Estrogen Receptor Gene Research Articles

An Optimized Workflow to Evaluate Estrogen Receptor Gene Mutations in Small Amounts of Cell-Free DNA

The detection of mutated genes in cell-free DNA (cfDNA) in plasma has emerged as an important minimally invasive way to obtain detailed information regarding tumor biology. Reliable determination of circulating tumor-derived DNA, often present at a low quantity amidst an excess of normal DNA in plasma, would be of added value for screening and monitoring of cancer patients and for hypothesis-generating studies in valuable retrospective cohorts. Our aim was to establish a workflow to simultaneously assess four hotspot estrogen receptor mutations (mESR1) in cfDNA isolated from only 200 μL of plasma by means of uniplex or multiplex pre-amplification combined with digital PCR. This workflow was then applied in metastatic breast cancer (MBC) patients receiving systemic therapies for MBC. In accordance with previous studies, estrogen receptor mutations were more frequently detected in endocrine-treated MBC patients at progressive disease [34.1% (15/44)] than before the start of endocrine therapy [3.9% (2/51); P=0.001]. For a subset of samples, results were compared with analysis of these mutations by Oncomine-targeted next-generation sequencing, which, although requiring a higher cfDNA input, yielded concordant results. The data establish development and validation of a digital PCR workflow for the simultaneous detection of several tumor-derived mutations in minute amounts of cfDNA and show the potential of this workflow for use on archived volume-limited blood samples.

Unliganded Progesterone Receptor Governs Estrogen Receptor Gene Expression by Regulating DNA Methylation in Breast Cancer Cells.

Breast cancer prognosis and response to endocrine therapy strongly depends on the expression of the estrogen and progesterone receptors (ER and PR, respectively). Although much is known about ERα gene (ESR1) regulation after hormonal stimulation, how it is regulated in hormone-free condition is not fully understood. We used ER-/PR-positive breast cancer cells to investigate the role of PR in ESR1 regulation in the absence of hormones. We show that PR binds to the low-methylated ESR1 promoter and maintains both gene expression and DNA methylation of the ESR1 locus in hormone-deprived breast cancer cells. Depletion of PR reduces ESR1 expression, with a concomitant increase in gene promoter methylation. The high amount of methylation in the ESR1 promoter of PR-depleted cells persists after the stable re-expression of PR and inhibits PR binding to this genomic region. As a consequence, the rescue of PR expression in PR-depleted cells is insufficient to restore ESR1 expression. Consistently, DNA methylation impedes PR binding to consensus progesterone responsive elements. These findings contribute to understanding the complex crosstalk between PR and ER and suggest that the analysis of ESR1 promoter methylation in breast cancer cells can help to design more appropriate targeted therapies for breast cancer patients.

Генетика мигрени: ассоциативные исследования

Статья посвящена анализу имеющихся на сегодняшний день данных генетических полиморфизмов, излучавшихся при мигрени. При мигрени гены-кандидаты мигрени сгруппированы в четыре функциональных семейства генов: гены нервной системы, гены сердечно-сосудистой системы, гормональные, воспалительные гены. Среди генов нервной системы значимость представляют гены, кодирующие структуру и функциональную активность калиевых каналов. Наибольший интерес представляют гены сердечно-сосудистой системы, которые как повышают риск мигрени, так и увеличивают риск коморбидных кардиоваскулярных заболеваний. Среди генов эндокринной системы роль в патогенезе мигрени играют полиморфизмы гена эстрогенового рецептора 1 типа. Для воспалительных генов циклооксегиназы 2, главного комплекса гистосовместимости HLA-DRB1, лимфотоксина альфа, фактора некроза опухоли альфа и бета найдены положительные ассоциации с мигренью. The article focused on analysis of currently available reports on genetic polymorphisms in migraine. The migraine candidate genes were grouped into four functional families: genes of the nervous system, genes of the cardiovascular system, hormonal genes, and inflammatory genes. Among the nervous system genes, the genes encoding the structure and functional activity of potassium channels are of a special significance. Of the greatest interest are the cardiovascular system genes, which increase the risk of both migraine and comorbid cardiovascular diseases. Among the endocrine system genes, polymorphisms of the type 1 estrogen receptor gene play a role in the pathogenesis of migraine. Positive associations with migraine were found for inflammatory genes of cyclooxygenase 2, the main histocompatibility complex HLA-DRB1, lymphotoxin alpha, and tumor necrosis factors alpha and beta.

Cartilaginous Fishes Provide Insights into the Origin, Diversification, and Sexually Dimorphic Expression of Vertebrate Estrogen Receptor Genes.

Vertebrate estrogen receptors (ERs) perform numerous cell signaling and transcriptional regulatory functions. ERɑ (Esr1) and ERβ (Esr2) likely evolved from an ancestral receptor that duplicated and diverged at the protein and cis-regulatory levels, but the evolutionary history of ERs, including the timing of proposed duplications, remains unresolved. Here we report on identification of two distinct ERs in cartilaginous fishes and demonstrate their orthology to ERα and ERβ. Phylogenetic analyses place the ERα/ERβ duplication near the base of crown gnathostomes (jawed vertebrates). We find that ERα and ERβ from little skate (Leucoraja erinacea) and mammals share key subtype-specific residues, indicating conserved protein evolution. In contrast, jawless fishes have multiple non-orthologous Esr genes that arose by parallel duplications. Esr1 and Esr2 are expressed in subtype-specific and sexually dimorphic patterns in skate embryos, suggesting that ERs might have functioned in sexually dimorphic development before the divergence of cartilaginous and bony fishes.

Association of GPER gene polymorphism with social function of children with attention deficit hyperactivity disorder

To assess the association of G protein-coupled estrogen receptor(GPER) gene polymorphism with social function of children with attention deficit hyperactivity disorder (ADHD). The social function of 135 children with ADHD were assessed by Weiss Functional Impairment Scale-Parent form (WFIRS-P). The coding region of GPER gene of all patients was subjected to Sanger sequencing. The association of polymorphisms with the social function of the ADHD children was analyzed. In the case group, the social function scores of Learning and School and Risky Activities of boys were significantly higher than those of girls (t=2.704, P=0.008; t=2.289, P=0.027). No significant difference was found in the genotypic frequencies of the c.-9T/C and c.789G/A loci between different genders. But the learning and school scores of those with a TC genotype for the c.-9T to C locus were significantly higher than those with a TT genotype (t= 2.159, P=0.033). For children with ADHD, the social function of Learning and School of those with a TC genotype of the GPER gene c.-9T/C locus is more severely damaged compared with those with a TT genotype.

Activating human epidermal growth factor receptor 2 (HER2) gene mutation in bone metastases from breast cancer.

In addition to amplification, point mutations of the human epidermal growth factor receptor 2 (HER2) gene (ERBB2) have been shown to activate the corresponding signaling pathway in breast cancer. The prevalence of ERBB2/HER2 mutation in bone metastasis of breast cancer and the associated phenotype are not known. In this study, bone metastases from breast cancer patients (n = 231) were analyzed for ERBB2/HER2 mutation. In 7 patients (3%; median age 70years, range 50-83years), gain-of-function mutations of ERBB2/HER2 were detected. The most frequent mutation was p.L755S (71%). In 29% of mutated cases, p.V777L was found. Lobular breast cancer was present in 71% of mutated cases (n = 5) and in 49% of all samples (n = 231; p = 0.275). Mutation frequency was 4.4% in the lobular subgroup and 17.4% in the pleomorphic subtype of lobular cancer (n = 23), respectively. All but one mutated lobular cancers were of the pleomorphic subtype (p = 0.006). Mutated cancers belonged either to the luminal (n = 4) or to the triple-negative types (n = 3). With regard to protein expression and gene amplification, HER2 was negative in all mutated cases. Among the 14% of metastatic luminal cancers with estrogen receptor gene (ESR1) mutation, conveying resistance against aromatase inhibitors, no concomitant ERBB2/HER2 mutation occurred. We conclude that activating HER2 mutation is present in about 3% of bone metastases from breast cancers, with significantly higher rates in the pleomorphic subtype of lobular cancer. Since mutated cases appear to be HER2-negative by conventional testing, the opportunity for specific anti-HER2 therapy may be missed.

Role of genetic factors in the development of premenstrul syndrome

To identify risks of development of any disease is a priority of modern medicine. The article deals with ESR1 gene polymorphisms and its role in the development of premenstrual. The objective: of this study was to investigate the frequency of polymorphic variants of A-351G gene estrogen receptor ESR1 in patients with various forms of premenstrual syndrome. Materials and methods. Molecular genetic analysis of ESR1 gene polymorphism was determines in 50 women with premenstrual syndrome (25 women of them had edematous form of disease, 25 – neuropsychical one; 25 suffered from mild form, 25 – severe one). 25 women without diagnosis of premenstrual syndrome were examined as controls. Results. The study of A-351G polymorphism estrogen gene ESR1 demonstrated no statistically significant differences in the frequency of distribution of genotypes and alleles between women with premenstrual syndrome and without this pathology. However, the frequency of GG genotype in women with severe PMS was significantly higher in 8.0 times compared with healthy women (χ2=4.87; p=0.03) and in women with edematous form of PMS – in 7.0 times (χ2=3.72; p=0.05). Conclusion. Thus, a polymorphic variant of A-351G estrogen receptor gene ESR1 can be regarded as a marker for the development of premenstrual syndrome. Pathological variant GG genotype is significantly associated with the presence of edematous and severe forms of the disease. Key words: premenstrual syndrome, genetic factors, development.

Zearalenone-Promoted Follicle Growth through Modulation of Wnt-1/β-Catenin Signaling Pathway and Expression of Estrogen Receptor Genes in Ovaries of Postweaning Piglets.

Feedstuffs are severely contaminated by zearalenone (ZEA) worldwide. A specific dietary level of ZEA could cause malformations of the reproductive organs of sows, false estrus, decreased litter size, and abortion. However, the underlying mechanisms are still not clear. The objectives of the present study were to assess the effects of ZEA on morphology, distribution, and expression of estrogen receptors (ERα and ERβ) in the ovaries of postweaning piglets. Furthermore, the relationship between ERs/glycogen synthase kinase (GSK)-3β-dependent pathways mediated by ZEA and the Wnt-1/β-catenin signaling pathway was examined. Forty healthy weaning piglets were allocated to the following four treatment groups: piglets fed with basal diet only (control), and ZEA0.5, ZEA1.0, and ZEA1.5, which were fed basal diets supplemented with ZEA at 0.5, 1.0, and 1.5 mg·kg-1, respectively. Then, the expression of GSK-3β, ERα, ERβ, and Wnt-1/β-catenin were examined histomorphologically and immunohistochemically. Results showed that the proportion of primordial follicles (PrF's) decreased ( p < 0.001) but that of atretic primordial follicles (APFs) increased ( p < 0.001) with increasing dietary ZEA levels. More interestingly, the immunopositivity of ERβ in the ovaries was stronger than that of ERα with the same treatment. The relative mRNA and protein expression levels of ERα, ERβ, Wnt-1, β-catenin, and GSK-3β in the ovaries of postweaning gilts increased linearly ( p < 0.05) as dietary ZEA concentrations increased. Moreover, the accumulation of Wnt-1 and β-catenin in the ovaries indicated that ZEA activated the Wnt-1/β-catenin pathway, mediated by ERs/GSK-3β. Our results strongly suggested that ovarian follicles in the ZEA (0.5-1.5 mg·kg-1)-treated groups were highly proliferative state, indicating that ZEA promoted ovarian development. The results also suggested that ZEA activates the ERs/GSK-3β-dependent Wnt-1/β-catenin signaling pathway, indicating its important role in accelerating development of the ovaries.

Association between estrogen receptor gene polymorphisms and susceptibility to hepatocellular carcinoma: A meta-analysis

Association between estrogen receptor gene polymorphisms and susceptibility to hepatocellular carcinoma: A meta-analysis

Associations of ESR1 and ESR2 gene polymorphisms with metabolic syndrome and its components in postmenopausal women

ObjectivesMetabolic syndrome (MS) affects a quarter of Polish people and is associated with diabetes mellitus type 2 and ischemic heart disease. The prevalence of MS in postmenopausal women can be increased by the lack of protective effects of oestrogens. In the near future, because of the general increase in life expectancy, the number of postmenopausal women will rise substantially. Therefore, investigating both the environmental and the genetic factors predisposing to MS may have a great impact on women’s health. The aim of this study was to determine whether particular oestrogen receptor (ESR) gene polymorphisms can predispose to the development of MS in women after menopause. Study designThe sample consisted of 147 postmenopausal women. In addition to collecting medical history and analyzing body composition using the TANITA scale, patient’s waist size, blood pressure, serum lipids, glucose, insulin, C-reactive protein and adiponectin were measured. The analysis of ESR gene polymorphisms was performed using the Sequenom MassARRAY platform. ResultsThree out of ten analyzed polymorphisms in the ESR1 gene (rs2234693, rs6902771, rs7774230) and one out of eight analyzed polymorphisms in the ESR2 gene (rs3020449) were associated with MS. The ESR1 rs2234693, rs6902771 and rs7774230 polymorphisms were associated with serum concentrations of high-density lipoproteins. The ESR2 rs3020449 polymorphism was associated with serum concentrations of total cholesterol and low-density lipoprotein. Four ESR1 polymorphisms (rs1709183, rs2234693, rs6902771, rs7774230) were associated with total fat tissue content. ConclusionsBearing the particular alleles at the ESR gene polymorphisms may impact the development of MS and some of the ESR polymorphisms may influence serum cholesterol concentrations in women after menopause.

Abstract PL01-01: Liquid biopsy: Novel technologies and clinical applications

Abstract PL01-01: Liquid biopsy: Novel technologies and clinical applications

Epigenomic and Transcriptomic Characterization of Secondary Breast Cancers.

Molecular alterations impact tumor prognosis and response to treatment. This study was designed to identify transcriptomic and epigenomic signatures of breast cancer (BC) tumors from patients with any prior malignancy. RNA-sequencing and genome-wide DNA methylation profiles from BCs were generated in the Cancer Genome Atlas project. Patients with secondary breast cancer (SBC) were separated by histological subtype and matched to primary breast cancer controls to create two independent cohorts of invasive ductal (IDC, n = 36) and invasive lobular (ILC, n = 40) carcinoma. Differentially expressed genes, as well as differentially methylated genomic regions, were integrated to identify epigenetically regulated abnormal gene pathways in SBCs. Differentially expressed genes were identified in IDC SBCs (n = 727) and in ILC SBCs (n = 261; Wilcoxon's test; P < 0.05). In IDC SBCs, 105 genes were upregulated and hypomethylated, including an estrogen receptor gene, and 73 genes were downregulated and hypermethylated, including genes involved in antigen presentation and interferon response pathways (HLA-E, IRF8, and RELA). In ILC SBCs, however, only 17 genes were synchronously hypomethylated and upregulated, whereas 46 genes hypermethylated and downregulated. Interestingly, the SBC gene expression signatures closely corresponded with each histological subtype with only 1.51% of genes overlapping between the two histological subtypes. Differential gene expression and DNA methylation signatures are seen in both IDC and ILC SBCs, including genes that are relevant to tumor growth and proliferation. Differences in gene expression signatures corresponding with each histological subtype emphasize the importance of disease subtype-specific evaluations of molecular alterations.

Clinical application and utility of genomic assays in early-stage breast cancer: key lessons learned to date.

Early-stage hormone receptor-positive breast cancer is the most common subtype and stage presenting in countries with organized screening programs. Standard clinical and pathologic factors are routinely used to support prognosis and decisions about adjuvant therapies. Hormone receptor and her2 status are essential for decision-making about the use of adjuvant hormonal and anti-her2 therapies respectively. Genomic assays are now commercially available to aid in either further prognostication or in refining the potential benefit of adjuvant chemotherapy. The current genomic assays all generally quantify estrogen receptor and proliferation gene sets (among others) by rna expression, although the specific genes assayed are quite discordant. The present review focuses on the pivotal studies in which each assay attempted to demonstrate clinical utility, with an emphasis on prospective trial data for each assay, if available. Using genomic assays, health care providers will increasingly be able to individualize therapy or de-escalate therapy, optimizing clinic benefit while minimizing toxicities from systemic therapies.

Polymorphism FSHR (-29G/A) as a genetic agent together with ESRI (XbaIG/A) in women with poor response to controlled ovarian hyperstimulation.

One of the major problems in IVF is the poor response of the ovary to gonadotropins. ESRI and FSHR are two effective genes on controlled ovarian hyperstimulation (COH). Evaluating the correlation of alleles and genotypes of polymorphism (-29G/A) located in the FSH receptor gene and polymorphism (XbaI G/A) located in Estrogen receptor genes with the ovary's response would help to anticipate the results of ovulation in IVF cycles. In the present study, two hundred (200) blood samples were taken from infertile women aged 20 to 39 who were under IVF therapy. After DNA extraction from the samples, real-time PCR was performed using a specific probe-primer. Statistical analysis revealed that the frequency of alleles and genotypes of polymorphisms (-29G/A) and (XbaI A/G) in women with normal to poor response did not have significant correlation.

The Impact of ESR1 Mutations on the Treatment of Metastatic Breast Cancer.

After nearly 20years of research, it is now established that mutations within the estrogen receptor (ER) gene, ESR1, frequently occur in metastatic breast cancer and influence response to hormone therapy. Though early studies presented differing results, sensitive sequencing techniques now show that ESR1 mutations occur at a frequency between 20 and 40% depending on the assay method. Recent studies have focused on several "hot spot mutations," a cluster of mutations found in the hormone-binding domain of the ESR1 gene. Throughout the course of treatment, tumor evolution can occur, and ESR1 mutations emerge and become enriched in the metastatic setting. Sensitive techniques to continually monitor mutant burden in vivo are needed to effectively treat patients with mutant ESR1. The full impact of these mutations on tumor response to different therapies remains to be determined. However, recent studies indicate that mutant-bearing tumors may be less responsive to specific hormonal therapies, and suggest that aromatase inhibitor (AI) therapy may select for the emergence of ESR1 mutations. Additionally, different mutations may respond discretely to targeted therapies. The need for more preclinical mechanistic studies on ESR1 mutations and the development of better agents to target these mutations are urgently needed. In the future, sequential monitoring of ESR1 mutational status will likely direct personalized therapeutic regimens appropriate to each tumor's unique mutational landscape.

Prenatal exposure to benzophenone-3 (BP-3) induces apoptosis, disrupts estrogen receptor expression and alters the epigenetic status of mouse neurons

Current evidence indicates that benzophenone-3 (BP-3) can pass through the placental and blood–brain barriers and thus can likely affect infant neurodevelopment. Despite widespread exposure, data showing the effects of BP-3 on the developing nervous system are scarce. This study revealed for the first time that prenatal exposure to BP-3 led to apoptosis and neurotoxicity, altered the levels of estrogen receptors (ERs) and changed the epigenetic status of mouse neurons. In the present study, subcutaneous injections of pregnant mice with BP-3 at 50 mg/kg, which is an environmentally relevant dose, evoked activation of caspase-3 and lactate dehydrogenase (LDH) release as well as substantial loss of mitochondrial membrane potential in neocortical cells of their embryonic offspring. Apoptosis-focused microarray analysis of neocortical cells revealed up-regulation of 22 genes involved in apoptotic cell death. This effect was supported by increased BAX and CASP3 mRNA and protein levels, as evidenced by qPCR, ELISAs and western blots. BP-3-induced apoptosis and neurotoxicity were accompanied by decreases in the mRNA and protein expression levels of ESR1 and ESR2 (also known as ERα and ERβ), with a simultaneous increase in GPER1 (also known as GPR30) expression. In addition to the demonstration that treatment of pregnant mice with BP-3 induced apoptosis, caused neurotoxicity and altered ERs expression levels in neocortical cells of their embryonic offspring, we showed that prenatal administration of BP-3 inhibited global DNA methylation as well as reduced DNMTs activity. BP-3 also caused specific hypomethylation of the genes Gper1 and Bax, an effect that was accompanied by increased mRNA and protein expression levels. In addition, BP-3 caused hypermethylation of the genes Esr1, Esr2 and Bcl2, which could explain the reduced mRNA and protein levels of the estrogen receptors. This study demonstrated for the first time that prenatal exposure to BP-3 caused severe neuronal apoptosis that was accompanied by impaired ESR1/ESR2 expression, enhanced GPER1 expression, global DNA hypomethylation and altered methylation statuses of apoptosis-related and ERs genes. We suggest that the effects of BP-3 in embryonic neurons may be the fetal basis of the adult onset of nervous system disease.

Elevated seminal plasma estradiol and epigenetic inactivation of ESR1 and ESR2 is associated with CP/CPPS.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is associated with urinary tract symptoms and hormonal imbalances amongst others. The heterogeneous clinical presentation, unexplored molecular background and lack of prostate biopsies complicate therapy. Here, using liquid biopsies, we performed a comprehensive translational study on men diagnosed with CP/CPPS type III (n = 50; median age 39.8, range 23–65) and age-matched controls (n = 61; median age 36.8, range 20–69), considering biochemical parameters of blood and ejaculates, and epigenetic regulation of the estrogen receptor genes (ESR1 and ESR2) in leukocytes isolated from blood (systemic regulation) and in somatic cells isolated from ejaculates (local regulation). We found elevated 17β-estradiol (E2) levels in seminal plasma, but not in blood plasma, that was significantly associated with CP/CPPS and impaired urinary tract symptoms. In ejaculated somatic cells of CP/CPPS patients we found that ESR1 and ESR2 were both significantly higher methylated in CpG-promoters and expressionally down-regulated in comparison to controls. Mast cells are reported to contribute to CP/CPPS and are estrogen responsive. Consistent with this, we found that E2 –treatment of human mast cell lines (HMC-1 and LAD2) resulted in altered cytokine and chemokine expression. Interestingly, in HMC-1 cells, possessing epigenetically inactivated ESR1 and ESR2, E2 –treatment led to a reduced transcription of a number of inflammatory genes. Overall, these data suggest that elevated local E2 levels associate with an epigenetic down-regulation of the estrogen receptors and have a prominent role in CP/CPPS. Investigating E2 levels in semen could therefore serve as a promising biomarker to select patients for estrogen targeted therapy.

Thermo‐responsive Estrogen Receptor‐fusions for Spatiotemporal Control of Signaling and Gene Regulation

Many diseases are caused by aberrant cell signaling, leading to a dysregulation of proteins responsible for cell maintenance and repair. Understanding these events require a precise manipulation of the cells' machinery, but many technologies used to study signaling and transcription lack the temporal control to rapidly modulate these biologic processes. This limitation could be addressed by the introduction of elastin‐like polypeptides (ELPs) which are synthetic, thermally responsive biopolymers that self‐assemble into microdomains of ELP‐fusions within cells. Our research group recently discovered that these fusion proteins can modulate precise processes involved in cell trafficking. They are now being investigated to modulate estrogen receptor (ER) signaling and gene regulation. ER has been implicated in a number of diseases such as breast cancer and Sjögren's Syndrome and although this nuclear hormone receptor has been well studied, the lack of drug therapy and resistance, requires attention to develop a deeper understanding of the underlying mechanisms. Thus, ELP‐ERɑ fusions have been generated and successfully expressed in CHO, HeLa and 293T cell lines. They coalesce in the nucleus at physiological temperatures and rapidly solubilize at room temperature. These preliminary data indicate that these fusions retain ELPs self‐assembling properties and may control ER signaling and gene regulation. The ability to rapidly and reversibly, with spatiotemporal control and precision, may allow this fusion to serve as a powerful tool to study ER‐mediated biological processes.Support or Funding InformationThis funding is supported by the National Institutes of Health (NIGMS 1R01GM114839‐02) and the American College of Clinical Pharmacy Research Institute Futures Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Defensin γ‐Thionin from Capsicum chinense Induces Apoptosis in the Human Breast Cancer Cell Line MCF‐7 and Regulate Histone H3 Epigenetic Modifications

Cellular homeostasis is regulated by genetic and epigenetic mechanisms controlling cell proliferation and differentiation; dysregulation in these functions has been associated to cancer. Breast cancer is considered the first worldwide cause of cancer death in women. Antimicrobial peptides (AMPs) have showed cytotoxic activity against diverse cancer cells through different mechanisms; however, only the effects of animal AMPs have been well characterized. Therefore, the aim of this work was to determine the cytotoxic effect of the plant defensin γ‐thionin on MCF‐7 breast cancer cells and to characterize its mechanism of action.The cytotoxic effect of γ‐thionin on MCF‐7 cells was assessed by trypan blue exclusion assays. This AMP was cytotoxic in a concentration‐dependent manner, with an IC50=117.29 μg/ml. Interestingly, viability of normal bovine mammary epithelial cells and peripheral blood mononuclear cells was unaffected. Additionally, this AMP induced apoptosis in MCF‐cells and did not affect the membrane integrity or cytosolic calcium. Further, γ‐thionin IC50 increased the expression of apoptosis related genes such as caspase 7, 8, 9, Apaf‐1 and cytochrome c. Transcriptional regulation has been linked to H3 epigenetic marks, for this reason, the global levels of H3 modifications were determined by western blotting. Data showed that γ‐thionin IC50 increases the mark of active transcription H3K9ac after 24 h of treatment. Intriguingly, the level of H3S10ph in cells treated with the defensin was higher at 12 h, which suggests an active proliferation mark. The mRNA expression of desacetylases HDAC1, HDAC2 and demethylase KDM4A was increased after 6 h of γ‐thionin treatment, while the expression of the kinase AURKB was induced at 12 h. Also, the expression of estrogen receptor gene (ESR1) was increased at 6 h and decreased at 24 h. Taken together, these results show that γ‐thionin induces apoptosis in MCF‐7 breast cancer cells and regulates changes in genetic expression, which could be associated with H3 epigenetic modifications. This is the first study of a plant AMP inducing cytotoxicity through epigenetic mechanisms in a hormone response breast cancer model.Support or Funding InformationCIC‐UMSNH 14.5This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Immune-Specific Expression and Estrogenic Regulation of the Four Estrogen Receptor Isoforms in Female Rainbow Trout (Oncorhynchus mykiss).

Genomic actions of estrogens in vertebrates are exerted via two intracellular estrogen receptor (ER) subtypes, ERα and ERβ, which show cell- and tissue-specific expression profiles. Mammalian immune cells express ERs and are responsive to estrogens. More recently, evidence became available that ERs are also present in the immune organs and cells of teleost fish, suggesting that the immunomodulatory function of estrogens has been conserved throughout vertebrate evolution. For a better understanding of the sensitivity and the responsiveness of the fish immune system to estrogens, more insight is needed on the abundance of ERs in the fish immune system, the cellular ratios of the ER subtypes, and their autoregulation by estrogens. Consequently, the aims of the present study were (i) to determine the absolute mRNA copy numbers of the four ER isoforms in the immune organs and cells of rainbow trout, Oncorhynchus mykiss, and to compare them to the hepatic ER numbers; (ii) to analyse the ER mRNA isoform ratios in the immune system; and, (iii) finally, to examine the alterations of immune ER mRNA expression levels in sexually immature trout exposed to 17β-estradiol (E2), as well as the alterations of immune ER mRNA expression levels in sexually mature trout during the reproductive cycle. All four ER isoforms were present in immune organs—head kidney, spleen-and immune cells from head kidney and blood of rainbow trout, but their mRNA levels were substantially lower than in the liver. The ER isoform ratios were tissue- and cell-specific, both within the immune system, but also between the immune system and the liver. Short-term administration of E2 to juvenile female trout altered the ER mRNA levels in the liver, but the ERs of the immune organs and cells were not responsive. Changes of ER gene transcript numbers in immune organs and cells occurred during the reproductive cycle of mature female trout, but the changes in the immune ER profiles differed from those in the liver and gonads. The correlation between ER gene transcript numbers and serum E2 concentrations was only moderate to low. In conclusion, the low mRNA numbers of nuclear ER in the trout immune system, together with their limited estrogen-responsiveness, suggest that the known estrogen actions on trout immunity may be not primarily mediated through genomic actions, but may involve other mechanisms, such as non-genomic pathways or indirect effects.