with no direct interference with GPER levels, miR-206 mimics have the potential to act as pathway-selective antagonists and deserve further testing as RNA therapeutics in estrogen-dependent EOC.

Inhibition of estrogen signaling in myeloid cells increases tumor immunity in melanoma.

Immune checkpoint blockade (ICB) therapies have significantly prolonged patient survival across multiple tumor types, particularly in melanoma. Interestingly, sex-specific differences in response to ICB have been observed, with males receiving a greater benefit from ICB than females, although the mechanism or mechanisms underlying this difference are unknown. Mining published transcriptomic data sets, we determined that the response to ICBs is influenced by the functionality of intratumoral macrophages. This puts into context our observation that estrogens (E2) working through the estrogen receptor α (ERα) stimulated melanoma growth in murine models by skewing macrophage polarization toward an immune-suppressive state that promoted CD8+ T cell dysfunction and exhaustion and ICB resistance. This activity was not evident in mice harboring macrophage-specific depletion of ERα, confirming a direct role for estrogen signaling within myeloid cells in establishing an immunosuppressed state. Inhibition of ERα using fulvestrant, a selective estrogen receptor downregulator (SERD), decreased tumor growth, stimulated adaptive immunity, and increased the antitumor efficacy of ICBs. Further, a gene signature that determines ER activity in macrophages predicted survival in patients with melanoma treated with ICB. These results highlight the importance of E2/ER signaling as a regulator of intratumoral macrophage polarization, an activity that can be therapeutically targeted to reverse immune suppression and increase ICB efficacy.

Farnesoid X Receptor Agonist GW4064 Inhibits Aromatase and ERβ Expression in Human Endometriotic Stromal Cells

Endometriosis is an estrogen-dependent disease. Farnesoid X receptor (FXR) activation has been shown to inhibit estrogen signaling in breast cancer and testicular tumors. However, the role of FXR in endometriosis is still poorly understood. Here, we aimed to investigate whether FXR activation by its synthetic agonist GW4064 has a therapeutic effect on endometriosis and the underlying molecular mechanisms. We found that the expression of FXR (encoded by the NR1H4 gene) in endometriotic tissues and stromal cells (ESCs) was higher than that in eutopic endometrial tissues and stromal cells. The GW4064 treatment led to a dose-dependent decrease in aromatase and estrogen receptor β (ERβ) expression and induced ERK1/2, p38, AMPK, and Stat3 activation in ESCs. In contrast, ERK1/2 inhibitor reversed the GW4064-induced reduction in aromatase expression. In addition, treatment with p38, AMPK, and Stat3 inhibitors or small interfering RNAs could also reverse the GW4064-induced reduction of ERβ expression in ESCs. The GW4064 treatment markedly increased Stat3 phosphorylation, enhancing the binding of Stat3 to the ESR2 promoter, which resulted in the downregulation of ERβ. Coimmunoprecipitation assay and chromatin immunoprecipitation analysis revealed that FXR was able to compete with cyclic AMP response element-binding (CREB) protein for binding to a common sequence on the aromatase promoter region after GW4064 treatment in ESCs. Moreover, treatment of endometriosis xenografts with GW4064 suppressed aromatase and ERβ expression in nude mice. Our results suggest that FXR may represent a potential therapeutic target for future therapy.

Using short-term bioassays to evaluate the endocrine disrupting capacity of the pesticides linuron and fenoxycarb

Several short-term whole-organism bioassays based on transgenic aquatic models are now under validation by the OECD (Organization for Economic Co-operation and Development) to become standardized test guidelines for the evaluation of the endocrine activity of substances. Evaluation of the endocrine disrupting capacity of pesticides will be a domain of applicability of these future reference tests. The herbicide linuron and the insecticide fenoxycarb are two chemicals commonly used in agricultural practices. While numerous studies indicate that linuron is likely to be an endocrine disruptor, there is little information available on the effect of fenoxycarb on vertebrate endocrine systems. Using whole-organism bioassays based on transgenic Xenopus laevis tadpoles and medaka fry we assessed the potential of fenoxycarb and linuron to disrupt thyroid, androgen and estrogen signaling. In addition we used in silico approach to simulate the affinity of these two pesticides to human hormone receptors. Linuron elicited thyroid hormone-like activity in tadpoles at all concentrations tested and, showed an anti-estrogenic activity in medaka at concentrations 2.5mg/L and higher. Our experiments suggest that, in addition to its previously established anti-androgenic action, linuron exhibits thyroid hormone-like responses, as well as acting at the estrogen receptor level to inhibit estrogen signaling. Fenoxycarb on the other hand, did not cause any changes in thyroid, androgen or estrogen signaling at the concentrations tested.

Abstract P3-07-05: CDK8 inhibition improves the efficacy of ER- and HER2-targeted drugs in breast cancer

Abstract Over 70% of breast cancer patients are estrogen receptor (ER) positive and 25% of patients over-express HER2 making these patients susceptible to therapeutic intervention with ER- and HER2-targeted therapies, respectively. However, intrinsic and acquired resistance to targeted therapies is a significant clinical issue and new therapeutic approaches aimed to preventing and overcoming resistance are urgently needed. We have previously shown that high expression of CDK8, a transcription regulating kinase, is associated with shorter relapse free survival in both ER and HER2 positive breast cancer. We have found that CDK8 inhibition by a selective small molecule inhibitor (Senexin B), by shRNA knockdown or by CRISPR/CAS9 knockout, strongly inhibits estrogen signaling in ER-positive breast cancer cells. Senexin B produces a synergistic growth inhibitory effect with an antiestrogen fulvestrant in all the tested ER-positive breast cancer cell lines in vitro and in MCF7 xenograft model in vivo. Senexin B treatment also inhibited invasive growth of MCF7 xenografts. CDK8 inhibition suppressed the emergence of estrogen independence upon long-term estrogen deprivation. A highly synergistic growth inhibitory effect occurred when Senexin B was combined with an anti-HER2 monoclonal antibody (a biosimilar of trastuzumab) or with the HER2/EGFR small molecule inhibitor lapatinib. These synergistic effects were observed in all HER2 positive breast cancer cell lines tested including those that exhibit innate and acquired resistance to HER2 targeting therapy. Furthermore, combining lapatinib with Senexin B completely abrogated the emergence of acquired lapatinib resistance. Taken together these results suggest that CDK8 inhibition, when combined with either ER- or HER2-targeted therapies, offers a rational approach to improving the efficacy of targeted drugs in breast cancer. Citation Format: McDermott MS, Chumanevich A, Liang J, Chen M, Altilia S, Hennes C, Roninson IB, Broude EV. CDK8 inhibition improves the efficacy of ER- and HER2-targeted drugs in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-05.

Localization of tamoxifen in human breast cancer tumors by MALDI mass spectrometry imaging.

BackgroundTamoxifen is used in endocrine treatment of breast cancer to inhibit estrogen signaling. A set of stratified ER-positive and ER-negative tumor sections was subjected to manual deposition of tamoxifen solution in order to investigate its spatial distribution upon exposure to interaction within thin tissue sections.MethodsThe localization of tamoxifen in tumor sections was assessed by matrix assisted laser deposition/ionization mass spectrometry imaging. The images of extracted ion maps were analyzed for comparison of signal intensity distributions.ResultsThe precursor ion of tamoxifen (m/z 372.233) displayed heterogeneous signal intensity distributions in histological compartments of tumor tissue sections. The levels of tamoxifen in tumor cells compared with stroma were higher in ER-positive tissues, whereas ER-negative tissue sections showed lower signal intensities in tumor cells.ConclusionsThe experimental model was successfully applied on frozen tumor samples allowing for differentiation between ER groups based on distribution of tamoxifen.Electronic supplementary materialThe online version of this article (doi:10.1186/s40169-016-0090-9) contains supplementary material, which is available to authorized users.

The pitfall of the transient, inconsistent anticancer capacity of antiestrogens and the mechanism of apparent antiestrogen resistance.

Although antiestrogens have been available for breast cancer therapy since the early 1970s, neither their inconsistent anticancer capacity nor the developing antiestrogen resistance of tumors can be fully understood. Although clinical and experimental investigations revealed many tiny details concerning the link between estrogen signaling and tumor development, they yielded fairly controversial findings. Estrogen receptor (ER) overexpression in tumor cells induced by estrogen treatment was erroneously regarded as a promoter of DNA damage, genomic instability, and tumor growth. Similarly, compensatory ER overexpression caused by antiestrogen treatment or estrogen withdrawal was mistakenly evaluated as a key for rapid tumor growth attributed to acquired antiestrogen resistance. Nevertheless, ER upregulation induced by estrogen treatment is a physiologic process even in tumor cells, whereas in the case of antiestrogen administration, it is a contraregulatory action to defend the endangered estrogen signaling. Upregulation of estrogen signaling displays a unique dichotomy, ensuring the survival and safe proliferative activity of healthy cells, while inducing apoptotic death of malignant tumor cells. Analysis of the fairly controversial results justifies that whatever type of available endocrine therapies may be used, including estrogen, antiestrogen treatment, or oophorectomy, an extreme upregulation of ER signaling seems to be the crucial mechanism of successful prevention and treatment for breast cancer. The inconsistent therapeutic effects of antiestrogen administration may be explained by the different genetic capacities of patients for the compensatory upregulation of ER and aromatase enzyme expressions. The weaker the defensive counteraction against the inhibition of estrogen signaling, the poorer is the prognosis of the disease. De novo or acquired antiestrogen resistance of tumors may be associated with the missing capacity of patients for the extreme upregulation of estrogen signaling or with the exhaustion of defensive counteractions in cases that previously showed good reactivity. High-dose estrogen treatment is capable of restoring ER signaling and anticancer capacity even after heavy exposure to antiestrogen therapy.

Epigenetic Regulation of miR-22 in a BPA-exposed Human Hepatoma Cell

Bisphenol A (BPA), an widely used environmental chemical, is encircled to human life. However, we generally do not know whether or not it can cause negative health effects. One of the representative epigenetic changes that inhibit gene expression is DNA methylation, which has been very well studied in association with cancer and development. Gene function is changed by DNA methylation; however, its genetic code does not change. Our study hypothesized that a post-transcriptional change in DNA occurs due to exposure to BPA. These changes then cause regulation of microRNA and gene expression. To identify these successional regulations, we conducted microarraybased assays. For validation, also we conducted bisulfite sequencing, quantitative real-time PCR, miRNA inhibitor assay, and Western blotting. We found 1,751 hypo-methylation changed regions, and several micro- RNA had included methylated-regions. miR-22 was also hypomethylated (chr17:1563947-1564031) by BPA-exposure, and expression of miR-22 was up-regulated in an miRNA array and real-time PCR. miR- 22 has been reported to inhibit estrogen signaling by direct targeting of the estrogen receptor alpha mRNA. Taking notice of this point, we analyzed gene expression profiles that included its predicted targets. In the present study, we found the cause of hypomethylation of miR-22 and negative regulation of its apoptosisrelated target gene expression by BPA-exposure. These results suggests that BPA can alter sequential genomic appearances in HepG2 cells, a potentially affection of BPA toxicity. Also, the results of our study support that toxicology study need to integrated analysis of array-based assays for help in understanding of the molecular action of environmental toxicants.

Abstract 3473: Identification of progesterone-dependent genes regulated by ablation of mitogen-inducible gene-6 (Mig-6) in the murine uterus

Abstract Mitogen-inducible gene-6 (Mig-6) is an important mediator of progesterone signaling to inhibit estrogen signaling in the uterus. Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. Although Mig-6 is an important tumor suppressor in endometrial cancer, relatively little is known about the molecular mechanisms of Mig-6 in endometrial cancer. To identify the molecular pathways regulated by Mig-6 in a steroid-hormone dependent manner, we performed microarray analysis on the uterus of ovariectomized PRcre/+Mig-6f/f (Mig-6d/d) mice treated with progesterone for 6 hours. In the analysis of GeneChip® Mouse Genome 430 2.0 Array, 2,034 genes, 1,062 genes were up-regulated and 972 genes were down-regulated, were altered by P4 treatment. To determine which pathways are regulated by P4 treatment, we performed pathway analysis using Ingenuity Systems Software. Genes that involved in Estrogen Receptor, IGF-1, PI3K/AKT, JAK/Stat, ERK/MAPK, ILK, p53, PTEN, and Wnt/β-catenin signaling were found to be P4 response. In addition, genes that regulated the molecular mechanisms of cancer were disproportionated. And in microarray, 2,236 genes, 1,883 genes were up-regulated and 356 genes were down-regulated, were regulated by Mig-6 ablation on P4 treatment. Genes involved in ILK, p53, PTEN, PI3K/AKT, and Wnt/β-catenin signaling were found to be deregulated upon Mig-6 ablation on P4 response. Furthermore 324 genes of the 2,034 genes were regulated by P4 treatment were dys-regulated in Mig-6d/d mice treated with P4 compared to Mig-6f/f control mice. These results showed that gene expression pattern was altered by the deletion of Mig-6 and the change of these genes was dependent on P4 in uterus. Thus the further investigation should be completed to identify molecules associated with Mig-6 and the identification of relation between these responsive genes will contribute to define the molecular pathways regulated by Mig-6 in uterine biology. (This work was supported by NIH, R01 HD057873 and American Cancer Society Research Grant, RSG-12-084-01-TBG to J.W.J.) Citation Format: Tae Hoon Kim, Jung-Yoon Yoo, Jae-Wook Jeong. Identification of progesterone-dependent genes regulated by ablation of mitogen-inducible gene-6 (Mig-6) in the murine uterus. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3473. doi:10.1158/1538-7445.AM2014-3473

Inhibition of estrogen signaling through depletion of estrogen receptor alpha by ursolic acid and betulinic acid from Prunella vulgaris var. lilacina

Extracts of Prunella vulgaris have been shown to exert antiestrogenic effects. To identify the compounds responsible for these actions, we isolated the constituents of P. vulgaris and tested their individual antiestrogenic effects. Rosmarinic acid, caffeic acid, ursolic acid (UA), oleanolic acid, hyperoside, rutin and betulinic acid (BA) were isolated from the flower stalks of P. vulgaris var. lilacina Nakai (Labiatae). Among these constituents, UA and BA showed significant antiestrogenic effects, measured as a decrease in the mRNA level of GREB1, an estrogen-responsive protein; the effects of BA were stronger than those of UA. UA and BA were capable of suppressing estrogen response element (ERE)-dependent luciferase activity and expression of estrogen-responsive genes in response to exposure to estradiol, further supporting the suppressive role of these compounds in estrogen-induced signaling. However, neither UA nor BA was capable of suppressing estrogen signaling in cells ectopically overexpressing estrogen receptor α (ERα). Furthermore, both mRNA and protein levels of ERα were reduced by treatment with UA or BA, suggesting that UA and BA inhibit estrogen signaling by suppressing the expression of ERα. Interestingly, both compounds enhanced prostate-specific antigen promoter activity. Collectively, these findings demonstrate that UA and BA are responsible for the antiestrogenic effects of P. vulgaris and suggest their potential use as therapeutic agents against estrogen-dependent tumors.

An Activin A/BMP2 chimera, AB215, blocks estrogen signaling via induction of ID proteins in breast cancer cells.

BackgroundOne in eight women will be affected by breast cancer in her lifetime. Approximately 75% of breast cancers express estrogen receptor alpha (ERα) and/or progesterone receptor and these receptors are markers for tumor dependence on estrogen. Anti-estrogenic drugs such as tamoxifen are commonly used to block estrogen-mediated signaling in breast cancer. However, many patients either do not respond to these therapies (de novo resistance) or develop resistance to them following prolonged treatment (acquired resistance). Therefore, it is imperative to continue efforts aimed at developing new efficient and safe methods of targeting ER activity in breast cancer.MethodsAB215 is a chimeric ligand assembled from sections of Activin A and BMP2. BMP2’s and AB215’s inhibition of breast cancer cells growth was investigated. In vitro luciferase and MTT proliferation assays together with western blot, RT_PCR, and mRNA knockdown methods were used to determine the mechanism of inhibition of estrogen positive breast cancer cells growth by BMP2 and AB215. Additionally in vivo xenograft tumor model was used to investigate anticancer properties of AB215.ResultsHere we report that AB215, a chimeric ligand assembled from sections of Activin A and BMP2 with BMP2-like signaling, possesses stronger anti-proliferative effects on ERα positive breast cancer cells than BMP2. We further show that AB215 inhibits estrogen signaling by inducing expression of inhibitor of DNA binding proteins (IDs). Specifically, we demonstrate that knockdown of ID proteins attenuates the anti-estrogen effects of AB215. Remarkably, we find that AB215 is more effective than tamoxifen in suppressing tumor growth in a xenograft model.ConclusionThis study shows that IDs have profound role to inhibit estrogen signaling in ERα positive breast cancer cells, and that engineered TGF-beta ligands may have high therapeutic value.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-549) contains supplementary material, which is available to authorized users.

Oral Contraceptives and Nicotine Synergistically Exacerbate Cerebral Ischemic Injury in the Female Brain

Oral contraceptives (OC) and smoking-derived nicotine (N) are known to synergistically increase the risk and severity of cerebral ischemia in women. Although it has been known for some time that long-term use of OC and nicotine will have an increased risk of peripheral thrombus formation, little is known about how the combination of OC and nicotine increases severity of brain ischemia. Recent laboratory studies simulating the conditions of nicotine exposure produced by cigarette smoking and OC regimen of women in female rats confirms that the severity of ischemic hippocampal damage is far greater in female rats simultaneously exposed to OC than to nicotine alone. These studies also demonstrated that the concurrent exposure of OC and nicotine reduces endogenous 17β-estradiol levels and inhibits estrogen signaling in the brain of female rats. The endogenous 17β-estradiol plays a key role in cerebrovascular protection in women during their pre-menopausal life and loss of circulating estrogen at reproductive senescence increases both the incidence and severity of cerebrovascular diseases. Therefore, OC and nicotine induced severe post-ischemic damage might be a consequence of lack of estrogen signaling in the brain. In the present review we highlight possible mechanisms by which OC and nicotine inhibits estrogen signaling that could be responsible for severe ischemic damage in females.

Zinc finger protein 131 inhibits estrogen signaling by suppressing estrogen receptor α homo-dimerization

Steroid hormone estrogen elicits various physiological functions, many of which are mediated through two structurally and functionally distinct estrogen receptors, ERα and ERβ. The functional role of zinc finger protein 131 (ZNF131) is poorly understood, but it is assumed to possess transcriptional regulation activity due to the presence of a DNA binding motif. A few recent reports, including ours, revealed that ZNF131 acts as a negative regulator of ERα and that SUMO modification potentiates the negative effect of ZNF131 on estrogen signaling. However, its molecular mechanism for ERα inhibition has not been elucidated in detail. Here, we demonstrate that ZNF131 directly interacts with ERα, which consequently inhibits ERα-mediated trans-activation by suppressing its homo-dimerization. Moreover, we show that the C-terminal region of ZNF131 containing the SUMOylation site is necessary for its inhibition of estrogen signaling. Taken together, these data suggest that ZNF131 inhibits estrogen signaling by acting as an ERα-co-repressor.

Abstract B66: Characterization of bis-phenazine compounds that inhibit estrogen signaling through direct interaction with estrogen response elements

Abstract Hormone therapies interfere with estrogen signaling by targeting Estrogen Receptors (ERs) and are routinely used for the prevention and treatment of ER-expressing breast cancers. However, current therapies have significant limitations related to inherent or acquired resistance. Thus, there is a need to develop therapeutic compounds that do not depend on competitive binding to the ER or on depleting cells of endogenous estrogen. Previous data suggested that certain bis-phenazines, such as our lead compound XR5944, possess potent antiestrogenic properties through a novel mechanism of action: they inhibit gene transcription via direct intercalation with DNA at estrogen response elements (EREs) which are located in estrogen-responsive gene promoters. While these studies have demonstrated the potent antiestrogenic activity of XR5944, they have also demonstrated its capacity to bind to alternative DNA motifs. In order to improve upon ERE target specificity, we have identified XR5944 derivatives that retain potent antiestrogenic properties but may have greater specificity for ERE sequences, to be developed as new therapies for the treatment of estrogen-driven cancers. Such compounds may also be effective in overcoming resistance to current antiestrogen therapies. Here, we characterize DNA sequence interactions and transcriptional effects of five XR5944 derivatives that differ from the parent compound in the composition of their linker chains (compounds 26357 and 26373), cyclic side rings (26576), or both (26536 and 27402). We utilized 1D 1H NMR titration experiments to monitor compound:DNA binding properties to a canonical ERE (AGGTCAcggTGACCT) containing a CGG trinucleotide spacer (ERECGG), electrophoretic mobility shift assays (EMSA) to assess the ability of the derivatives to disrupt ER:ERECGG interactions, and luciferase reporter assays to evaluate their potency in blocking estrogen-dependent transactivation through ERECGG. Our NMR results indicated that alteration of either linker chain or side ring structures of XR5944 reduced the affinity and specificity of compound:ERECGG interactions. These findings correlated with the ability of the compounds to inhibit ER:ERECGG binding in the order of XR5944>26576>26536>26373>26357>27402, as evaluated by EMSA. To further determine the effects of the compounds on ERECGG-driven transactivation, we performed luciferase reporter assays utilizing ERα-expressing MCF-7 breast cancer cells transfected with an ERECGG-luciferase reporter construct. Unlike the EMSA results, there was not a strict concordance between the compound-dependent potency in inhibiting estrogen-driven transcriptional responses and the specificity of interaction with DNA as determined by NMR. Specifically, inhibition of reporter activity was more potent with compounds that had the same cyclic side rings but altered linker chains as XR5944 (26373 and 26357) and was less effective with compounds possessing altered side rings (26576, 26536, and 27402). Together, our findings indicate that the cyclic side chain structures appear to be the most important components of bis-phenazines in disrupting the transcriptional potential of ER:ERE interactions. NMR can be useful for predicting the relative potency of XR5944 derivatives in blocking ER binding to EREs in vitro, but fails to predict their efficacy in inhibiting ERE-mediated transactivation. This information should prove valuable for the rational drug design of new antiestrogenic compounds with improved specificity for ERE sequences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B66.

Polyphenols, isothiocyanates, and carotenoid derivatives enhance estrogenic activity in bone cells but inhibit it in breast cancer cells

While exposure to estrogens is a major risk factor of breast and endometrial cancer, it well established that estrogens are beneficial for bone health. We have previously shown that carotenoids inhibit estrogen signaling in breast and endometrial cancer cells. The aim of this study was to compare the effects of various phytonutrients, (carotenoid derivatives, polyphenols, isothiocyanates) on estrogenic activity in breast cancer cells and osteoblast-like cells. All the tested phytonutrients inhibited estrogen response element (ERE) transactivation in breast cancer cells. In contrast, these compounds either did not affect or enhanced ERE activity and the expression of several bone-forming genes. These results were obtained using two osteoblast-like cell lines, MG-63 human osteosarcoma cells stably transfected with estrogen receptor-α (ERα) and MC3T3-E1 mouse calvaria-derived cells expressing endogenous ER. Phytonutrients-induced ERE inhibition in breast cancer cells, and its potentiation in osteoblast-like cells were associated both with a decrease and a rise in total and nuclear ERα levels, respectively. Phytonutrients activated the electrophile/antioxidant response element (EpRE/ARE) transcription system to various extents in both cancer and bone cell lines. Overexpression of Nrf2, the major EpRE/ARE activating transcription factor, mimicked the effects of phytonutrients, causing inhibition and enhancement of ERE transactivation in breast cancer cells and in osteoblast-like cells, respectively. Moreover, reduction in Nrf2 levels by RNAi led to a decrease in the phytonutrient potentiation of ERE activity transactivation in osteoblast-like cells. These findings suggest that the enhancement and inhibition of estrogen signaling by phytonutrients in bone-derived cells and breast cancer cells, respectively, is partially mediated by the activation of the Nrf2/ARE system.

Estrogen-Related Fecundity Reduction of Lymnaea ollula Following Fasciola gigantica Infection

Infection by trematode parasites generally affects life history traits of their intermediate hosts. Reduction in life expectancy and reproductive capacity have previously been documented in Fasciola gigantica-infected Lymnaea ollula, but the influence of the endocrine system on this specific host–parasite interaction has not been previously examined. In the present study, we observed survival, growth pattern, and reproductive output of L. ollula following exposure to F. gigantica. Both the survival and the growth pattern of infected snails were similar to those of the non-infected control group. However, a significant difference was apparent in fecundity, as infected snails consistently showed lower levels of egg and embryo production throughout a 7-wk observation period. Semi-quantitative RT-PCR also revealed down-regulation of estrogen receptor expression in infected snails during the first 4 wk of infection. Nonetheless, the inhibition of estrogen signaling was transient, as they regained expression in the later phase of infection. It is, therefore, suggested that other hormones of the complex endocrine system may be involved in the reduced fecundity of L. ollula following F. gigantica infection.

MiR-22 Inhibits Estrogen Signaling by Directly Targeting the Estrogen Receptor α mRNA

miR-22 Inhibits Estrogen Signaling by Directly Targeting the Estrogen Receptor α mRNA

MiR-22 Inhibits Estrogen Signaling by Directly Targeting the Estrogen Receptor α mRNA

Estrogen receptor alpha (ER alpha) is a ligand-regulated transcription factor with a broad range of physiological functions and one of the most important classifiers in breast cancer. MicroRNAs (miRNAs) are small noncoding RNAs that have emerged as important regulators of gene expression in a plethora of physiological and pathological processes. Upon binding the 3' untranslated region (UTR) of target mRNAs, miRNAs typically reduce their stability and/or translation. The ER alpha mRNA has a long 3' UTR of about 4.3 kb which has been reported to reduce mRNA stability and which bears evolutionarily conserved miRNA target sites, suggesting that it might be regulated by miRNAs. We have performed a comprehensive and systematic assessment of the regulatory role of all miRNAs that are predicted to target the 3' UTR of the ER alpha mRNA. We found that miR-22 represses ER alpha expression most strongly and by directly targeting the ER alpha mRNA 3' UTR. Of the three predicted miR-22 target sites in the 3' UTR, the evolutionarily conserved one is the primary target. miR-22 overexpression leads to a reduction of ER alpha levels, at least in part by inducing mRNA degradation, and compromises estrogen signaling, as exemplified by its inhibitory impact on the ER alpha-dependent proliferation of breast cancer cells.

Characterization of Antiestrogenic Activity of the Chinese Herb,Prunella vulgaris, Using In Vitro and In Vivo (Mouse Xenograft) Models1

Prunella vulgaris (PV), a commonly used Chinese herb, also known as Self-heal, has a wide range of reported medicinal activities. By screening multiple herbs using the endometrial cancer cell line, ECC-1, and an alkaline phosphatase detection assay, we found that PV displayed significant antiestrogenic activity. We investigated the possible usefulness of antiestrogenic activity using both in vitro and in vivo models of endometrial function. Using the well-differentiated, hormone-responsive endometrial cell line, ECC-1, PV extract, at concentrations that were not toxic to the cells, significantly reduced alkaline phosphatase activity and cell proliferation in response to estrogen in a dose-dependent manner. The expression of CYR61, an estrogen-induced protein, was blocked in ECC-1 cells by both the antiestrogen ICI 182,780 and PV extract. Interestingly, PV extract did not appear to directly inhibit estrogen signaling. Rather, we found that its activities were probably related to an ability to function as an aryl hydrocarbon receptor (AHR) agonist in ECC-1 cells. In support of this hypothesis, we noted that PV induced CYP1A1, CYP1B1, and AHR repressor expression in a dose-dependent manner--responses that were blocked by small interfering RNA treatment to reduce AHR and specific AHR antagonists. Ovariectomized immunodeficient RAG-2/gamma(c) knockout mice implanted with human endometrial xenografts developed implants only when treated with estrogen. Mice treated with estrogen and PV tea in their drinking water had fewer and smaller xenograft implants compared with their estrogen-treated counterparts that drank only water (P < 0.05). Analysis of the resulting implants by immunohistochemistry demonstrated persistent estrogen receptor (ER), but reduced proliferation and CYR61 expression. Mouse uterine tissue weight in PV-treated mice was not different from controls, and cycle fecundity of intact C57 female mice was unaffected by PV tea treatment. PV, or Self-heal, exhibits significant antiestrogenic properties, both in vitro and in vivo. This activity is likely due to the ability of PV-activated AHR to interfere with estrogen. This herb may be useful as an adjunct for the treatment of estrogen-dependent processes like endometriosis and breast and uterine cancers. Full characterization of this herb will likely provide new insights into the crosstalk between AHR and ESR1, with potential for therapeutic applications in women.

Lycopene and other carotenoids inhibit estrogenic activity of 17β-estradiol and genistein in cancer cells

Epidemiological evidence suggests that carotenoids prevent several types of cancer, including mammary and endometrial cancers. On the other hand, such studies have also shown that estrogens are the most important risk factors for these cancer types. Genistein, the phytoestrogen mainly found in soy, also shows significant estrogenic activity when tested at concentrations found in human blood. The aim of this study was to determine whether carotenoids inhibit signaling of steroidal estrogen and phytoestrogen which could explain their cancer preventive activity. Similar to the known effect of 17beta-estradiol (E(2)), treatment of breast (T47D and MCF-7) and endometrial (ECC-1) cancer cells with phytoestrogens induced cell proliferation, cell-cycle progression and transactivation of the estrogen response element (ERE). However, each of the tested carotenoids (lycopene, phytoene, phytofluene, and beta-carotene) inhibited cancer cell proliferation induced by either E(2) or genistein. The inhibition of cell growth by lycopene was accompanied by slow down of cell-cycle progression from G1 to S phase. Moreover, the carotenoids inhibited estrogen-induced transactivation of ERE that was mediated by both estrogen receptors (ERs) ERalpha and ERbeta. The possibility that this inhibition results from competition of carotenoid-activated transcription systems on a limited pool of shared coactivators with the ERE transcription system was tested. Although cotransfection of breast and endometrial cancer cells with four different coactivators (SRC-1, SRC-2, SRC-3, and DRIP) strongly stimulated ERE reporter gene activity, it did not oppose the inhibitory effect of carotenoids. These results suggest that dietary carotenoids inhibit estrogen signaling of both 17beta-estradiol and genistein, and attenuate their deleterious effect in hormone-dependent malignancies.