Nuclear Estrogen Receptor Research Articles

Abstract 453: The G Protein-coupled Estrogen Receptor GPER/GPR30 is a Potential Therapeutic Target for Cardiovascular Inflammation

Abstract: Neutrophil extracellular traps (NETs), DNA-based structures that play an important role in pathogen killing/capture, are also thought to contribute to inflammatory disease and have been implicated in thrombosis. Pharmacological agents that can modulate the formation of extracellular traps are as-yet unknown. We hypothesized that targeting G protein-coupled receptors (GPCRs) may provide such agents and thereby could aid in treating inflammation in the cardiovascular system. Here, we investigated the role of the G protein-coupled estrogen receptor (GPER/GPR30), which has recently been shown to play a role in innate immune function (Cabas et al. J Immunol . 191: 4628-39 [2013]). Quantitative PCR revealed that GPER is highly expressed in human neutrophils isolated from healthy male and female donors. Fluorescent quantification of extracellular DNA using PicoGreen revealed that both selective (G-36) and general (raloxifene) antagonists of GPER, but not selective antagonists for the nuclear estrogen receptors ERa (MPP) or ERb (PHTPP) inhibited phorbol myristate acetate (PMA)-induced NET production by human neutrophils. These results were verified using high throughput imaging of fixed, SytoxGreen-stained neutrophils. The activity of GPER antagonists appeared to be independent of Erk1/2 signaling and reactive oxygen species (ROS) generation, pathways that are mediators of NET production. Immunocytochemistry revealed that G-36 treatment of neutrophils raises their concentration of cAMP, which can inhibit production of NETs. Taken together, our data suggest that GPER may be a novel therapeutic target for modulating NET production and inflammation in the cardiovascular system.

Molecular cloning and characterization of hagfish estrogen receptors

One or more distinct forms of the nuclear estrogen receptor (ER) have been isolated from many vertebrates to date. To better understand the molecular evolution of ERs, we cloned and characterized er cDNAs from the inshore hagfish, Eptatretus burgeri, a modern representative of the most primitive vertebrates, the agnathans. Two er cDNAs, er1 and er2, were isolated from the liver of a reproductive female hagfish. A phylogenetic analysis placed hagfish ER1 into a position prior to the divergence of vertebrate ERs. Conversely, hagfish ER2 was placed at the base of the vertebrate ERβ clade. The tissue distribution patterns of both ER subtype mRNAs appeared to be different, suggesting that each subtype has different physiological roles associated with estrogen actions. An estrogen responsive-luciferase reporter assay using mammalian HEK293 cells was used to functionally characterize these hagfish ERs. Both ER proteins displayed estrogen-dependent activation of transcription. These results clearly demonstrate that the hagfish has two functional ER subtypes.

A role for multiple estrogen receptors in immune regulation of common carp

Estrogens are important for bi-directional neuroendocrine-immune interaction. They act via nuclear estrogen receptors (ERα and ERβ) and/or G-protein coupled receptor - GPR30.We found expression of ERα, ERβ and GPR30 in carp lymphoid tissues and head kidney monocytes/macrophages, neutrophils and lymphocytes. Interestingly, ERβ is also expressed in some head kidney lymphocytes but not in naive PBLs. Immune stimulation altered the cell type specific profile of expression of these receptors, which depends on both activation and maturation stage.This implies direct leukocyte responsiveness to estrogen stimulation and therefore in vitro effects of 17β-estradiol (E2) on reactive oxygen species (ROS) production in monocytes/macrophages were determined. Short-time incubation with E2 increased ROS production in PMA-stimulated cells. Results comply with mediation by GPR30, partially functioning via phosphoinositide 3-kinase activation.These results furthermore demonstrate that neuroendocrine-immune communication via estrogen receptors is evolutionary conserved.

The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens.

MP57-18 EXPRESSION OF STEROID HORMONE RECEPTORS IN RESIDUAL CANCER AND STROMAL CELLS AFTER NEOADJUVANT CHEMOHORMONAL THERAPY WITH DOCETAXAL FOR HIGH-RISK LOCALIZED PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Localized: Surgical Therapy III1 Apr 2016MP57-18 EXPRESSION OF STEROID HORMONE RECEPTORS IN RESIDUAL CANCER AND STROMAL CELLS AFTER NEOADJUVANT CHEMOHORMONAL THERAPY WITH DOCETAXAL FOR HIGH-RISK LOCALIZED PROSTATE CANCER Shintaro Narita, Taketoshi Nara, Mingguo Huang, Koichiro Takayama, Kazuyuki Numakura, Hiroshi Tsuruta, Atsushi Maeno, Mitsuru Saito, Takamitsu Inoue, Norihiko Tsuchiya, Hiroshi Nanjo, Shigeru Satoh, and Tomonori Habuchi Shintaro NaritaShintaro Narita More articles by this author , Taketoshi NaraTaketoshi Nara More articles by this author , Mingguo HuangMingguo Huang More articles by this author , Koichiro TakayamaKoichiro Takayama More articles by this author , Kazuyuki NumakuraKazuyuki Numakura More articles by this author , Hiroshi TsurutaHiroshi Tsuruta More articles by this author , Atsushi MaenoAtsushi Maeno More articles by this author , Mitsuru SaitoMitsuru Saito More articles by this author , Takamitsu InoueTakamitsu Inoue More articles by this author , Norihiko TsuchiyaNorihiko Tsuchiya More articles by this author , Hiroshi NanjoHiroshi Nanjo More articles by this author , Shigeru SatohShigeru Satoh More articles by this author , and Tomonori HabuchiTomonori Habuchi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.672AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent studies suggest that activation of steroid hormone receptors (SHRs) is associated with PCa progression and resistance to chemohormonal therapy. In this study, we investigated SHR expressions in human PCa tissues in patients who underwent radical prostatectomy (RP) with and without neoadjuvant chemohormonal therapy. METHODS A tissue microarray with 210 cores from 70 high-risk localized PCa patients who underwent RP without neoadjuvant therapy (NNA), with neoadjuvant hormonal therapy (NHT), or with neoadjuvant chemohormonal therapy with docetaxel (CHT) was used to assess the epithelial and stromal expressions of four SHRs including androgen receptor (AR), glucocorticoid receptor (GR), estrogen receptor (ER) alpha, and progesterone receptor (PR). Expression levels of SHRs between the three groups were statistically assessed. Multivariate analyses were performed to investigate the prognostic factors of BCR in patients who underwent surgery with CHT. RESULTS AR and GR were expressed in both residual cancer and stromal cells, whereas ER alpha and PR were mainly expressed in stromal cells. The mean nuclear AR immunoreactivity score in residual cancer cells was significantly higher in the NNA group than in the NHT and CHT groups. The mean nuclear GR immunoreactivity score in residual cancer cells and mean nuclear ER alpha and PR in stromal cells were significantly higher in the CHT group than in the NNA and NHT groups. The patients with a low PR immunoreactivity score in stromal cells had a significantly higher BCR rate than those with a high nuclear immunoreactivity score (p = 0.011). In multivariate analysis, the number of risk factors constituting the classification of high-risk, a preoperative PSA level, pathological chemotherapy effect, and low nuclear PR immunoreactivity score in stromal cells were independent prognostic factors for BCR. CONCLUSIONS Stromal PR expression may be a potential prognostic marker for BCR in high-risk PCa patients who underwent radical prostatectomy after neoadjuvant chemohormonal therapy. The expression of SHRs in both residual cancer and stromal cells may play an important role in PCa resistance to chemohormonal therapy. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e759-e760 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Shintaro Narita More articles by this author Taketoshi Nara More articles by this author Mingguo Huang More articles by this author Koichiro Takayama More articles by this author Kazuyuki Numakura More articles by this author Hiroshi Tsuruta More articles by this author Atsushi Maeno More articles by this author Mitsuru Saito More articles by this author Takamitsu Inoue More articles by this author Norihiko Tsuchiya More articles by this author Hiroshi Nanjo More articles by this author Shigeru Satoh More articles by this author Tomonori Habuchi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Estrogenic Effects of Several BPA Analogs in the Developing Zebrafish Brain

Important set of studies have demonstrated the endocrine disrupting activity of Bisphenol A (BPA). The present work aimed at defining estrogenic-like activity of several BPA structural analogs, including BPS, BPF, BPAF, and BPAP, on 4- or 7-day post-fertilization (dpf) zebrafish larva as an in vivo model. We measured the induction level of the estrogen-sensitive marker cyp19a1b gene (Aromatase B), expressed in the brain, using three different in situ/in vivo strategies: (1) Quantification of cyp19a1b transcripts using RT-qPCR in wild type 7-dpf larva brains exposed to bisphenols; (2) Detection and distribution of cyp19a1b transcripts using in situ hybridization on 7-dpf brain sections (hypothalamus); and (3) Quantification of the cyp19a1b promoter activity in live cyp19a1b-GFP transgenic zebrafish (EASZY assay) at 4-dpf larval stage. These three different experimental approaches demonstrated that BPS, BPF, or BPAF exposure, similarly to BPA, significantly activates the expression of the estrogenic marker in the brain of developing zebrafish. In vitro experiments using both reporter gene assay in a glial cell context and competitive ligand binding assays strongly suggested that up-regulation of cyp19a1b is largely mediated by the zebrafish estrogen nuclear receptor alpha (zfERα). Importantly, and in contrast to other tested bisphenol A analogs, the bisphenol AP (BPAP) did not show estrogenic activity in our model.

STX, a Novel Membrane Estrogen Receptor Ligand, Protects Against Amyloid-β Toxicity.

Because STX is a selective ligand for membrane estrogen receptors, it may be able to confer the beneficial effects of estrogen without eliciting the deleterious side effects associated with activation of the nuclear estrogen receptors. This study evaluates the neuroprotective properties of STX in the context of amyloid-β (Aβ) exposure. MC65 and SH-SY5Y neuroblastoma cell lines, as well as primary hippocampal neurons from wild type (WT) and Tg2576 mice, were used to investigate the ability of STX to attenuate cell death, mitochondrial dysfunction, dendritic simplification, and synaptic loss induced by Aβ. STX prevented Aβ-induced cell death in both neuroblastoma cell lines; it also normalized the decrease in ATP and mitochondrial gene expression caused by Aβ in these cells. Notably, STX also increased ATP content and mitochondrial gene expression in control neuroblastoma cells (in the absence of Aβ). Likewise in primary neurons, STX increased ATP levels and mitochondrial gene expression in both genotypes. In addition, STX treatment enhanced dendritic arborization and spine densities in WT neurons and prevented the diminished outgrowth of dendrites caused by Aβ exposure in Tg2576 neurons. These data suggest that STX can act as an effective neuroprotective agent in the context of Aβ toxicity, improving mitochondrial function as well as dendritic growth and synaptic differentiation. In addition, since STX also improved these endpoints in the absence of Aβ, this compound may have broader therapeutic value beyond Alzheimer's disease.

Immunohistochemical Detection of Estrogen Receptors in Canine Mammary Tumors

Abstract Mammary tumors are among the most common neoplasms in intact female dogs. They have a complex morphology, usually affecting middle age and older bitches. Almost 50% of the mammary tumors in dogs are malignant neoplasms. Prognosis is based on several factors: stage, age, tumor size, metastasis, histopathology, ovariectomy status and hormone-receptor activity. Immunohistochemical (IHC) measurement has become increasingly an important diagnostic and prognostic parameter, with the development of monoclonal antibodies against nuclear estrogen and progestin receptors. The aim of this study was to detect the presence of ER receptors in malignant canine mammary tumors and to identify their association with the clinical course of the tumor. Mammary tumor samples have been obtained by mastectomy from dogs presented at our clinic. Detailed clinical examination, CBC and basic serum biochemical profile were performed in all patients. Surgery was the only treatment. Histopathological examination and immunohistochemical detection of estrogen α receptors (ERα) was performed on 8 formalin-fixed, paraffin-embedded tissue samples, using the PT LINK immunoperoxidase technique. Histopathological examination of the mammary tumor samples (n=11) revealed tubular adenocarcinoma (n=6,54.5%) and ductal adenocarcinoma (n=3, 27.3%), one patient with benign adenoma and one with mastitis. Patients with positive ER tumors are alive, without remission, while 3 of the patients that were ER negative died due to lung metastases. According to our results, it can be concluded that the appearance and development of canine mammary tumors is highly connected with ovarian steroid hormones and that immunostaining of the tumors may be used as a good prognostic parameter in these patients.

Abstract B17: High expression of the transcription factor Kaiso correlates with aggressive breast cancers (BC) and racial disparity in BC outcomes in women of African ancestry

Abstract The highly metastatic triple negative breast cancers (TNBC) lack expression of estrogen-receptor (ER), progesterone-receptor (PR) and human epidermal growth factor receptor-2 (HER2), and thus have limited patient treatment options and prognosis. Intriguingly TNBC is most prevalent in young women of African Ancestry (WAA) who, despite having lower BC incidence rates than Caucasian women, have higher mortality rates. This racial disparity appears unrelated to socio-economic status and increasing evidence hints at an ancestral genetic predisposition to TNBC development and high mortality rates. Recently, a significant correlation was found between nuclear expression of the transcription factor Kaiso, ERα negativity, basal/TNBCs and invasive ductal carcinoma in African American (AA) women. Emerging evidence from our lab also found that high Kaiso expression is associated with shorter metastasis-free survival in aggressive breast cancers. Based on these findings we hypothesized that Kaiso may play a crucial role in the metastatic spread of advanced breast cancers, and perhaps contribute to the racial disparity in TNBC prevalence and BC outcomes in WAA. To gain insight into Kaiso's role in aggressive breast cancers and the racial disparity associated with BC outcomes, we generated stable Kaiso-depleted TNBC cells (MDA-231) and then performed xenograft studies in immuno-compromised mice. Kaiso-depleted MDA-231 cells exhibited no or negligible metastases to the lungs and liver, compared to control MDA-231 cells, which formed extensive metastases in the lungs and liver of immuno-compromised mice. We also analysed Kaiso expression levels and subcellular localization in a tissue microarray (TMA) comprised of breast tumor tissues from ~100 Caucasian and ~100 AA women. Intriguingly, we observed a significant correlation between high nuclear Kaiso expression and ER(-) status in WAA, but surprisingly we found that high nuclear Kaiso expression is associated with ER(+) status and better overall survival in women of European descent. Collectively our findings implicate high Kaiso expression in breast cancer aggressiveness and suggest a context-dependent role for Kaiso in BC outcomes in women of different ethnicities. Ongoing studies are focused on clarifying the role of Kaiso in TNBC outcomes in WAA, and will incorporate analysis of a clinically annotated TMA comprised of ~200 TNBC tissues obtained from Barbadian and Nigerian women in conjunction with mechanistic studies to elucidate the signaling pathways involved in Kaiso-mediated tumor metastasis. Our findings may unravel a therapeutic role for Kaiso as a useful biomarker for the prognosis of TNBCs especially in WAA. Citation Format: Blessing I. Bassey-Archibong, Lyndsay G. A. Rayner, Kevin Gardner, Juliet Daniel. High expression of the transcription factor Kaiso correlates with aggressive breast cancers (BC) and racial disparity in BC outcomes in women of African ancestry. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B17.

Alteration of nuclear factor-kappaB pathway promote neuroinflammation depending on the functions of estrogen receptors in substantia nigra after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment

The simultaneous role of neuroprotective estrogen and neurodegenerative inflammation during the progression of Parkinson’s disease (PD) is still remaining elusive. The novel importance of the present study in MPTP mediated mouse model of Parkinson’s disease (PD) is—to investigate the status of neuronal and glial cells in a time chase experiment; to explore which pathway of NF-kappaB exist to proceed the neuroinflammation; to investigate the status of estrogen and the activation pattern of nuclear or cytosolic estrogen receptors in either sexes of Swiss albino mice during MPTP mediated progressive neurodegeneration in the substantia nigra. After MPTP intoxication, the nigral molecular anatomy was changed differently in separate time interval during the progression of neurodegeneration with/without association of glial cells and functional (via its nuclear and cytosolic receptors) estrogen level. Both the canonical and/or non-canonical pathways of NF-kappaB exist in the substantia nigra of both the sexes after MPTP treatment that is why inspite of presence of estrogen, neuroinflammation progresses. The homodimeric or heterodimeric form of ER-beta binds with NF-kappaB molecules p65 and RelB differently, but the canonical or non-canonical pathways of NF-kappaB molecules could not be stopped or may be promoted.

Prostate Cancer Expression Profiles of Cytoplasmic ERβ1 and Nuclear ERβ2 are Associated with Poor Outcomes following Radical Prostatectomy

Existing data regarding the expression of estrogen receptors (ERs) and prostate cancer outcomes have been limited. We evaluated the relationship of expression profiles of ERβ subtypes and the ER GPR30 (G-protein-coupled receptor-30) with patient factors at diagnosis and outcomes following radical prostatectomy. Tissue microarrays constructed using samples from 566 men with long-term clinical followup were analyzed by immunohistochemistry targeting ERβ1, ERβ2, ERβ5 and GPR30. An experienced pathologist scored receptor distribution and staining intensity. Tumor staining characteristics were evaluated for associations with patient characteristics, recurrence-free survival and prostate cancer specific mortality following radical prostatectomy. Prostate cancer cells had unique receptor subtype staining patterns. ERβ1 demonstrated predominantly nuclear localization while ERβ2, ERβ5 and GPR30 were predominantly cytoplasmic. After controlling for patient factors intense cytoplasmic ERβ1 staining was independently associated with time to recurrence (HR 1.7, 95% CI 1.1-2.6, p = 0.01) and prostate cancer specific mortality (HR 6.6, 95% CI 1.8-24.9, p = 0.01). Intense nuclear ERβ2 staining was similarly independently associated with prostate cancer specific mortality (HR 3.9, 95% CI 1.1-13.4, p = 0.03). Patients with cytoplasmic ERβ1 and nuclear ERβ2 co-staining had significantly worse 15-year prostate cancer specific mortality than patients with expression of only cytoplasmic ERβ1, only nuclear ERβ2 and neither ER (16.4%, 4.3%, 0.0% and 2.0 %, respectively, p = 0.001). Increased cytoplasmic ERβ1 and nuclear ERβ2 expression is associated with worse cancer specific outcomes following radical prostatectomy. These findings suggest that tumor ERβ1 and ERβ2 staining patterns provide prognostic information on patients treated with radical prostatectomy.

GPER/GPR30 Knockout Mice: Effects of GPER on Metabolism.

Endogenous estrogens, predominantly 17β-estradiol (E2), mediate various diverse effects throughout the body in both normal physiology and disease. Actions include development (including puberty) and reproduction as well as additional effects throughout life in the metabolic, endocrine, musculoskeletal, nervous, cardiovascular, and immune systems. The actions of E2 have traditionally been attributed to the classical nuclear estrogen receptors (ERα and ERβ) that largely mediate transcriptional/genomic activities. However, more recently the G protein-coupled estrogen receptor GPER/GPR30 has become recognized as an essential mediator of certain, and particularly rapid, signaling events in response to E2. Murine genetic knockout (KO) models represent an important approach to understand the mechanisms of E2 action in physiology and disease. Studies of GPER KO mice over the last years have revealed functions for GPER in the regulation of obesity, insulin resistance and glucose intolerance, among other areas of (patho)physiology. This chapter focuses on methods for the evaluation of metabolic parameters in vivo and ex vivo with an emphasis on glucose homeostasis and metabolism through the use of glucose and insulin tolerance tests, pancreatic islet and adipocyte isolation and characterization.

Generation of an estrogen receptor beta-iCre knock-in mouse.

A novel knock-in mouse that expresses codon-improved Cre recombinase (iCre) under regulation of the estrogen receptor beta (Esr2) promoter was developed for conditional deletion of genes and for the spatial and/or temporal localization of Esr2 expression. ESR2 is one of two classical nuclear estrogen receptors and displays a spatiotemporal expression pattern and functions that are different from the other estrogen receptor, ESR1. A cassette was constructed that contained iCre, a polyadenylation sequence, and a neomycin selection marker. This construct was used to insert iCre in front of the endogenous start codon of the Esr2 gene of a C57BL/6J embryonic stem cell line via homologous recombination. Resulting Esr2-iCre mice were bred with ROSA26-lacZ and Ai9-RFP reporter mice to visualize cells of functional iCre expression. Strong expression was observed in the ovary, the pituitary, the interstitium of the testes, the head and tail but not body of the epididymis, skeletal muscle, the coagulation gland (anterior prostate), the lung, and the preputial gland. Additional diffuse or patchy expression was observed in the cerebrum, the hypothalamus, the heart, the adrenal gland, the colon, the bladder, and the pads of the paws. Overall, Esr2-iCre mice will serve as a novel line for conditionally ablating genes in Esr2-expressing tissues, identifying novel Esr2-expressing cells, and differentiating the functions of ESR2 and ESR1.

Tissue responsiveness to estradiol and genistein in the sea bass liver and scale

As in mammals, estrogens in fish are essential for reproduction but also important regulators of mineral homeostasis. Fish scales are a non-conventional target tissue responsive to estradiol and constitute a good model to study mineralized tissues effects and mechanisms of action of estrogenic compounds, including phytoestrogens. The responsiveness to estradiol and the phytoestrogen genistein, was compared between the scales and the liver, a classical estrogenic target, in sea bass (Dicentrarchus labrax). Injection with estradiol and genistein significantly increased circulating vitellogenin (for both compounds) and mineral levels (estradiol only) and genistein also significantly increased scale enzymatic activities suggesting it increased mineral turnover. The repertoire, abundance and estrogenic regulation of nuclear estrogen receptors (ESR1, 2a and 2b) and membrane G-protein receptors (GPER and GPER-like) were different between liver and scales, which presumably explains the tissue-specific changes detected in estrogen-responsive gene expression. In scales changes in gene expression mainly consisted of small rapid increases, while in liver strong, sustained increases/decreases in gene expression occurred. Similar but not overlapping gene expression changes were observed in response to both estradiol and genistein. This study demonstrates for the first time the expression of membrane estrogen receptors in scales and that estrogens and phytoestrogens, to which fish may be exposed in the wild or in aquaculture, both affect liver and mineralized tissues in a tissue-specific manner.

Immunohistochemical Performance of Estrogen and Progesterone Receptor Antibodies on the Dako Omnis Staining Platform: Evaluation in Multicenter Studies.

The analysis of estrogen receptor (ER) and progesterone receptor (PR) expression levels by immunohistochemistry is an important part of the initial evaluation of breast cancer and critically important in treatment planning. Anti-ERα (clone EP1) and anti-PR (clone PgR 1294) antibodies are in development for the Dako Omnis automated staining platform. These antibodies are not yet commercially available and are in performance evaluation, including the 4 international, multicenter studies reported here. For each antibody, a reproducibility study and a method comparison study was done in a randomized manner in order to test the antibodies under conditions closest to real-world user conditions. The reproducibility studies included 5 staining runs on the Dako Omnis with 20 formalin-fixed and paraffin-embedded human breast carcinoma specimens in 3 independent laboratories, and the method comparison studies included several hundred specimens stained on the Dako Omnis and on the Autostainer Link 48 platforms. Stained slides were evaluated for nuclear ER or PR expression according to American Society of Clinical Oncology/College of American Pathologists guidelines (≥1% cut-off for positive) by pathologists who were blinded from the staining method and specimen ID. For both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis, high reproducibility agreement rates were obtained on the interrun, interlaboratory, and interobserver endpoints. High concordance rates were observed between the specimens stained on the Dako Omnis platform and the Autostainer Link 48 platform. Staining quality was excellent for both anti-ERα (clone EP1) and anti-PR (clone PgR 1294) on the Dako Omnis. These results suggest that these antibodies are reliable and reproducible tools for immunohistochemistry analysis of ER and PR expression levels in formalin-fixed and paraffin-embedded breast carcinoma tissues on the Dako Omnis platform.

Messenger RNA profile analysis deciphers new Esrrb responsive genes in prostate cancer cells.

BackgroundOrphan nuclear receptor estrogen related receptor β (Esrrb or ERRβ) is well known in stem cells and early embryonic development. However, little is known about its function in cancer.MethodWe investigated the mRNA profile alterations induced by Esrrb expression and its synthetic ligand DY131 in human prostate cancer DU145 cells via RNA-Seq analysis.ResultsWe distinguished 67 mRNAs differentially expressed by Esrrb alone. Although DY131 alone did not change any gene, treatment of DY131 in the presence of Esrrb altered 1161 mRNAs. These observations indicated Esrrb had both ligand-independent and ligand-dependent activity. When Esrrb was expressed, DY131 treatment further regulated 15 Esrrb-altered mRNAs. DY131 acted as an antagonist for 11 of 15 mRNAs (wdr52, f13a1, pxdn, spns2, loc100506599, tagln, loc441454, tkel1, sema3f, zcwpw2, sdc2) and as an agonist for 4 of the 15 mRNAs (rarres3, oasl, padi2, ddx60). Gene ontology analyses showed altered genes are related to transcription and translation regulation, cell proliferation and apoptosis regulation, and cellular metabolism.ConclusionOur results characterized mRNA profiles in DU145 prostate cancer cells driven by Esrrb expression and Esrrb ligand DY131, and provided multiple markers to characterize Esrrb’s function in Esrrb research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12867-015-0049-1) contains supplementary material, which is available to authorized users.

Application of multispectral imaging in quantitative immunohistochemistry study of breast cancer: a comparative study.

Multispectral imaging (MSI) based on imaging and spectroscopy, as relatively novel to the field of histopathology, has been used in biomedical multidisciplinary researches. We analyzed and compared the utility of multispectral (MS) versus conventional red–green–blue (RGB) images for immunohistochemistry (IHC) staining to explore the advantages of MSI in clinical-pathological diagnosis. The MS images acquired of IHC-stained membranous marker human epidermal growth factor receptor 2 (HER2), cytoplasmic marker cytokeratin5/6 (CK5/6), and nuclear marker estrogen receptor (ER) have higher resolution, stronger contrast, and more accurate segmentation than the RGB images. The total signal optical density (OD) values for each biomarker were higher in MS images than in RGB images (all P < 0.05). Moreover, receiver operator characteristic (ROC) analysis revealed that a greater area under the curve (AUC), higher sensitivity, and specificity in evaluation of HER2 gene were achieved by MS images (AUC = 0.91, 89.1 %, 83.2 %) than RGB images (AUC = 0.87, 84.5, and 81.8 %). There was no significant difference between quantitative results of RGB images and clinico-pathological characteristics (P > 0.05). However, by quantifying MS images, the total signal OD values of HER2 positive expression were correlated with lymph node status and histological grades (P = 0.02 and 0.04). Additionally, the consistency test results indicated the inter-observer agreement was more robust in MS images for HER2 (inter-class correlation coefficient (ICC) = 0.95, r s = 0.94), CK5/6 (ICC = 0.90, r s = 0.88), and ER (ICC = 0.94, r s = 0.94) (all P < 0.001) than that in RGB images for HER2 (ICC = 0.91, r s = 0.89), CK5/6 (ICC = 0.85, r s = 0.84), and ER (ICC = 0.90, r s = 0.89) (all P < 0.001). Our results suggest that the application of MS images in quantitative IHC analysis could obtain higher accuracy, reliability, and more information of protein expression in relation to clinico-pathological characteristics versus conventional RGB images. It may become an optimal IHC digital imaging system used in quantitative pathology.

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

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

Differential expression of upstream stimulatory factor (USF) 2 variants in eutopic endometria from women with endometriosis: estradiol regulation.

BackgroundEndometriosis, pro-inflammatory and invasive benign disease estrogen dependent, abnormally express in endometria the enzyme P450Arom, positively regulated by steroid factor-1 (SF-1). Our objective was to study the nuclear protein contents of upstream stimulating factor 2 (USF2a and USF2b), a positive regulator of SF-1, throughout the menstrual cycle in eutopic endometria from women with and without (control) endometriosis and the involvement of nuclear estrogen receptors (ER) and G-coupled protein estrogen receptor (GPER)-1.ResultsUpstream stimulating factor 2 protein contents were higher in mid (USF2b) and late (USF2a and USF2b) secretory phase in eutopic endometria from endometriosis than control (p < 0.05). In isolated control epithelial cells incubated with E2 and PGE2, to resemble the endometriosis condition, the data showed: (a) significant increase of USF2a and USF2b nuclear protein contents when treated with E2, PPT (specific agonist for ERα) or G1 (specific agonist for GPER1); (b) no increase in USF2 binding to SF-1 E-Box/DNA consensus sequence in E2-treated cells; (c) USF2 variants protein contents were not modified by PGE2; (d) SF-1 nuclear protein content was significantly higher than basal when treated with PGE2, E2 or G1, stimulation unaffected by ICI (nuclear ER antagonist); and (e) increased (p < 0.05) cytosolic protein contents of P450Arom when treated with PGE2, E2, PPT or G1 compared to basal, effect that was additive with E2 + PGE2 together. Nevertheless, in endometriosis cells, the high USF2, SF-1 and P450Arom protein contents in basal condition were unmodified.ConclusionThese data strongly suggest that USF2 variants and P450Arom are regulated by E2 through ERα and GPER1, whereas SF-1 through GPER1, visualized by the response of the cells obtained from control endometria, being unaffected the endogenously stimulated cells from endometriosis origin. The lack of E2 stimulation on USF2/SF-1 E-Box/DNA-sequence binding and the absence of PGE2 effect on USF2 variants opposite to the strong induction that they exert on SF1 and P450 proteins suggest different mechanisms and indirect regulations. The sustained USF2 variants protein expression during the secretory phase in eutopic endometria from women with endometriosis may participate in the pathophysiology of this disease strongly associated with infertility and its characteristic endometrial invasion to ectopic sites in the pelvic cavity.

The Activation Function-1 of Estrogen Receptor Alpha Prevents Arterial Neointima Development Through a Direct Effect on Smooth Muscle Cells.

17β-Estradiol (E2) exerts numerous beneficial effects in vascular disease. It regulates gene transcription through nuclear estrogen receptor α (ERα) via 2 activation functions, AF1 and AF2, and can also activate membrane ERα. The role of E2 on the endothelium relies on membrane ERα activation, but the molecular mechanisms of its action on vascular smooth muscle cells (VSMCs) are not fully understood. The aim of this study was to determine which cellular target and which ERα subfunction are involved in the preventive action of E2 on neointimal hyperplasia. To trigger neointimal hyperplasia of VSMC, we used a mouse model of femoral arterial injury. Cre-Lox models were used to distinguish between the endothelial- and the VSMC-specific actions of E2. The molecular mechanisms underlying the role of E2 were further characterized using both selective ERα agonists and transgenic mice in which the ERαAF1 function had been specifically invalidated. We found that (1) the selective inactivation of ERα in VSMC abrogates the neointimal hyperplasia protection induced by E2, whereas inactivation of endothelial and hematopoietic ERα has no effect; (2) the selective activation of membrane ERα does not prevent neointimal hyperplasia; and (3) ERαAF1 is necessary and sufficient to inhibit postinjury VSMC proliferation. Altogether, ERαAF1-mediated nuclear action is both necessary and sufficient to inhibit postinjury arterial VSMC proliferation, whereas membrane ERα largely regulates the endothelial functions of E2. This highlights the exquisite cell/tissue-specific actions of the ERα subfunctions and helps to delineate the spectrum of action of selective ER modulators.