Estrogen Receptor Protein Research Articles

Patterns of sex hormone receptor expression in stimulated endometrium from oocyte donors

In patients with high serum E2 embryo transfer is often postponed, as high E2 levels adversely affect embryo transfer outcome. We aimed to determine if stratified serum oestradiol (E2) and progesterone (P4) levels differentially affect endometrial histology and endometrial oestrogen and progesterone receptor protein levels. Endometrial biopsies were collected from oocyte donors. Samples were divided based on peak serum E2 levels into three groups: (i) low-E2 (n = 33) E2≤2999pg/mL; (ii) mid-E2 (n = 40) E2 3000–4999 pg/mL; and (iii) high-E2 (n = 15) E2≥5000 pg/mL. Oestrogen receptor alpha (ERα) and progesterone receptors A and B (PR) protein levels in endometrial stroma (S), glandular (GE) and luminal (LE) epithelia were assessed by immunohistochemistry. Samples in high-E2 group demonstrated strongest association with accelerated endometrial maturation (2 (1–2); 2 (1–3); and 3 (2.8–3) median days of advancement of endometrial maturation respectively in low, mid, high-E2 groups, p = 0.046). There were significant differences in ERα and PR immunoexpression in S, GE and LE among the groups (p < 0.05). Higher E2 levels were associated with decreased ERα expression (p < 0.017) in GE and LE, and increased PR expression in S and GE (p < 0.011 and p < 0.0001, respectively). Higher serum E2 levels were associated with impaired endometrial steroid hormone receptor expression, higher serum P4 and more advancement of endometrial maturation.

3D Visualization of the Dynamic Bidirectional Talk Between ER/PR and Her2 Pathways.

Background: Extensive amounts of archived formalin fixed paraffin embedded (FFPE) human tumor tissues are the ultimate resource to investigate signaling network underlying tumorigenesis in human. Yet, their usage is severely limited for lacking of suitable protein techniques. In this study, a quantitative, objective, absolute, and high throughput immunoblot method, quantitative dot blot (QDB), was explored to address this issue by investigating the putative relationship between estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (Her2) pathways in breast cancer tumorigenesis. Methods: In this descriptive observational retrospective study, ER, PR, Her2, and Ki67 protein levels were measured absolutely and quantitatively in 852 FFPE breast cancer tissues using the QDB method. ER, PR, and Her2 levels were charted on the X, Y, and Z-axes to observe samples distribution in a 3D scatterplot. Results: A “seesaw” relationship between ER/PR and Her2 pathways was observed in ER–PR–Her2 space, characterized by the expression levels of these 3 proteins. Specimens with strong expressions of ER/PR proteins were found spreading along the ER/PR floor while those with strong Her2 expression were found wrapping around the Her2 axis. Those lacking strong expressions of all 3 proteins were found accumulating at the intersection of the ER, PR, and Her2 axes. Few specimens floated in the ER–PR–Her2 space to suggest the lack of co-expression of all 3 proteins simultaneously. Ki67 levels were found to be significantly reduced in specimens spreading in the ER–PR space. Conclusions: The unique distribution of specimens in ER–PR–Her2 space prior to any clinical intervention provided visual support of bidirectional talk between ER/PR and Her2 pathways in breast cancer specimens. Clinical interventions to suppress these 2 pathways alternatively warrant further exploration for breast cancer patients accordingly. Our study also demonstrated that the QDB method is an effective tool to analyze archived FFPE cancer specimens in biomedical research.

FGFR1 Is Associated With Tamoxifen Resistance and Poor Prognosis of ER-Positive Breast Cancers by Suppressing ER Protein Expression.

Fibroblast growth factor receptor 1 (FGFR1) is widely recognized as a key player in mammary carcinogenesis and associated with the prognosis and therapeutic response of breast cancers. With the aim of investigating the correlation between FGFR1 expression and estrogen receptor (ER) and exploring the effect of FGFR1 on endocrine therapy response and ER+ breast cancer prognosis, we examined the FGFR1 protein expression among 184 ER-positive breast cancers by the immunohistochemistry (IHC) method, analyzed the association between FGFR1 expression and disease characters using the Pearson’s chi-square test, and assessed the prognostic role of FGFR1 among breast cancers using Cox regression and Kaplan-Meier analyses. Moreover, in vitro assays were conducted to confirm the correlation between FGFR1 and ER expression and investigate the effect of FGFR1 on tamoxifen (TAM) sensitivity in ER+ breast cancer. The results showed that ER expression was negatively correlated with FGFR1 expression (P = 0.011, r = -0.221). Moreover, FGFR1 expression was one of the prognostic factors of ER-positive breast cancer (OR = 1.974, 95% CI = 1.043-3.633), and high FGFR1 expression was correlated with decreased breast cancer overall survival. In addition, knocking down FGFR1 inhibited cell proliferation and enhanced TAM sensitivity in TAM-resistant cells. In conclusion, we found that there was a significant negative correlation between FGFR1 and ER levels in ER+ breast cancers, high FGFR1 protein expression was associated with poor breast cancer prognosis, down-regulating FGFR1 could elevate ER expression and is associated with enhanced TAM sensitivity in ER+ breast cancers.

ESR1 genetic alterations and their association with clinicopathologic characteristics in advanced breast cancer: a single academic institution experience

Estrogen receptor (ER) alpha, a ligand-dependent nuclear transcription factor encoded by the ESR1 gene, is expressed in 70% of breast carcinomas (BCs) and is used as a target for endocrine-based therapies. However, some patients develop resistance to endocrine-based therapies due to ESR1 mutation, which leads to constitutive activation in the absence of ligand. We retrospectively analyzed 223 clinically advanced BCs using the FoundationOne CDX assay and found 13.9% (31/223) of cases had ESR1 genetic alterations (26 mutations and 5 amplifications). All ESR1 mutations occurred within the ligand binding domain, with the most prevalent being Y537S (42.3%) and D538G (38.5%), and all ESR1-mutated cases had a history of aromatase inhibitor use. No significant difference in clinicopathologic features was identified between ESR1-mutated and ESR1-amplified cases except higher frequency of HER2 positivity and TP53 mutations in ESR1-amplified cases. The prevalence of ESR1 mutations in ER-positive BCs was 19.1% (26/136). In comparison to ESR1-nonmutated ER-positive cases, ESR1-mutated cases demonstrated significantly higher percentage of tumor cells with ER and progesterone receptor expression, an increased tendency for overall distant metastasis and liver metastasis, higher frequency of FGF3/4/19 mutations, lower frequency of TP53 mutation, but no difference in overall survival and metastatic/recurrent intervals. In conclusion, our findings suggest that development of ESR1 mutations are selected for under the influence of estrogen deprivation, and a positive correlation between ESR1 mutations and ER protein expression may exist.

Early-Life Body Adiposity and the Breast Tumor Transcriptome.

Cumulative epidemiologic evidence has shown that early-life adiposity is strongly inversely associated with breast cancer risk throughout life, independent of adult obesity. However, the molecular mechanisms remain poorly understood. We assessed the association of early-life adiposity, defined as self-reported body size during ages 10-20 years from a validated 9-level pictogram, with the transcriptome of breast tumor (N = 835) and tumor-adjacent histologically normal tissue (N = 663) in the Nurses' Health Study. We conducted multivariable linear regression analysis to identify differentially expressed genes in tumor and tumor-adjacent tissue, respectively. Molecular pathway analysis using Hallmark gene sets (N = 50) was further performed to gain biological insights. Analysis was stratified by tumor estrogen receptor (ER) protein expression status (n = 673 for ER+ and 162 for ER- tumors). No gene was statistically significantly differentially expressed by early-life body size after multiple comparison adjustment. However, pathway analysis revealed several statistically significantly (false discovery rate < 0.05) upregulated or downregulated gene sets. In stratified analyses by tumor ER status, larger body size during ages 10-20 years was associated with decreased cellular proliferation pathways, including MYC target genes, in both ER+ and ER- tumors. In ER+ tumors, larger body size was also associated with upregulation in genes involved in TNFα/NFkB signaling. In ER- tumors, larger body size was additionally associated with downregulation in genes involved in interferon α and interferon γ immune response and Phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling; the INFγ response pathway was also downregulated in ER- tumor-adjacent tissue, though at borderline statistical significance (false discovery rate = 0.1). These findings provide new insights into the biological and pathological underpinnings of the early-life adiposity and breast cancer association.

Механізми кардіо- та вазопротекторної дії ресвератролу

У статті наведено огляд сучасних даних щодо механізмів реалізації кардіо- та вазопротекторної дії ресвератролу. Наведені результати експериментальних і клінічних досліджень, що підтверджують можливість включення ресвератролу в фармакотерапію серцево-судинних захворювань. Вплив ресвератролу на серцево-судинну систему полягає в підвищенні продукції NO, зниженні синтезу ендотеліну-1, проявів оксидативного стресу, запобіганні проліферації клітин гладеньких м’язів, ремоделюванні судин та посиленні жорсткості артерій. Крім того, ресвератрол знижує інфільтрацію імунних клітин у судинну стінку та зменшує запалення судинної стінки. Ресвератрол має значну біологічну активність та може застосовуватися не тільки як кардіопротекторний, але й як протизапальний, антидіабетичний, нейротропний, протипухлинний засіб.

Immunohistological expression of oestrogen receptor, progesterone receptor, mammaglobin, human epidermal growth factor receptor 2 and GATA-binding protein 3 in non-small-cell lung cancer.

Non-small-cell lung cancer (NSCLC) and breast cancer are common entities. Staining for oestrogen receptor (ER), progesterone receptor (PgR), mammaglobin (MAMG) and GATA-binding protein 3 (GATA3) is frequently performed to confirm a mammary origin in the appropriate diagnostic setting. However, comprehensive data on the immunohistological expression of these markers in NSCLC are limited. Therefore, the aim of this study was to analyse a large cohort of NSCLCs and correlate the staining results with clinicopathological variables. A tissue microarray was stained for ER, PgR, MAMG, human epidermal growth factor receptor 2 (HER2), and GATA3, and included 636 adenocarcinomas (ADCs), 536 squamous cell carcinomas (SqCCs), 65 large-cell-carcinomas, 34 pleomorphic carcinomas, and 20 large-cell neuroendocrine carcinomas. HER2 status was determined for immunohistochemically positive cases with chromogenic in-situ hybridisation. Markers with a proportion of ≥5% positive cases in ADC and SqCC were considered for survival analysis. Among ADCs, 62 (10%), 17 (3%), one (<1%), seven (1%), and 49 (8%) cases were positive for ER, PgR, MAMG, HER2, and GATA3, respectively. Among SqCCs, 10 (2%), 14 (3%), two (<1%) and 109 (20%) cases were positive for ER, PgR, HER2, and GATA3, but none of the samples showed positivity for MAMG. ER positivity was associated with ADC, female sex, smaller tumour size, and lower clinical stage. None of the markers had an impact on survival. We report on ER, PgR, MAMG, HER2 and GATA3 expression in a large cohort of NSCLCs. Interpretation of these markers in the differential diagnostic setting should be based on a multimarker panel.

NDV-D90 inhibits 17β-estradiol-mediated resistance to apoptosis by differentially modulating classic and nonclassic estrogen receptors in breast cancer cells.

Newcastle disease virus (NDV) is endowed with the oncolytic ability to kill tumor cells, while rarely causing side effects in normal cells. Both estrogen receptor α (ERα) and the G protein estrogen receptor (GPER) modulate multiple biological activities in response to estrogen, including apoptosis in breast cancer (BC) cells. Here, we investigated whether NDV-D90, a novel strain isolated from natural sources in China, promoted apoptosis by modulating the expression of ERα or the GPER in BC cells exposed to 17β-estradiol (E2). We found that NDV-D90 significantly killed the tumor cell lines MCF-7 and BT549 in a time- and dose-dependent manner. We also found that NDV-D90 exerted its effects on the two cell lines mainly by inducing apoptosis but not necrosis. NDV-D90 induced apoptosis via the intrinsic and extrinsic signaling pathways in MCF-7 cells (ER-positive cells) during E2 exposure not only by disrupting the E2/ERα axis and enhancing GPER expression but also by modulating the expression of several apoptosis-related proteins through ERα-and GPER-independent processes. NDV-D90 promoted apoptosis via the intrinsic signaling pathway in BT549 cells (ER-negative cells), possibly by impairing E2-mediated GPER expression. Furthermore, NDV-D90 exerted its antitumor effects in vivo by inducing apoptosis. Overall, these results demonstrated that NDV-D90 promotes apoptosis by differentially modulating the expression of ERα and the GPER in ER-positive and negative BC cells exposed to estrogen, respectively, and can be utilized as an effective approach to treating BC.

Bisphenol A impairs reproductive fitness in zebrafish ovary: Potential involvement of oxidative/nitrosative stress, inflammatory and apoptotic mediators

Bisphenol A (BPA) is a highly pervasive chemical in consumer products with its ascribed endocrine-disrupting properties. Several studies have shown the cytotoxic, genotoxic, and carcinogenic property of BPA over a multitude of tissues. Although BPA exposure has earlier been implicated in female infertility, the underlying molecular mechanisms explaining the toxicity of BPA in the ovary remains less understood. In the present study, a plausible correlation between redox balance or inflammatory signaling and reproductive fitness upon BPA exposure has been examined in zebrafish (Danio rerio) ovary. Congruent with significant alteration of major antioxidant enzymes (SOD1, SOD2, catalase, GPx1α, GSTα1) at the transcript level, 30 d BPA exposure at environmentally relevant concentrations (1, 10 and 100 μg L−1) promotes ovarian ROS/RNS synthesis, lipid peroxidation but attenuates catalase activity indicating elevated stress response. BPA promotes a sharp increase in ovarian p38 MAPK, NF-κB phosphorylation (activation), inducible nitric oxide synthase (Nos2a), and pro-inflammatory cytokines (TNF-α and IL-1β) expression, the reliable markers for inflammatory response. Congruent to an increased number of atretic follicles, BPA-exposed zebrafish ovary reveals elevated Bax/Bcl2 ratio, activation of caspase-8, -3 and DNA breakdown suggesting heightened cell death. Importantly, significant alteration in nuclear estrogen receptor (ER) transcripts (esr1, esr2a, and esr2b) and proteins (ERα, ERβ), gonadotropin receptors, and markers associated with steroidogenesis and growth factor gene expression in BPA-exposed ovary correlates well with impaired ovarian functions and maturational response. Collectively, elevated oxidative/nitrosative stress-mediated inflammatory response and altered ER expression can influence ovarian health and reproductive fitness in organisms exposed to BPA environment.

Accurate assessment of estrogen receptor profiles in non-functioning pituitary adenomas using RT-digital PCR and immunohistochemistry

BackgroundNon-functioning pituitary adenomas (NFPAs) are common pituitary tumors, and surgery is generally the only treatment option. Few attempts have been made to explore target molecules for the development of NFPA pharmacological treatments. MethodWe quantitatively assessed the expression profiles of estrogen receptor (ER) transcripts and proteins in NFPA samples, using reverse transcription-digital polymerase chain reaction (RT-dPCR) and immunohistochemistry, and further investigated the correlations between the expression levels of ER and those of downstream responsive genes. All patients had undergone surgery at the same high-volume hospital. A total of 20 patients with NFPAs were included. All patients were new-onset, and none were diagnosed with intratumoral hemorrhages or cysts. ResultsNFPA samples exhibited a bimodal ESR1 expression pattern and were categorized into significantly different high- and low-ESR1 expression level groups (P < 0.05). In contrast, expression levels of ESR1 variants and ESR2 could barely be detected. Similar results were obtained through the immunohistochemical staining of NFPAs, using well-validated antibodies against ERs. The expression levels of ESR1 positively correlated with those of GREB1, an estrogen-responsive gene [correlation coefficient (r) = 0.623, P = 0.003]. ConclusionsESR1 expression levels in NFPAs exhibited a bimodal pattern and were positively correlated with GREB1 expression levels. The accurate assessment of ER expression levels may further advance future NFPA-related research.

Endocrine Resistance in Breast Cancer: The Role of Estrogen Receptor Stability.

Therapy of hormone receptor positive breast cancer (BCa) generally targets estrogen receptor (ER) function and signaling by reducing estrogen production or by blocking its interaction with the ER. Despite good long-term responses, resistance to treatment remains a significant issue, with approximately 40% of BCa patients developing resistance to ET. Mutations in the gene encoding ERα, ESR1, have been identified in BCa patients and are implicated as drivers of resistance and disease recurrence. Understanding the molecular consequences of these mutations on ER protein levels and its activity, which is tightly regulated, is vital. ER activity is in part controlled via its short protein half-life and therefore changes to its stability, either through mutations or alterations in pathways involved in protein stability, may play a role in therapy resistance. Understanding these connections and how ESR1 alterations could affect protein stability may identify novel biomarkers of resistance. This review explores the current reported data regarding posttranslational modifications (PTMs) of the ER and the potential impact of known resistance associated ESR1 mutations on ER regulation by affecting these PTMs in the context of ET resistance.

Response to growth hormone in patients with RNPC 3 mutations

Corrigendum7 September 2020Open Access Response to growth hormone in patients with RNPC3 mutations Gabriel Á Martos-Moreno Gabriel Á Martos-Moreno Search for more papers by this author Lourdes Travieso-Suárez Lourdes Travieso-Suárez Search for more papers by this author Jesús Pozo-Román Jesús Pozo-Román Search for more papers by this author María T Muñoz-Calvo María T Muñoz-Calvo Search for more papers by this author Julie A Chowen Julie A Chowen Search for more papers by this author Mikko J Frilander Mikko J Frilander Search for more papers by this author Luis A Pérez-Jurado Luis A Pérez-Jurado Search for more papers by this author Federico G Hawkins Federico G Hawkins Search for more papers by this author Jesús Argente Jesús Argente orcid.org/0000-0001-5826-0276 Search for more papers by this author Gabriel Á Martos-Moreno Gabriel Á Martos-Moreno Search for more papers by this author Lourdes Travieso-Suárez Lourdes Travieso-Suárez Search for more papers by this author Jesús Pozo-Román Jesús Pozo-Román Search for more papers by this author María T Muñoz-Calvo María T Muñoz-Calvo Search for more papers by this author Julie A Chowen Julie A Chowen Search for more papers by this author Mikko J Frilander Mikko J Frilander Search for more papers by this author Luis A Pérez-Jurado Luis A Pérez-Jurado Search for more papers by this author Federico G Hawkins Federico G Hawkins Search for more papers by this author Jesús Argente Jesús Argente orcid.org/0000-0001-5826-0276 Search for more papers by this author Author Information Gabriel Á Martos-Moreno, Lourdes Travieso-Suárez, Jesús Pozo-Román, María T Muñoz-Calvo, Julie A Chowen, Mikko J Frilander, Luis A Pérez-Jurado, Federico G Hawkins and Jesús Argente EMBO Mol Med (2020)12:e13132https://doi.org/10.15252/emmm.202013132 This article corrects the following: Response to growth hormone in patients with RNPC3 mutations06 June 2018 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The authors recently noticed that one of the reported mutations, chr1:104093621C>A/p.P474T, was incorrectly listed as c.1320C>A at the cDNA level. The correct mutation is c.1420C>A; the nomenclature at the protein level was correct. This genetic variant is correctly listed in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/variation/587367/), has an allele frequency of 0.00003185 in gnomAD (https://gnomad.broadinstitute.org/variant/1-104093621-C-A?dataset=gnomad_r2_1) and has been reported in a second family with growth hormone deficiency due RNPC3 dysfunction (Verberne et al, 2020). References Verberne EA, Faries S, Mannens MMAM, Postma AV, van Haelst MM (2020) Expanding the phenotype of biallelic RNPC3 variants associated with growth hormone deficiency. Am J Med Genet A https://doi.org/10.1002/ajmg.a.61632Wiley Online LibraryWeb of Science®Google Scholar Previous ArticleNext Article Read MoreAbout the coverClose modalView large imageVolume 12,Issue 9,07 September 2020This month's cover highlights the article Characterization of circulating breast cancer cells with tumorigenic and metastatic capacity by Claudia Koch, Andra Kuske, Klaus Pantel and colleagues. New estrogen receptor‐positive breast cancer cell line CTC‐ITB‐01 enables functional studies of circulating tumor cells. Cytoplasmatic keratin (orange) and nuclear estrogen receptor (green) protein expression in CTC‐ITB‐01 cells. DAPI nuclear staining (blue). (Scientific image by Claudia Koch et al., Institute of Tumor Biology, University Medical Center Hamburg, Eppendorf, Hamburg, Germany) Volume 12Issue 97 September 2020In this issue ReferencesRelatedDetailsLoading ...

Abstract 1750: Combination of ZEN-3694 with CDK4/6 inhibitors reverses resistance in ER-positive breast cancer

Abstract CDK4/6 inhibitors have been shown to significantly prolong progression-free survival in patients with advanced hormone receptor (HR)-positive, HER2-negative breast cancer. In spite of the great successes with the currents lines of therapies, the patients still acquire resistance over time and, the development of additional novel therapeutic strategies is needed. The bromodomain and extra-terminal domain (BET) proteins are key epigenetic regulators that interact with acetylated lysine (AcLys) residues of histones or transcription factors, leading to the regulation of gene transcription. BET proteins have also been shown to be directly involved in the transcription of the estrogen receptor (ER) mRNA as well as cell cycle and therefore, BET inhibitors can potentially offer new strategies in the treatment of CDK4/6i- resistant and endocrine resistant ER+ breast cancer. ZEN-3694 is an orally bioavailable small molecule inhibitor of BET proteins currently being evaluated in Phase 1/2 clinical trials in metastatic castration-resistant prostate cancer (NCT02711956) and triple negative breast cancer (NCT03901469). We have shown previously that ZEN-3694 inhibits proliferation and induces apoptosis in ER+ cell lines. To assess the effects of ZEN-3694 in the combination with CDK4/6 inhibitors as a potential therapy in a CDK4/6i resistant population, we have developed a panel of ER+ cell lines resistant to palbociclib or abemaciclib by continuous stepwise exposure to increasing concentrations of CDK4/6 inhibitors. These cell lines have demonstrated cross-resistance to all three CDK4/6 inhibitors. Here, we describe that the combination treatment of ZEN-3694 with CDK4/6 inhibitors potently inhibits proliferation and induces apoptosis in all CDK4/6i resistant cell lines. The resistance to both palbociclib and abemaciclib was associated with the strong upregulation of CDK6 and CCND1protein levels in MCF7 which is consistent with their clinical mechanism of resistance. ZEN-3694 showed efficacy in all CDK4/6i-resistant models leading to downregulation of ER and CDK6 protein levels. Furthermore, our RNAseq data revealed several potential pathways involved in CDK4/6i resistance, including upregulation of cell cycle pathway and decrease of interferon signaling and demonstrated that the mechanisms of resistance to abemaciclib and palbociclib are similar but not identical. Additionally, we elucidated the mechanism of action of ZEN-3694 and in combinations with CDK4/6 inhibitors alleviating these resistance mechanisms. The pathway analysis demonstrated that the combination of ZEN-3694 with CDK4/6 inhibitors led to the strong downregulation of multiple pathways including cell cycle regulation, signaling by Rho family GTPases, STAT3, IL6 and other pathways. We conclude that ZEN-3694 has therapeutic potential in a combination with CDK4/6 inhibitors in ER+ breast cancer patients that developed resistance to endocrine therapies and/or CDK4/6 inhibitors. Citation Format: Olesya A. Kharenko, Reena G. Patel, Cyrus Calosing, Edward H. van der Horst. Combination of ZEN-3694 with CDK4/6 inhibitors reverses resistance in ER-positive breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1750.

The IL1\u03b2-IL1R signaling is involved in the stimulatory effects triggered by hypoxia in breast cancer cells and cancer-associated fibroblasts (CAFs)

BackgroundHypoxia plays a relevant role in tumor-related inflammation toward the metastatic spread and cancer aggressiveness. The pro-inflammatory cytokine interleukin-1β (IL-β) and its cognate receptor IL1R1 contribute to the initiation and progression of breast cancer determining pro-tumorigenic inflammatory responses. The transcriptional target of the hypoxia inducible factor-1α (HIF-1α) namely the G protein estrogen receptor (GPER) mediates a feedforward loop coupling IL-1β induction by breast cancer-associated fibroblasts (CAFs) to IL1R1 expression by breast cancer cells toward the regulation of target genes and relevant biological responses.MethodsIn order to ascertain the correlation of IL-β with HIF-1α and further hypoxia-related genes in triple-negative breast cancer (TNBC) patients, a bioinformatics analysis was performed using the information provided by The Invasive Breast Cancer Cohort of The Cancer Genome Atlas (TCGA) project and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) datasets. Gene expression correlation, statistical analysis and gene set enrichment analysis (GSEA) were carried out with R studio packages. Pathway enrichment analysis was evaluated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. TNBC cells and primary CAFs were used as model system. The molecular mechanisms implicated in the regulation of IL-1β by hypoxia toward a metastatic gene expression profile and invasive properties were assessed performing gene and protein expression studies, PCR arrays, gene silencing and immunofluorescence analysis, co-immunoprecipitation and ChiP assays, ELISA, cell spreading, invasion and spheroid formation.ResultsWe first determined that IL-1β expression correlates with the levels of HIF-1α as well as with a hypoxia-related gene signature in TNBC patients. Next, we demonstrated that hypoxia triggers a functional liaison among HIF-1α, GPER and the IL-1β/IL1R1 signaling toward a metastatic gene signature and a feed-forward loop of IL-1β that leads to proliferative and invasive responses in TNBC cells. Furthermore, we found that the IL-1β released in the conditioned medium of TNBC cells exposed to hypoxic conditions promotes an invasive phenotype of CAFs.ConclusionsOur data shed new light on the role of hypoxia in the activation of the IL-1β/IL1R1 signaling, which in turn triggers aggressive features in both TNBC cells and CAFs. Hence, our findings provide novel evidence regarding the mechanisms through which the hypoxic tumor microenvironment may contribute to breast cancer progression and suggest further targets useful in more comprehensive therapeutic strategies.

Downregulation of gap junctional intercellular communication and connexin 43 expression by bisphenol A in human granulosa cells.

Gap junctional intercellular communication (GJIC) is the transfer of ions, metabolites, and second messengers between neighboring cells through intercellular junctions. Connexin 43 (Cx43) was found to be the type of gap junction protein responsible for human granulosa cells (GCs) and oocyte communication, which is required for folliculogenesis and oocyte maturation. Bisphenol A (BPA), an estrogenic-like endocrine-disrupting chemical, is one of the most widely produced chemicals around the world. There are reports that the chemical might cause endometrial tumorigenesis and several female reproductive disorders. This study demonstrated that cell culture medium, containing antioxidants (N-acetyl-l-cysteine and l-ascorbic acid-2-phosphate), was able to enhance the survival and self-renewal of GCs. In addition, we found that BPA at environmentally relevant concentration (10-7 M) reduced Cx43 expression and GJIC in GCs through estrogen receptor and mitogen-activated protein kinase pathways. The results of this study not only reveal the reproductive toxicity of BPA but also provide possible mechanisms by which BPA inhibited GJIC in GCs.

Abstract B29: Ovarian hormones regulate C/EBPD induced EMT/MET transition in the human fallopian tube epithelia

Abstract Introduction: The histologically normal BRCA1 mutation carrier fallopian tube epithelia (FTE), compared to controls, showed that CEBPD was upregulated in the luteal phase of the ovulatory cycle. CEBPD is involved with the maintenance of genomic stability, promoting cellular differentiation, and regulating the cell cycle in response to cytotoxic stressors. In breast epithelial cells, CEBPD protein expression correlated with estrogen receptor (ER) and progesterone receptor (PR) and was found to be associated with increased progression-free survival in breast cancer patients. In fallopian tube epithelia (FTE), CEBPD was also found to modulate the epithelial-to-mesenchymal (EMT)/mesenchymal-to-epithelial transition (MET) by modulating target genes of this pathway. Given the hormonal response of this gene and its function in modulating an EMT/MET, the objective of this study was to determine whether sex hormones influence CEBPD regulation of EMT/MET in the fallopian tube and thus ovarian cancer. Methods: Fresh fallopian tube (FTE) tissues were obtained from patients approved for collection by IRB. Immunohistochemical profiling on normal fallopian tube tissue and HGSC was performed using CEBPD, ER, and PR protein markers. FTE cell lines with a p53 mutation (R175H) were subjected to estradiol (50nM and 100nM) and 4-hydroxytamoxifen (10nM) and assayed using qRT-PCR and PCR. ANOVA and t-tests were conducted in GraphPad Prism software with significance set at p&amp;lt;0.05. Results: E-cadherin in normal fallopian tube tissue was highly expressed in both the luteal and follicular phase whereas vimentin was highly expressed in the follicular phase but showed a range of expression (low expression to high expression) in the luteal phase. CEBPD overexpression increased SNAIL expression (p&amp;lt;0.0001), consistent with previous findings; treatment with 50nM estradiol (E2) resulted in increased SNAIL and SLUG mRNA expression in FTE cell lines overexpressing CEBPD (p&amp;lt;0.0001) relative to controls and decreased ZEB1 and ZEB2 mRNA expression. Addition of tamoxifen to cells overexpressing CEBPD increased SNAIL mRNA expression compared to cells without tamoxifen (p&amp;lt;0.0001); however, a combination of both tamoxifen and estradiol added to these cells decreased SNAIL expression relative to controls. IL6 mRNA expression level was increased in CEBPD overexpressing cells compared to controls (p&amp;lt;0.0001), which was further increased by E2 (p&amp;lt;0.0001). Conclusion: Together these results demonstrate a role for CEBPD in modulating EMT/MET in normal FTE in a hormonally regulated manner, which during cancer formation and spread is critical for anoikis and metastasis. Furthermore, these data will facilitate an understanding of the early events of carcinogenesis in fallopian tube epithelia. Citation Format: Ramlogan Sowamber, Leah V. Dodds, Patricia Shaw, Sophia H.L. George. Ovarian hormones regulate C/EBPD induced EMT/MET transition in the human fallopian tube epithelia [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B29.

MiR-18a activates Wnt pathway in ER-positive breast cancer and is associated with poor prognosis.

Despite the established benefits of long‐term endocrine therapy, women with hormone receptor‐positive breast cancer remain at risk for late relapse. The basis of this is multi‐factorial including genetic, epigenetic, and host factors. In this study we have explored the epigenetic regulation of estrogen receptor (ER)‐dependent molecular and cellular phenotype by hsa‐miR‐18a‐5p using well‐established human ER‐positive (ER+) breast cancer cell lines. miR‐18a was overexpressed in MCF7 and ZR‐75‐1 and this led to an increase in the proliferative ability of the cells and concurrently resulted in decreased expression of luminal markers and higher expression of the basal marker, cytokeratin 14. The cells became more migratory with a significant repression of E‐cadherin and activation of the Wnt noncanonical pathway. We observed an activation of the planar cell polarity (PCP) pathway with increased activation of JNK pathway and eventually change in actin dynamics. There was increased F‐actin polymerization in cells with higher expression of miR‐18a. Examination of miR‐18a expression in a set of human ER+ breast cancer specimens showed a negative correlation between miR‐18a and ESR1 transcripts as well as ER protein. Kaplan‐Meier survival analysis of the cohort stratified by tumor hsa‐miR‐18a‐5p levels produced significant differences in disease‐free survival (log rank P < .05). This observation was independently validated in the METABRIC cohort. These data provide support for a role of hsa‐miR‐18a‐5p in altering the proliferative and migratory behavior of ER+ cells and its potential utility as a prognostic marker in clinical ER+ breast cancers.

Loss of Estrogen Efficacy Against Hippocampus Damage in Long-Term OVX Mice Is Related to the Reduction of Hippocampus Local Estrogen Production and Estrogen Receptor Degradation.

Postmenopausal women experience a higher risk for neurodegenerative diseases, including cognitive impairment and ischemic stroke. Many preclinical studies have indicated that estrogen replacement therapy (ERT) may provide protective effects against these neurological diseases. However, the results of Women's Health Initiative (WHI) studies have led to the proposal of "critical period hypothesis," which states that there is a precise window of opportunity for administering beneficial hormone therapy following menopause. However, the underlying molecular mechanisms require further characterization. Here, we explored the effects of ERT on cognition decline and global cerebral ischemia (GCI)-induced hippocampal neuronal damage in mice that had experienced both short-term (ovariectomized (OVX) 1week) and long-term (OVX 10weeks) estrogen deprivation. We also further explored the concentration of 17β-estradiol (E2) in the circulation and hippocampus and the expression of aromatase and estrogen receptors (ERα, ERα-Ser118, and ERβ). We found that the neuroprotective effectiveness of ERT against hippocampus damage exhibited in OVX1w mice was totally absent in OVX10w mice. Interestingly, the concentration of hippocampal E2 was irreversibly reduced in OVX10w mice, which was related to the decrease of aromatase expression in the hippocampus. In addition, long-term estrogen deprivation (LTED) led to a decrease in estrogen receptor proteins in the hippocampus. Thus, we concluded that the loss of ERT neuroprotection against hippocampus injury in LTED mice was related to the reduction in hippocampus E2 production and estrogen receptor degradation. These results provide several intervention targets to restore the effectiveness of ERT neuroprotection in elderly post-menopausal women.

A retrospective single-center study investigating the clinical significance of grade in triple-negative breast cancer.

e13108 Background: Triple negative breast cancer is defined as estrogen (ER), progesterone (PR), and human epidermal growth factor receptor (HER-2) proteins negative. The grade is the degree of similarity of tumor cells to normal cells under microscope and is an important biomarker of overall patient outcomes or prognosis with higher grades having a poor prognosis. As recent chemotherapy trials noted moderately undifferentiated grade 2 tumors showing higher rates of relapse, we hypothesize that grade can also be a predictive biomarker or determinant of response to specific treatment. Methods: We reviewed 305 patient charts of triple negative breast cancer patients from 2004-2017 at Windsor Regional Cancer Center analyzing the significance of grade with respect to oncological variables, survival-time, and time to relapse. Statistical analysis was performed using Fleming-Harrington, Pairwise Testing, and COX regression, where applicable. Results: Univariate analysis showed statistically significance difference in chemotherapy type (P = 0.008) and a marginal one in ER &amp; hormone therapy status (P ~0.09) between the grades. The overall survival rates were 90.12%, 64.4%, and 77.2%, for grade 1, 2, 3 respectively. The overall difference in survival among the three groups was statistically significant, based on Fleming-Harrington test (P = 0.019). Comparing only between grade 2 and grade 3, we found that after five years, grade 2 patients had a 5.5-fold increased risk of death (HR = 5.5; 95% CI 1.2-25.6) and 2-folds higher risk of relapse (HR = 1.9; 95% CI 1.1-3.2). Grade 3 does significantly better than grade 2 in time to relapse with relapse rates of 70%, 55.6 %, and 75.6%, respectively for grades 1, 2, and 3 (P = 0.04). Conclusions: Tumor grade has a significant positive predictive value in determining relapse with grade 2 tumors demonstrating poorer disease-free survival as compared to grade 1 &amp; 3, less time to relapse, and increased risk of death. This has implications in stratifying triple negative breast cancer patients by grade in future clinical trials while ongoing research yields new targets for chemotherapy.

A Mechanistic High-Content Analysis Assay Using a Chimeric Androgen Receptor That Rapidly Characterizes Androgenic Chemicals.

Human health is at risk from environmental exposures to a wide range of chemical toxicants and endocrine-disrupting chemicals (EDCs). As part of understanding this risk, the U.S. Environmental Protection Agency (EPA) has been pursuing new high-throughput in vitro assays and computational models to characterize EDCs. EPA models have incorporated our high-content analysis-based green fluorescent protein estrogen receptor (GFP-ER): PRL-HeLa assay, which allows direct visualization of ER binding to DNA regulatory elements. Here, we characterize a modified functional assay based on the stable expression of a chimeric androgen receptor (ARER), wherein a region containing the native AR DNA-binding domain (DBD) was replaced with the ERα DBD (amino acids 183-254). We demonstrate that the AR agonist dihydrotestosterone induces GFP-ARER nuclear translocation, PRL promoter binding, and transcriptional activity at physiologically relevant concentrations (<1 nM). In contrast, the AR antagonist bicalutamide induces only nuclear translocation of the GFP-ARER receptor (at μM concentrations). Estradiol also fails to induce visible chromatin binding, indicating androgen specificity. In a screen of reference chemicals from the EPA and the Agency for Toxic Substances and Disease Registry, the GFP-ARER cell model identified and mechanistically grouped activity by known (anti-)androgens based on the ability to induce nuclear translocation and/or chromatin binding. Finally, the cell model was used to identify potential (anti-)androgens in environmental samples in collaboration with the Houston Ship Channel/Galveston Bay Texas A&M University EPA Superfund Research Program. Based on these data, the chromatin-binding, in vitro assay-based GFP-ARER model represents a selective tool for rapidly identifying androgenic activity associated with drugs, chemicals, and environmental samples.