Including Estrogen Receptor Alpha Research Articles

Androgen regulation of behavioral stress responses and the hypothalamic-pituitary-adrenal axis

Testosterone is a powerful steroid hormone that can impact the brain and behavior in various ways, including regulating behavioral and neuroendocrine (hypothalamic-pituitary-adrenal (HPA) axis) stress responses. Early in life androgens can act to alter development of brain regions associated with stress regulation, which ultimately impacts the display of stress responses later in life. Adult circulating androgens can also influence the expression of distinct genes and proteins that regulate stress responses. These changes in the brain are hypothesized to underlie the potent effects of androgens in regulating behaviors related to stress and stress-induced activation of the HPA axis. Androgens can induce alterations in these functions through direct binding to the androgen receptor (AR) or following conversion to estrogens and subsequent binding to estrogen receptors including estrogen receptor alpha (ERα), beta (ERβ), and G protein-coupled estrogen receptor 1 (GPER1). In this review, we focus on the role of androgens in regulating behavioral and neuroendocrine stress responses at different stages of the lifespan and the sex hormone receptors involved in regulating these effects. We also review the specific brain regions and cell phenotypes upon which androgens are proposed to act to regulate stress responses with an emphasis on hypothalamic and extended amygdala subregions. This knowledge of androgen effects on these neural systems is critical for understanding how sex hormones regulate stress responses.

Reduction of daily-use parabens and phthalates reverses accumulation of cancer-associated phenotypes within disease-free breast tissue of study subjects

Estrogenic overstimulation is carcinogenic to the human breast. Personal care products (PCPs) commonly contain xenoestrogens (XE), such as parabens and phthalates. Here, we identified the adverse effects of persistent exposure to such PCPs directly within human estrogen responsive breast tissue of subjects enrolled in a regimen of reduced XE use (REDUXE). Pre- and post-intervention fine needle aspirates (FNAs) of the breast were collected from healthy volunteers who discontinued the use of paraben and phthalate containing PCPs over a 28 d period. Based on high-dimensional gene expression data of matched FNA pairs of study subjects, we demonstrate a striking reversal of cancer-associated phenotypes, including the PI3K-AKT/mTOR pathway, autophagy, and apoptotic signaling networks within breast cells of REDUXE compliant subjects. These, and other altered phenotypes were detected together with a significant reduction in urinary parabens and phthalate metabolites. Moreover, in vitro treatment of paired FNAs with 17β-estradiol (E2), displayed a ‘normalizing’ impact of REDUXE on gene expression within known E2-modulated pathways, and on functional endpoints, including estrogen receptor alpha: beta ratio, and S-phase fraction of the cell cycle. In a paradigm shifting approach facilitated by community-based participatory research, REDUXE reveals unfavorable consequences from exposure to XEs from daily-use PCPs. Our findings illustrate the potential for REDUXE to suppress pro-carcinogenic phenotypes at the cellular level towards the goal of breast cancer prevention.

Combination of Estrogen Receptor Alpha and Histological Type Helps to Predict Lymph Node Metastasis in Patients with Stage IA2 to IIA2 Cervical Cancer.

ObjectiveThis study aimed to identify a subset of patients with stage IA2 to IIA2 cervical cancer who are at low risk of lymph node metastasis (LNM) using pathological parameters including estrogen receptor alpha (ERα) and progesterone receptor (PR).MethodsThe clinical data of patients with stage IA2 to IIA2 cervical cancer who underwent radical surgery between 2014 and 2015 were retrospectively reviewed. Immunohistochemical staining was used to determine the expression of ERα and PR. A low-risk criterion for LNM was identified using logistic regression analysis, and its performance was estimated through receiver-operating characteristic curve analysis.ResultsOf 263 patients, 57 (21.7%) had pathological LNM. ERα (adjusted odds ratio [aOR], 7.582; 95% confidence interval [CI], 2.991–19.222; P < 0.001) and squamous cell carcinoma (aOR, 3.520; 95% CI, 1.887–6.568; P < 0.001) were identified as independent predictors for no LNM by multivariate logistic regression analysis, while PR had no effect on LNM. The rate of LNM was 1.4% for low-risk patients (n = 73) identified as ERα positive with squamous cell carcinoma. The 5-year disease-free survival in low-risk patients was significantly greater than in those negative for ERα and/or those with non-squamous cell carcinoma (96.9% vs 80.1%, P = 0.002).ConclusionERα positivity and squamous cell carcinoma are associated with a low risk of LNM in patients with stage IA2 to IIA2 cervical cancer. Hence, those patients without a low risk of LNM could be considered for definitive chemoradiotherapy to avoid unnecessary surgery.

A set of accessible enhancers enables the initial response of breast cancer cells to physiological progestin concentrations

Here, we report that in T47D breast cancer cells 50 pM progestin is sufficient to activate cell cycle entry and the progesterone gene expression program. At this concentration, equivalent to the progesterone blood levels found around the menopause, progesterone receptor (PR) binds only to 2800 genomic sites, which are accessible to ATAC cleavage prior to hormone exposure. These highly accessible sites (HAs) are surrounded by well-organized nucleosomes and exhibit breast enhancer features, including estrogen receptor alpha (ERα), higher FOXA1 and BRD4 (bromodomain containing 4) occupancy. Although HAs are enriched in RAD21 and CTCF, PR binding is the driving force for the most robust interactions with hormone-regulated genes. HAs show higher frequency of 3D contacts among themselves than with other PR binding sites, indicating colocalization in similar compartments. Gene regulation via HAs is independent of classical coregulators and ATP-activated remodelers, relying mainly on MAP kinase activation that enables PR nuclear engagement. HAs are also preferentially occupied by PR and ERα in breast cancer xenografts derived from MCF-7 cells as well as from patients, indicating their potential usefulness as targets for therapeutic intervention.

17-β estradiol signalling affects cardiovascular and cancer pathogenesis by regulating the crosstalk between transcription factors and EC-miRNAs

17β-estradiol (E2) or the bioactive form of estrogen has wide range of impact in regulating human physiology. It not only regulates organ development but also controls immune responses. E2 impacts the recruitment of immune cells to endothelium by regulating the expression of pattern recognition receptors. As endothelium is central to human physiology, it becomes a good target for many cancer and cardiovascular diseases. A broad range of genes get dysregulated during the course of pathophysiological events and many of them are regulated via E2 signalling. Recent evidence shows that E2 regulate the expression of many endothelial cell specific microRNAs (EC-miRNA), thereby affecting expression and stability of many factors including estrogen receptor alpha. In this study, we analysed two distinct datasets of different endothelial cell models to discern a set of genes that potentially play very crucial role in regulating general physiological as well as pathophysiological events. GEO2R and Cytoscape were used to analyze the datasets GSE51535 and GSE16683. DAVID and ClueGO functional annotation of the genes revealed association with genetically associated disease classes such as cancer and cardiovascular. KEGG pathways, Reactome pathways and Wiki-pathways were used for pathway enrichment analysis. One of the enriched pathways includes the synthesis of pri-miRNA by RNA polymerase II. Endothelial cell specific miRNA and target gene association revealed key miRNAs and their involvement in regulatory pathways. Overall, our study highlights the complexity of miRNA mediated E2 regulation of genes in endothelial cells and provides potential target genes for therapeutics in cancer and cardiovascular pathophysiological conditions.

The association between estrogen receptor 2 gene polymorphism and complexity of coronary artery disease: an analysis in elective percutaneous coronary intervention patients

BackgroundOne of the most common causes of death in the world is coronary artery disease (CAD). Estrogen, the most important early sex hormones in women, plays an important role in the risk reduction of cardiovascular disease (CVD). Expression of estrogen as well as its receptors including estrogen receptor alpha (ER1) and estrogen receptor beta (ER2) might have an association with the severity or the complexity of CAD. Since most articles have focused on the relationship between ER1 gene polymorphism and CAD, in this study, we aimed to evaluate the association of two ER2 gene polymorphisms, rs4986938 (AluI) and rs1256049 (RsaI), with the severity of CAD.Methods148 patients with confirmed CAD who underwent elective percutaneous coronary intervention (PCI) were included in this study. Blood samples were collected before coronary angiography and ER2 gene polymorphisms were analyzed by the PCR–RFLP method. The STNTAX Score (SS), grading system for CAD complexity, was evaluated by an interventional cardiologist who was blinded to other data.Results110 men and 38 women were participated in this study. Our results revealed a statistically significant relationship between SS and rs4986938 polymorphism of ER2 in men. In contrast, there was no association between rs1256049 genotypes and SS after performing regression analysis.ConclusionsBesides to the estrogen level, the genetic variation of its receptors might play an important role in the severity or the complexity of CAD. According to our results, rs4986938 polymorphism of ER2 gene may assert a pivotal role in the severity of CAD in men; however, this assumption needs to be proved in studies with a larger population.

The Role of Estrogen Receptors in Cardiovascular Disease.

Cardiovascular Diseases (CVDs) are the leading cause of death globally. More than 17 million people die worldwide from CVD per year. There is considerable evidence suggesting that estrogen modulates cardiovascular physiology and function in both health and disease, and that it could potentially serve as a cardioprotective agent. The effects of estrogen on cardiovascular function are mediated by nuclear and membrane estrogen receptors (ERs), including estrogen receptor alpha (ERα), estrogen receptor beta (ERβ), and G-protein-coupled ER (GPR30 or GPER). Receptor binding in turn confers pleiotropic effects through both genomic and non-genomic signaling to maintain cardiovascular homeostasis. Each ER has been implicated in multiple pre-clinical cardiovascular disease models. This review will discuss current reports on the underlying molecular mechanisms of the ERs in regulating vascular pathology, with a special emphasis on hypertension, pulmonary hypertension, and atherosclerosis, as well as in regulating cardiac pathology, with a particular emphasis on ischemia/reperfusion injury, heart failure with reduced ejection fraction, and heart failure with preserved ejection fraction.

Pharmacological Targets and the Biological Mechanisms of Formononetin for Alzheimer's Disease: A Network Analysis.

BackgroundThis study aimed to identify the pharmacological targets and mechanisms of action of the traditional Chinese medicine, formononetin, in the treatment of Alzheimer’s disease (AD) using network pharmacological analysis.Material/MethodsTargets of AD were obtained by using DisGeNET gene discovery platform, the herbal ingredients target (HIT) database, the SuperPred, and the SwissTargetPrediction compound target prediction platforms. Pathogenic and therapeutic targets were imported to the STRING biological database, and Cytoscape network integration software was used to construct component-target and disease-target interaction networks. Core targets were identified by topological analysis and were further tested to identify the biological processes and signaling pathways.ResultsSeven key target genes for formononetin in the treatment of patients with AD were identified, including estrogen receptor alpha (ESR1), peroxisome proliferator-activated receptor gamma (PPARG), tumor protein p53 (TP53), sirtuin 1 (SIRT1), tumor necrosis factor (TNF), cytochrome P450 19A1 (CYP19A1), and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2). The biological processes included hormone metabolism, regulation of nucleoside, nucleotide and nucleic acid metabolism, apoptosis, energy pathways, metabolism, cell communication, and signal transduction. The signaling pathways included histone acetylation and deacetylation (HDAC) class I, regulation of p38-alpha/beta, p38 mitogen-activated protein kinase (MAPK) signaling pathway, bone morphogenetic protein (BMP) receptor signaling, interleukin-1 (IL1) mediated signaling events, the tumor necrosis factor (TNF) receptor signaling pathway, and cytoplasmic and nuclear Smad2/3 signaling.ConclusionsPharmacological network analysis was used to identify the gene targets and mechanisms of formononetin treatment in patients with AD.

Characterizing steroid hormone receptor chromatin binding landscapes in male and female breast cancer

Male breast cancer (MBC) is rare and poorly characterized. Like the female counterpart, most MBCs are hormonally driven, but relapse after hormonal treatment is also noted. The pan-hormonal action of steroid hormonal receptors, including estrogen receptor alpha (ERα), androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) in this understudied tumor type remains wholly unexamined. This study reveals genomic cross-talk of steroid hormone receptor action and interplay in human tumors, here in the context of MBC, in relation to the female disease and patient outcome. Here we report the characterization of human breast tumors of both genders for cistromic make-up of hormonal regulation in human tumors, revealing genome-wide chromatin binding landscapes of ERα, AR, PR, GR, FOXA1, and GATA3 and enhancer-enriched histone mark H3K4me1. We integrate these data with transcriptomics to reveal gender-selective and genomic location-specific hormone receptor actions, which associate with survival in MBC patients.

Rapid effects of estradiol on aggression depend on genotype in a species with an estrogen receptor polymorphism

The white-throated sparrow (Zonotrichia albicollis) represents a powerful model in behavioral neuroendocrinology because it occurs in two plumage morphs that differ with respect to steroid-dependent social behaviors. Birds of the white-striped (WS) morph engage in more territorial aggression than do birds of the tan-striped (TS) morph, and the TS birds engage in more parenting behavior. This behavioral polymorphism is caused by a chromosomal inversion that has captured many genes, including estrogen receptor alpha (ERα). In this study, we tested the hypothesis that morph differences in aggression might be explained by differential sensitivity to estradiol (E2). We administered E2 non-invasively to non-breeding white-throated sparrows and quantified aggression toward a conspecific 10 min later. E2 administration rapidly increased aggression in WS birds but not TS birds, consistent with our hypothesis that differential sensitivity to E2 may at least partially explain morph differences in aggression. To query the site of E2 action in the brain, we administered E2 and quantified Egr-1 expression in brain regions in which expression of ERα is known to differ between the morphs. E2 treatment decreased Egr-1 immunoreactivity in nucleus taeniae of the amygdala, but this effect did not depend on morph. Overall, our results support a role for differential effects of E2 on aggression in the two morphs, but more research will be needed to determine the neuroanatomical site of action.

Polychlorinated Biphenyl-Related Alterations of the Expression of Essential Genes in Harbour Seals (Phoca vitulina) from Coastal Sites in Canada and the United States.

As long-lived marine mammals found throughout the temperate coastal waters of the North Pacific and Atlantic Oceans, harbour seals (Phoca vitulina) have become an invaluable sentinel of food-web contamination. Their relatively high trophic position predisposes harbour seals to the accumulation of harmful levels of persistent organic pollutants (POPs). We obtained skin/blubber biopsy samples from live-captured young harbour seals from various sites in the northeastern Pacific (British Columbia, Canada, and Washington State, USA) as well as the northwestern Atlantic (Newfoundland and Quebec, Canada). We developed harbour seal-specific primers to investigate the potential impact of POP exposure on the expression of eight important genes. We found correlations between the blubber mRNA levels of three of our eight target genes and the dominant persistent organic pollutant in seals [polychlorinated biphenyls (PCBs)] including estrogen receptor alpha (Esr1: r 2=0.12, p=0.038), thyroid hormone receptor alpha (Thra: r 2=0.16; p=0.028), and glucocorticoid receptor (Nr3c1: r 2=0.12; p=0.049). Age, sex, weight, and length were not confounding factors on the expression of genes. Although the population-level consequences are unclear, our results suggest that PCBs are associated with alterations of the expression of genes responsible for aspects of metabolism, growth and development, and immune function. Collectively, these results provide additional support for the use of harbour seals as indicators of coastal food-web contamination.

Estrogen receptor α and aryl hydrocarbon receptor independent growth inhibitory effects of aminoflavone in breast cancer cells.

BackgroundNumerous studies have implicated the aryl hydrocarbon receptor (AhR) as a potential therapeutic target for several human diseases, including estrogen receptor alpha (ERα) positive breast cancer. Aminoflavone (AF), an activator of AhR signaling, is currently undergoing clinical evaluation for the treatment of solid tumors. Of particular interest is the potential treatment of triple negative breast cancers (TNBC), which are typically more aggressive and characterized by poorer outcomes. Here, we examined AF’s effects on two TNBC cell lines and the role of AhR signaling in AF sensitivity in these model cell lines.MethodsAF sensitivity in MDA-MB-468 and Cal51 was examined using cell counting assays to determine growth inhibition (GI50) values. Luciferase assays and qPCR of AhR target genes cytochrome P450 (CYP) 1A1 and 1B1 were used to confirm AF-mediated AhR signaling. The requirement of endogenous levels of AhR and AhR signaling for AF sensitivity was examined in MDA-MB-468 and Cal51 cells stably harboring inducible shRNA for AhR. The mechanism of AF-mediated growth inhibition was explored using flow cytometry for markers of DNA damage and apoptosis, cell cycle analysis, and β-galactosidase staining for senescence. Luciferase data was analyzed using Student’s T test. Three-parameter nonlinear regression was performed for cell counting assays.ResultsHere, we report that ERα-negative TNBC cell lines MDA-MB-468 and Cal51 are sensitive to AF. Further, we presented evidence suggesting that neither endogenous AhR expression levels nor downstream induction of AhR target genes CYP1A1 and CYP1B1 is required for AF-mediated growth inhibition in these cells. Between these two ERα negative cell lines, we showed that the mechanism of AF action differs slightly. Low dose AF mediated DNA damage, S-phase arrest and apoptosis in MDA-MB-468 cells, while it resulted in DNA damage, S-phase arrest and cellular senescence in Cal51 cells.ConclusionsOverall, this work provides evidence against the simplified view of AF sensitivity, and suggests that AF could mediate growth inhibitory effects in ERα-positive and negative breast cancer cells, as well as cells with impaired AhR expression and signaling. While AF could have therapeutic effects on broader subtypes of breast cancer, the mechanism of cytotoxicity is complex, and likely, cell line- and tumor-specific.

Circulating tumour cells: insights into tumour heterogeneity

Tumour heterogeneity is a major barrier to cure breast cancer. It can exist between patients with different intrinsic subtypes of breast cancer or within an individual patient with breast cancer. In the latter case, heterogeneity has been observed between different metastatic sites, between metastatic sites and the original primary tumour, and even within a single tumour at either a metastatic or a primary site. Tumour heterogeneity is a function of two separate, although linked, processes. First, genetic instability is a hallmark of malignancy, and results in 'fixed' genetic changes that are almost certainly carried forward through progression of the cancer over time, with increasingly complex additional genetic changes in new metastases as they arise. The second type of heterogeneity is due to differential but 'plastic' expression of various genes important in the biology and response to various therapies. Together, these processes result in highly variable cancers with differential response, and resistance, to both targeted (e.g. endocrine or anti-human epithelial growth receptor type 2 (HER2) agents) and nontargeted therapies (e.g. chemotherapy). Ideally, tumour heterogeneity would be monitored over time, especially in relation to therapeutic strategies. However, biopsies of metastases require invasive and costly procedures, and biopsies of multiple metastases, or serially over time, are impractical. Circulating tumour cells (CTCs) represent a potential surrogate for tissue-based cancer and therefore might provide the opportunity to monitor serial changes in tumour biology. Recent advances have enabled accurate and reliable quantification and molecular characterization of CTCs with regard to a number of important biomarkers including oestrogen receptor alpha and HER2. Preliminary data have demonstrated that expression of these markers between CTCs in individual patients with metastatic breast cancer reflects the heterogeneity of the underlying tumours. Future studies are designed to determine the clinical utility of these novel technologies in either research or routine clinical settings.

Interplay between estrogen receptor and AKT in Estradiol-induced alternative splicing

BackgroundAlternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown.MethodsMCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively.ResultsWe identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF.ConclusionE2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.

Abstract P2-10-22: Phosphorylation of Steroid Receptor Coactivator 3 (SRC3) at Ser543 is a novel independent prognostic marker in breast cancer

Abstract Introduction: Steroid receptor coactivator 3 (SRC3) acts as a coactivator of nuclear receptors including estrogen receptor-alpha (ER). SRC3 has been implicated in the pathogenesis of breast cancer (BC) as well as in resistance to endocrine therapy. SRC3 is phosphorylated at a number of residues following the stimulation of growth factors or hormones. Tyr24 and Ser543 are both phosphorylated upon estrogen stimulation, while Tyr24 is modulated by JNK and Ser543 by both p38 and JNK. To date, the importance and potential role of phosphorylation at these residues has not been explored in BC. In this study we assessed the protein expression of SRC3, pTyr24 and pSer543 and association with clinico-pathological features and outcome in a well defined breast cancer series. Methods: The expressions of SRC3, pTyr24 and pSer543 were assessed in the Nottingham Tenovus Primary Breast Cancer Series which consists of 1650 cases of primary invasive. SRC3, pTyr24 and pSer543 were correlated with clinico-pathological data as well as outcome. Results: SRC3 expression was significantly associated with unfavourable clinicopathological features including ER -ve (p = 0.02), PR –ve (p = .038), HER2 overexpression (p &amp;lt; 0.0001), Triple negative phenotype (p = 0.001), high proliferation (p &amp;lt; 0.0001), high histological grade (p &amp;lt; 0.001), and lympho-vascular invasion. On contrast, the expression of pSer543 was significantly associated with a luminal phenotype, well differentiation, low proliferation (low mitotic index, low Ki67 and low SPAG5; p &amp;lt; 0.0001), hormonal receptors (ERα+ve/PR+ve/AR+ve), absence of both ER-B1 and ER-B5 (p &amp;lt; 0.01), high expression of ER-α associated proteins (cyclin D1 and Bcl2; p &amp;lt; 0.0001), high expression of c-jun (p &amp;lt; 0.0001), JNK (p &amp;lt; 0.0001), SRC3 (p &amp;lt; 0.0001), T24 (p &amp;lt; 0.001) and active p53 transcriptional pathways that regulate cell cycle progression and apoptosis (MDM2+ve, MDM4+ve and Bax+ve; p &amp;lt; 0.01) and absence of basal like phenotypes (p &amp;lt; 0.01). The absence of pSer543 was significantly associated with loss of expression of the key DNA repair proteins including XRCC1 (p &amp;lt; 0.0001), BRCA1 (p &amp;lt; 0.0001), ATM (p = 0.008) and TOP2A (p &amp;lt; 0.0001) reflecting a higher risk of genomic instability. Moreover, absence of pSer543 was more common in BC with a triple negative phenotype (p &amp;lt; 0.001). With regard to outcome, no association with outcome based on the expression of SRC3 either with or without tamoxifen was observed. However, expression of pSer543 was associated with significantly longer disease free survival (DFS) (p &amp;lt; 0.00001) and breast cancer specific survival (BCSS) (p = 0.0001). Furthermore, absence of pSer543 was associated with both a shorter DFS (p = 0.007) and BCSS (p = 0.01) in ER+ ve high risk BC. pSer534 was confirmed as an independent prognostic factor after adjustment for endocrine therapy and other validated prognostic factors and absent of pSer534 was associated with a two-fold increased risk of recurrence (HR = 1.9, CI 95%= 1.2–3.1). Data on Tyr24 will also be presented. Conclusion: Phosphorylation at Ser543 is associated with a luminal phenotype, positive prognostic factors and sensitivity to tamoxifen. Furthermore, it is an independent prognostic factor. pS543 is a novel prognostic marker in BC and warrants further investigation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-10-22.

Bisphenol A Inhibits Follicle Growth and Induces Atresia in Cultured Mouse Antral Follicles Independently of the Genomic Estrogenic Pathway1

Bisphenol A (BPA) is an estrogenic chemical used to manufacture many commonly used plastic and epoxy resin-based products. BPA ubiquitously binds to estrogen receptors throughout the body, including estrogen receptor alpha (ESR1) in the ovary. Few studies have investigated the effects of BPA on ovarian antral follicles. Thus, we tested the hypothesis that BPA alters cell cycle regulators and induces atresia in antral follicles via the genomic estrogenic pathway, inhibiting follicle growth. To test this hypothesis, we isolated antral follicles from 32- to 35-day-old control and Esr1-overexpressing mice and cultured them with vehicle control (dimethylsulfoxide [DMSO]) or BPA (1-100 μg/ml). Additionally, antral follicles were isolated from 32- to 35-day-old FVB mice and cultured with DMSO, BPA (1-100 μg/ml), estradiol (10 nM), ICI 182,780 (ICI; 1 μM), BPA plus ICI, or BPA plus estradiol. Follicles were measured for growth every 24 h for 96-120 h and processed either for analysis of estrogen receptor, cell cycle, and/or atresia factor mRNA expression, or for histological evaluation of atresia. Results indicate that estradiol and ICI do not protect follicles from BPA-induced growth inhibition and that estradiol does not protect follicles from BPA-induced atresia. Furthermore, overexpressing Esr1 does not increase susceptibility of follicles to BPA-induced growth inhibition. Additionally, BPA up-regulates Cdk4, Ccne1, and Trp53 expression, whereas it down-regulates Ccnd2 expression. BPA also up-regulates Bax and Bcl2 expression while inducing atresia in antral follicles. These data indicate that BPA abnormally regulates cell cycle and atresia factors, and this may lead to atresia and inhibited follicle growth independently of the genomic estrogenic pathway.

Abstract 2920: Transcriptional regulation of estrogen response through long-range chromatin interactions

Abstract Classical 17β-estradiol (E2) signaling requires the activity of the estrogen receptor at genomic regulatory regions. Differences in DNA sequence, ligand, receptor type and co-factor availability can affect this activity. However, the role of chromatin structure in the transmission of the estrogen signal is relatively unexplored. The 3D interaction between regulatory elements and promoters at long-range chromatin interaction sites presents a novel and dynamic mechanism of transcriptional regulation. Recent work to study such interactomes has identified relationships among histone modifications and loop boundaries [1], as well as among specific proteins, including estrogen receptor alpha (ERα), that appear to mediate the formation of chromatin loops [2-4]. We wish to identify the protein, DNA sequence, and epigenetic characteristics required for chromatin looping at ERα-bound sites. The publication of an ERα-bound interactome in the MCF-7 breast cancer cell line [2] has permitted us to identify the 67-kb loop region upstream of the krüppel-like factor 9 (KLF9) gene as an ideal candidate region for examination of the role of chromatin looping in breast cancer ERα-mediated transcriptional regulation. To study chromatin looping in situ, we have developed a model system which integrates a luciferase gene with the entire native regulatory locus of KLF9 on a single BAC. With this construct we can use reporter assays to screen for the elements required for long-distance chromatin interaction mediated, E2 stimulated transcriptional regulation. We detect early estrogen-induced activation of KLF9 in MCF-7 breast cancer cells and have validated 3/5 previously reported ERα binding sites [2, 5, 6] in this region by quantitative ChIP. Enrichment of ERα at these sites is E2 dependent. Additionally, we observe unique transcriptional responses from proximal and distal ER-bound sites in reporter assays. We hypothesize that the KLF9 loop is responsible for a rapid repression of transcriptional that occurs between 2-4 hours post E2 treatment. Chromatin looping is a regulatory mechanism common among most nuclear hormones and occurs at many estrogen-responsive genes; this process may present a novel mechanism for drug targeting in the treatment of many anti-endocrine resistant cancers and disorders. Future work will test our hypothesis of loop-based repression, identify the elements required for looping at the KLF9 locus and detect patterns in epigenetic modifications that may regulate or be regulated by the loop structure. Unique protein targets involved in looping will be examined for regulatory roles at a genome-wide level. 1. Handoko, L., et al., Nat Genet, 2011: p. 630-8. 2. Fullwood, M.J., et al., Nature, 2009: p. 58-64. 3. Tan, S.K., et al., EMBO J, 2011: p. 2569-81. 4. Kagey, M.H., et al., Nature, 2010: p. 430-435. 5. Carroll, J.S., et al., Nat Genet, 2006: p. 1289-1297. 6. Hua, S., et al., Mol Syst Biol, 2008. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2920. doi:1538-7445.AM2012-2920

Regulation of the Wip1 phosphatase and its effects on the stress response.

Wip1 (PPM1D) is a stress responsive PP2C phosphatase that plays a key role in stress signaling. Although originally identified as a gene induced by p53 after genotoxic stress, we now know that Wip1 expression is additionally regulated by other mechanisms. Wip1 is not only a target of p53, but is also a target of other transcription factors, including Estrogen Receptor-alpha and NF-kappaB. Additionally, Wip1 expression is regulated by post-transcriptional mechanisms such as mRNA stabilization and alternative splicing. Upon induction, Wip1 dampens the stress response by dephosphorylating and inactivating proteins such as p53, p38 MAPK, and ATM, usually as part of a negative feedback loop. As a result, Wip1 functions to abrogate cell cycle checkpoints and inhibit senescence, apoptosis, DNA repair, and the production of inflammatory cytokines. Furthermore, Wip1 is overexpressed in several types of human cancers and has oncogenic functions. The regulation of Wip1, the role of Wip1 in stress signaling, and the cooperation of Wip1 with oncogenes in promoting tumorigenesis will be discussed in this review.

FOXP1, an estrogen-inducible transcription factor, modulates cell proliferation in breast cancer cells and 5-year recurrence-free survival of patients with tamoxifen-treated breast cancer.

Breast cancer is primarily a hormone-dependent tumor that can be regulated by the status of steroid hormones, including estrogen and progesterone. Forkhead box P1 (FOXP1) is a member of the forkhead box transcription factor family and has been reported to be associated with various types of tumors. In the present study, we investigated the expression of FOXP1 in 133 human invasive breast cancers, obtained by core biopsy, by immunohistochemical analysis. Nuclear immunoreactivity of FOXP1 was detected in 89 cases (67%) and correlated positively with tumor grade and hormone receptor status, including estrogen receptor alpha (ERα) and progesterone receptor, and negatively with pathological tumor size. In ERα-positive MCF-7 breast cancer cells, we demonstrated that FOXP1 mRNA was upregulated by estrogen and increased ERα recruitment to ER binding sites identified by ChIP-on-chip analysis within the FOXP1 gene region. We also demonstrated that proliferation of MCF-7 cells was increased by exogenously transfected FOXP1 and decreased by FOXP1-specific siRNA. Furthermore, FOXP1 enhanced estrogen response element-driven transcription in MCF-7 cells. Finally, FOXP1 immunoreactivity was significantly elevated in relapse-free breast cancer patients treated with tamoxifen. These results suggest that FOXP1 plays an important role in proliferation of breast cancer cells by modulating estrogen signaling and that FOXP1 immunoreactivity could be associated with the estrogen dependency of clinical breast cancers, which may predict favorable prognosis in the patients treated with tamoxifen.

One-Step Preservation of Phosphoproteins and Tissue Morphology at Room Temperature for Diagnostic and Research Specimens

BackgroundThere is an urgent need to measure phosphorylated cell signaling proteins in cancer tissue for the individualization of molecular targeted kinase inhibitor therapy. However, phosphoproteins fluctuate rapidly following tissue procurement. Snap-freezing preserves phosphoproteins, but is unavailable in most clinics and compromises diagnostic morphology. Formalin fixation preserves tissue histomorphology, but penetrates tissue slowly, and is unsuitable for stabilizing phosphoproteins. We originated and evaluated a novel one-step biomarker and histology preservative (BHP) chemistry that stabilizes signaling protein phosphorylation and retains formalin-like tissue histomorphology with equivalent immunohistochemistry in a single paraffin block.ResultsTotal protein yield extracted from BHP-fixed, routine paraffin-embedded mouse liver was 100% compared to snap-frozen tissue. The abundance of 14 phosphorylated proteins was found to be stable over extended fixation times in BHP fixed paraffin embedded human colon mucosa. Compared to matched snap-frozen tissue, 8 phosphoproteins were equally preserved in mouse liver, while AMPKβ1 Ser108 was slightly elevated after BHP fixation. More than 25 tissues from mouse, cat and human specimens were evaluated for preservation of histomorphology. Selected tissues were evaluated in a multi-site, independent pathology review. Tissue fixed with BHP showed equivalent preservation of cytoplasmic and membrane cytomorphology, with significantly better nuclear chromatin preservation by BHP compared to formalin. Immunohistochemical staining of 13 non-phosphorylated proteins, including estrogen receptor alpha, progesterone receptor, Ki-67 and Her2, was equal to or stronger in BHP compared to formalin. BHP demonstrated significantly improved immunohistochemical detection of phosphorylated proteins ERK Thr202/Tyr204, GSK3-α/β Ser21/Ser9, p38-MAPK Thr180/Tyr182, eIF4G Ser1108 and Acetyl-CoA Carboxylase Ser79.ConclusionIn a single paraffin block BHP preserved the phosphorylation state of several signaling proteins at a level comparable to snap-freezing, while maintaining the full diagnostic immunohistochemical and histomorphologic detail of formalin fixation. This new tissue fixative has the potential to greatly facilitate personalized medicine, biobanking, and phospho-proteomic research.