Nuclear Estrogen Receptor Research Articles

4,4’- Isopropylidenediphenol – inhalable fraction. Documentation of proposed values of occupational exposure limits (OELs)

4,4’- Isopropylidenediphenol – inhalable fraction. Documentation of proposed values of occupational exposure limits (OELs)

Localization of estrogen receptor ERα, ERβ and GPR30 on myenteric neurons of the gastrointestinal tract and their role in motility

Estrogen is well known to have a modulatory role on gastrointestinal tract, particularly through its interaction with nuclear estrogen receptors (ERs), alpha and beta (ERα/β). Recent functional studies also indicate that estrogen can activate a G-protein coupled estrogen receptor, GPR30, or GPER1. The present study was designed to identify either the presence or absence of nuclear ERs and GPR30 in the myenteric plexus of the stomach, duodenum, jejunum, ileum and colon of female and male mice. Immunofluorescence staining revealed a high expression of GPR30 in the cytoplasm but not within the nucleus of enteric neurons in female and male mice. ERβ localization was similar to GPR30, where it was expressed in cytoplasm of enteric neurons, but was absent from nuclei, opening up the possibility that ERβ and GPR30 might work together to manifest estrogenic effects. Comparatively, ERα was mainly located in the nuclei of enteric neurons. ERα, ERβ and GPR30 were also expressed in the cytoplasm of glial cells in the stomach and small intestine, but levels were lower in the colon. The expression nuclear:cytoplasm ratio of ERα was higher in male than female mice, which might relate to sex-dependent translocation of ERα from cytoplasm to nucleus in response to known plasma levels of estrogen. A functional study using isolated ileal segments showed that ERα, ERβ and GPR30 are involved in the neuronal-mediated contractions in female tissues, but only ERα was involved in male tissues. This may indicate although expression level was similar between males and females, the downstream mechanisms of ERβ and GPR30 could be different between sexes. The present study provides a rationale for the action of estrogen to modulate gastrointestinal function in health and disease in different sexes.

Suppression of lymphoma growth by the xenoestrogens bisphenol A and genistein.

Well-defined physiological functions of estrogens are mediated via nuclear estrogen receptors α (ESR1) and β (ESR2). With regard to hematological malignancies, expression of ESR2 has been found in both B and T cell lymphomas. In addition to endogenous estrogens or selective ESR2 agonists, ESR2 signaling may be affected by both environmental synthetic estrogen-mimicking compounds and dietary phytoestrogens. In the present study, we demonstrate that oral exposure with either the synthetic compound bisphenol A (BPA) or the dietary phytoestrogen genistein reduced the growth of grafted murine T cell (EG7) and human B cell (Granta-519 mantle cell) lymphomas which both express ESR2. Suppression of lymphoma growth was due to reduced proliferation (BPA and genistein) and induction of apoptosis (genistein). Inhibition of lymphoma growth was seen at a BPA dose of 50 µg/kg body weight (BW)/day considered to be safe human exposure dose or a genistein dose of 1 mg/kg BW/day orally, which is reached in soy-rich diets. Thus, our study indicates that the environmental xenoestrogens BPA and genistein have anti-proliferative effects on ESR2-expressing lymphomas. Our data suggest that phytoestrogens may be considered as a dietary supplement for lymphoma patients and possibly for prevention of lymphoid malignancies.

Endocrine disruption through membrane estrogen receptors and novel pathways leading to rapid toxicological and epigenetic effects

Estrogen binding to estrogen receptors (ESR) triggers signaling cascades within cells. Historically, a major emphasis has been characterizing estrogen-induced genomic actions resulting from binding to nuclear estrogen receptor 1 (nESR1). However, recent evidence indicates the first receptors estrogens encounter as they enter a cell, membrane ESR1 (mESR1), also play crucial roles. Membrane and nuclear ESR are derived from the same transcripts but the former are directed to the membrane via palmitoylation. Binding and activation of mESR1 leads to rapid fluctuations in cAMP and Ca+2 and stimulation of protein kinase pathways. Endocrine disrupting chemicals (EDC) that mimic 17β-estradiol can signal through mESR1 and elicit non-genomic effects. Most current EDC studies have focused on genomic actions via nESR1. However, increasing number of studies have begun to examine potential EDC effects mediated through mESR1, and some EDC might have higher potency for signaling through mESR1 than nESR1. The notion that such chemicals might also affect mESR1 signaling via palmitoylation and depalmitoylation pathways has also begun to gain currency. Recent development of transgenic mice that lack either mESR1 or nESR1, while retaining functional ESR1 in the other compartment, will allow more precise in vivo approaches to determine EDC effects through nESR1 and/or mESR1. It is increasingly becoming apparent in this quickly evolving field that EDC directly affect mESR and estrogen signaling, but such chemicals can also affect proportion of ESR reaching the membrane. Future EDC studies should be designed to consider the full range of effects through mESR alone and in combination with nESR.

G protein-coupled receptor 30 mediates meiosis resumption and gap junction communications downregulation in goat cumulus-oocyte complexes by 17β-estradiol

Estrogen plays a critical role in the regulation of gap junctions between oocytes and granulosa cells in mammalian ovaries. G protein-coupled receptor 30 (GPR30) was identified as a membrane estrogen receptor, mediating rapid, nongenomic signaling events that might be responsible for the regulation of oocyte meiosis resumption and gap junction intercellular communications (GJICs). The present study aimed to determine the expression and localization of GPR30 and its role in oocyte meiotic progression and GJICs in goat cumulus-oocyte complexes (COCs). Immunofluorescence experiments revealed that GPR30 was primarily located in the plasma membrane of cumulus cells and oocytes in goats. 17β-estradiol could promote oocyte meiotic progression, which was blocked by G15 (a selective GPR30 antagonist) but not ICI182780 (a nuclear estrogen receptor inhibitor) in the early stage of in vitro culture. The effect of 17β-estradiol on the GJICs was quantified by lucifer yellow (LY) microinjection and calcein-AM fluorescent dye diffusion. 17β-estradiol treatment of goat COCs resulted in rapid downregulation of GJICs. The transfer of calcein from cumulus cells to oocytes could be significantly inhibited by carbenoxolone (a known gap junction blocker), 17β-estradiol or G1 (a GPR30 agonist), and this inhibition could be reversed by G15 but not ICI182780, indicating that GPR30 mediates the effect of 17β-estradiol on the rapid downregulation of GJICs. 17β-estradiol also stimulated the serine 368 phosphorylation of connexin 43 (Cx43) when COCs were in vitro cultured for 4 h, 6 h, and 8 h. More importantly, 17β-estradiol or G1 could separately promote the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and Cx43 significantly when COCs were cultured for 4 h. Furthermore, both ERK1/2 and Cx43 phosphorylation could be inhibited by G15 and the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor AG1478 or by the ERK1/2 inhibitor PD98059, indicating that EGFR-ERK1/2 signaling was involved in these events. These results supported the hypothesis that GPR30 mediated 17β-estradiol-stimulated meiotic resumption and GJIC reduction in goat COCs. Thus, the present study provides novel insights into elucidating the mechanisms for steroid hormone action in the regulation of oocyte maturation.

Differential involvement of the three nuclear estrogen receptors during oogenesis in European sea bass (Dicentrarchus labrax)†.

Estrogens are involved in a wide range of processes in vertebrate reproduction through ligand activation of their specific cognate receptors. In most teleosts, three nuclear estrogen receptor subtypes have been identified (Esr1, Esr2a, and Esr2b). Differences in ligand binding affinity and seasonal expression patterns in reproductive tissues among these Esr subtypes suggest distinct roles during oogenesis, vitellogenesis, and spermatogenesis. This study focuses on the role of the Esr subtypes in European sea bass (Dicentrarchus labrax) oogenesis and their endocrine regulation. The coding genes of the three Esr subtypes are highly expressed in reproduction-related tissues such as pituitary, gonad, and liver. Quantification of esr1, esr2a, and esr2b expression in the ovary and liver during a whole reproductive cycle showed different patterns depending on stage and subtype, suggesting differential roles of the three receptors in the regulation of oogenesis and vitellogenesis. Esr2a and Esr2b also showed differences in transcriptional activity and ligand affinity when functionally characterized in HEK293 cells. Finally, for the first time in teleosts, the localization of the three Esr subtypes in ovarian follicles and their regulation by gonadotropins is described. Immunodetection of the receptors revealed different distribution patterns in follicular cells and various subcellular locations of the oocyte. Gonadotropin stimulation of ovarian follicles in different stages of vitellogenesis showed a consistent induction of esrb2b expression by Fsh. All together, these data reinforce the hypothesis that each estrogen receptor plays a specific role in oogenesis.

Tamoxifen induces apoptosis and inhibits respiratory burst in equine neutrophils independently of estrogen receptors.

Neutrophils play an important role in the exacerbation and maintenance of severe equine asthma; persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues. Tamoxifen (TX) is a nonsteroidal estrogen receptor modulator with immunomodulatory effects and induces early apoptosis of blood and bronchoalveolar lavage neutrophils from horses with acute lung inflammation. This study investigated if the in vitro effects of tamoxifen are produced by its action on nuclear (α and β) and membrane (GPR30) estrogen receptors in healthy equine neutrophils. Results showed that TX inhibits neutrophil respiratory burst induced by opsonized zymosan in a dose-dependent manner. Nuclear (17-β-Estradiol) and GPR30 cell membrane (G1) estrogen receptor agonists and their antagonists (ICI 182,780 and G15, respectively) do not block or reproduce the effect of TX. Therefore, TX does not inhibit respiratory burst through estrogen receptors. TX (8.5μM) also increased phosphatidylserine translocation, a marker of early apoptosis, which did not occur with any of the estrogen receptor agonists or antagonists. Thus, tamoxifen generates dose-dependent inhibition of respiratory burst and increased early apoptosis in healthy equine neutrophils, independently of nuclear or membrane estrogen receptors. Further studies are necessary to explore the signaling pathways of tamoxifen-induced ROS inhibition and phosphatidylserine translocation.

Estrogen nuclear receptors affect cell migration by altering sublocalization of AQP2 in glioma cell lines

Glioblastomas are capable of infiltrating into neighboring brain tissues. The prognosis of a male patient is worse than that of women. Here, we demonstrate the effects of estrogen on invasion of glioma cells via regulating estrogen nuclear receptors (ERα and ERβ) combined with aquaporin 2 (AQP2). In our study, we conclude that AQP2 was located mainly in the nuclei of the glioma cell lines and is capable of inhibiting cell invasion. According to the gene ontology analysis, out of 138 screened genes, three genes of ankyrin repeat and FYVE domain containing 1 (ANKFY1), lymphocyte transmembrane adaptor 1 (LAX1), and latent transforming growth factor beta-binding protein 1 (LTBP1) were found to be regulating the ERα and ERβ. The expression of ERα was found to be high, whereas the expression of both ERβ and AQP2 was low in glioma cells from patient tissues and glioblastoma cell lines. The expression levels of AQP2, ANKFY1, LAX1, and LTBP1 were upregulated by both ERα small interfering RNA (siRNA) and overexpression of ERβ. AQP2 inhibition of cell invasion was inversely influenced by LAX1siRNA. The luciferase report system indicated that AQP2 promoted the transcriptional activity of LAX1 and inhibited cell invasion. These data suggest that ERβ may function as AQP promoter in the nucleus to sustain cells' stability by promoting AQP production, while ERα acts as an antagonist of AQP2. The ratio between ERα and ERβ is likely to affect the distribution of AQP2 in the nucleus. Low level of ERβ reduces the inhibition of invasion of glioma cells influenced by high level of LAX1 expression, leading to an increase in the invasion ability of glioma cells.

Estrogen receptor \u03b2 is associated with expression of cancer associated genes and survival in ovarian cancer

BackgroundIn ovarian cancer, the role of estrogen receptors (ERs), particularly of ERβ, being suggested as tumor suppressor in breast and prostate cancer, remains unclear. We examined the expression of nuclear and cytoplasmic ERβ in ovarian cancer and correlated it with expression of ovarian cancer markers CA125, CEA and CA72–4, steroid hormone receptors ERα and PR, cancer-associated genes EGFR, p53, HER2 and proliferation marker Ki-67. Additionally we examined to what extent expression of ERβ and the other proteins affects survival of ovarian cancer patients.MethodsWe established a tissue microarray from 171 ovarian cancer patients and performed immunohistochemical analyses of the mentioned proteins.ResultsNuclear ERβ was detected in 47.31% of the ovarian cancer tissues and cytoplasmic expression of this receptor was observed in 23.08%. Nuclear expression of ERβ was significantly decreased in the G3 subgroup compared to better differentiated cancers (p < 0.01) and correlated with ovarian cancer markers CEA (95% CI 0.1598–0.4465; p < 0.0001) and CA72–4 (95% CI 0.05953–0.3616; p < 0.01). Cytoplasmic ERβ expression correlated with EGFR levels (95% CI 0.1059–0.4049; p < 0.001). ERα expression was associated with expression of CA125 and PR. Overall survival of patients with tumors expressing cytoplasmic ERβ was significant longer compared to those with ERβ-negative ovarian cancer (chi-square statistic of the log-rank, p < 0.05). Progression-free survival was dependent on expression of PR (chi-square statistic of the log-rank, p < 0.05) and Ki-67 (p = 0.05).ConclusionsOur data suggest an important, but distinct role of nuclear and cytoplasmic ERβ expression in ovarian cancer and encourage further studies on its role in this cancer entity.

SIRT1 and Estrogen Signaling Cooperation for Breast Cancer Onset and Progression.

Breast cancer remains a significant female mortality cause. It constitutes a multifactorial disease for which research on environmental factors offers little help in predicting onset or progression. The pursuit for its foundations by analyzing hormonal changes as a motive for disease development, indicates that increased exposure to estrogens associates with increased risk. A prevalent number of breast cancer cases show dependence on the increased activity of the classic nuclear estrogen receptor (ER) for cell proliferation and survival. SIRT1 is a Type III histone deacetylase which is receiving increasing attention due to its ability to perform activities over relevant non-histone proteins and transcription factors. Interestingly, concomitant SIRT1 overexpression is commonly found in ER-positive breast cancer cases. Both proteins had been shown to directly interact, in a process related to altered intracellular signaling and aberrant transcription, then promoting tumor progression. Moreover, SIRT1 activities had been also linked to estrogenic effects through interaction with the G-protein coupled membrane bound estrogen receptor (GPER). This work aims to summarize present knowledge on the interplay between SIRT1 and ER/GPER for breast cancer onset and progression. Lastly, evidences on the ability of SIRT1 to interact with TGFß signaling, a concurrent pathway significantly involved in breast cancer progression, are reported. The potential of this research field for the development of innovative strategies in the assessment of orphan breast cancer subtypes, such as triple negative breast cancer (TNBC), is discussed.

Placental development during early pregnancy in sheep: nuclear estrogen and progesterone receptor mRNA expression in the utero-placental compartments

Sex steroid hormones are major regulators of uterine and placental growth and functions, as well as many other biological processes. To examine the mRNA expression of nuclear estrogen (ESR1 and 2) and progesterone (PGRAB and B) receptors in different compartments of the uterus and placenta, tissues were collected in experiment 1 on days 16, 20, and 28 after natural mating (NAT) and on day 10 after estrus (nonpregnant controls [NP]); and in experiment 2 on day 22 of NAT, and pregnancies established after transfer of embryos generated through mating of FSH-treated ewes (NAT-ET), in vitro fertilization (IVF), or in vitro activation (parthenotes). In experiment 1, ESR1 expression in endometrial stroma (ES), endometrial glands (EGs), and myometrial blood vessels (MBVs), ESR2 in endometrial blood vessels (EBV), PGRAB in ES, and PGRB in ES, EG, and MBV was greater in pregnant than NP ewes depending on the day of pregnancy. The day of pregnancy affected the expression of ESR1 in MBV, ESR2 in EBV and MBV, and PGRAB in ES. In experiment 2, ESR1, PGRAB, and PGRB in EG, but not in other compartments, was greater in NAT-ET than NAT, and PGRB was greater for NAT-ET than IVF. These data demonstrate that ESR and PGR expression differ in pregnant versus NP ewes in selected compartments and was affected by pregnancy stage or embryo origin in selected utero-placental compartments. Thus, sex steroid hormone mRNA expression is differentially regulated in a spatiotemporal manner in the uterus and placenta and is affected by the application of assisted reproductive technology in sheep.

Role of AhR and ARNT in recurrent spontaneous abortion

Objective To explore the relationship between aryl hydrocar-bon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (ARNT), estradiol (E2), estrogen receptor (ER) and recurrent spontaneous abortion (RSA) through observing the level of serum AhR, ARNT, E2, and AhR, ARNT, ER in decidua and chorionic tissues of the patients with recurrent spontaneous abortion. Methods 64 cases of RSA patients who induced abortion at the Shanxi Dayi hospital from May 2015 to September 2017 were chosen as RSA group, and 30 cases of healthy abortion women of the same period who had born full-term normal fetus were choosen as the normal group. The serum, villi and decidua of each case were collected during abortion. The level of AhR, ARNT, ER of both groups were detected by enzyme-linked immunosorbent assay (ELISA) method. The Serum E2 lever were detected by the method of chemical luminescence. The data of all the patients were analyzed. Results ⑴ The level of ARNT in peripheral blood of RSA group was significantly higher than that of normal group (P 0.05). The levels of AhR and ARNT in villi and decidua were significantly higher in RSA group than in normal group (P 0.05); the ratio of AhR/ER and ARNT/ER in the villi and decidua of the RSA group was higher than that of the normal group (P<0.05). Conclusions Overexpression of AhR and ARNT and low expression of ER in villi and decidua tissues may be related to the occurrence of RSA. Key words: Abortion, habitual/ME; Receptors, aryl hydrocarbon/ME; Aryl hydrocarbon receptor nuclear translocator/ME; Estradiol/ME; Receptors, estrogen/ME

Estrogen/GPR30 Signaling Contributes to the Malignant Potentials of ER-Negative Cervical Adenocarcinoma via Regulation of Claudin-1 Expression

Cervical adenocarcinomas are believed to lose estrogen response on the basis of no expression of a nuclear estrogen receptor such as ERα in clinical pathology. Here, we demonstrated that cervical adenocarcinoma cells respond to a physiological concentration of estrogen to upregulate claudin-1, a cell surface molecule highly expressed in cervical adenocarcinomas. Knockout of claudin-1 induced apoptosis and significantly inhibited proliferation, migration, and invasion of cervical adenocarcinoma cells and tumorigenicity in vivo. Importantly, all of the cervical adenocarcinoma cell lines examined expressed a membrane-bound type estrogen receptor, G protein–coupled receptor 30 (GPR30/GPER1), but not ERα. Estrogen-dependent induction of claudin-1 expression was mediated by GPR30 via ERK and/or Akt signaling. In surgical specimens, there was a positive correlation between claudin-1 expression and GPR30 expression. Double high expression of claudin-1 and GPR30 predicts poor prognosis in patients with cervical adenocarcinomas. Mechanism-based targeting of estrogen/GPR30 signaling and claudin-1 may be effective for cervical adenocarcinoma therapy.

Low-concentration BPF induced cell biological responses by the ERα and GPER1-mediated signaling pathways in MCF-7 breast cancer cells

Bisphenol F (BPF), one of the alternatives to bisphenol A (BPA), can induce proliferation through the nuclear estrogen receptor ERα (estrogen receptor alpha) pathway in human breast cancer MCF-7 cells. However, the roles of membrane estrogen receptor GPER1 (G-protein-coupled receptor 1)-mediated signaling pathways in MCF-7 cell proliferation caused by BPF are unclear. The influence of BPF on MCF-7 cells was evaluated in terms of cell proliferation, intracellular calcium (Ca2+) fluctuations, and reactive oxygen species (ROS) generation. The molecular mechanisms of the cellular responses to low doses of BPF were studied through detecting the activations of ERα and GPER1-regulated PI3K/PKB or AKT (phosphatidylinotidol 3-kinase/protein kinase B) and ERK1/2 (extracellular-signa1-regulated kinase 1/2) signals. At 0.01–1 μM, BPF significantly promoted cell proliferation and elevated the levels of intracellular ROS and Ca2+. At these concentrations, BPF also significantly upregulated protein expressions of ERα, GPER1, c-myc, and cyclin D and phosphorylations of PKB and ERK1/2. Specific signal inhibitors decreased PKB and ERK1/2 phosphorylations and attenuated the effects of BPF. Silencing of GPER1 also significantly decreased BPF-induced cell proliferation. These results indicate that activating the GPER1-PI3K/PKB and ERK1/2 signals by low doses of BPF can regulate the response of MCF-7 cells and that ERα also influences the effects of exposure to BPF on the cells. The present study suggests a new mechanism by which BPF exerts relevant estrogenic action in cancer cells and also highlights the potential risks in using BPF as an alternative to BPA.

G Protein-Coupled Estrogen Receptor Is Not Required for Sex Determination or Ovary Function in Zebrafish.

Estrogens regulate vertebrate development and function through binding to nuclear estrogen receptors α and β (ERα and ERβ) and the G protein-coupled estrogen receptor (GPER). Studies in mutant animal models demonstrated that ERα and ERβ are required for normal ovary development and function. However, the degree to which GPER signaling contributes to ovary development and function is less well understood. Previous studies using cultured fish oocytes found that estradiol inhibits oocyte maturation in a GPER-dependent manner, but whether GPER regulates oocyte maturation in vivo is not known. To test the hypothesis that GPER regulates oocyte maturation in vivo, we assayed ovary development and function in gper mutant zebrafish. We found that homozygous mutant gper embryos developed into male and female adults with normal sex ratios. Adult mutant fish exhibited normal secondary sex characteristics and fertility. Additionally, mutant ovaries were histologically normal. We observed no differences in the number of immature versus mature oocytes in mutant versus wild-type ovaries from both young and aged adults. Furthermore, expression of genes associated with sex determination and ovary function was normal in gper mutant ovaries compared with wild type. Our findings suggest that GPER is not required for sex determination, ovary development, or fertility in zebrafish.

Combinatorial anti-proliferative effects of tamoxifen and naringenin: The role of four estrogen receptor subtypes

Breast cancer is the most diagnosed diseases and the second-leading cause of death in females, among which the estrogen receptor positive (ER+) patients are more common of all cases. In present study, we selected MCF-7 as an in vitro model and investigated the combinatorial anti-proliferative effects of tamoxifen and naringenin on ER+ breast cancer, and then explored the potential mechanisms involved in mitochondrial dysfunction and oxidative stress mediated by several estrogen receptor subtypes. Six assessment endpoints including cell viability, cell migration, cell cycle, cell apoptosis, mRNA, and protein expression were estimated. Tamoxifen and naringenin were shown to inhibit the cell growth of MCF-7 cells at higher concentrations, and co-exposure with them significantly inhibited cell proliferation in an additive manner. Results from a wound healing assay indicated that the combined treatment of tamoxifen and naringenin markedly suppressed cell migration compared with the single exposure by downregulating the expression of MMP-9 and MMP-2. Flow cytometry analysis revealed that the combined treatment of tamoxifen and naringenin blocked cell cycle in G2/M phase through suppressing the transcription of cell cycle regulation proteins. Simultaneously, co-treatment with them also induced cell apoptosis by regulating the expression of mitochondrial apoptotic proteins as well as by simulating the production of reactive oxygen species (ROS). Real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting (WB) analysis results further demonstrated that the two nuclear estrogen receptors—ERα66 and ERβ, as well as the two membrane estrogen receptors—ERα36 and GPR30 were downregulated when cells were exposed to single tamoxifen, whereas naringenin treatment group not only downregulated the expression of ERα66 and GPR30 but also upregulated ERβ and ERα36. Interestingly, co-treatment group resulted in significant downregulation of ERα66, ERα36 and GPR30 but upregulation of ERβ. Taken together, co-treatment of tamoxifen and naringenin could inhibit cell proliferation more effectively in ER+ breast cancer cells, which was associated with a modulation of the expression levels of several estrogen receptors.

Prognostic significance of tumor-infiltrating lymphocytes in ductal carcinoma in situ of the breast

Tumor-infiltrating lymphocytes (TILs) provide prognostic value in invasive breast cancer and guidelines for their assessment have been published. This study aims to evaluate: (a) methods of TILs assessment, and (b) their prognostic significance in breast ductal carcinoma in situ (DCIS). Hematoxylin and eosin sections from two clinically annotated DCIS cohorts; a training set (n = 150 pure DCIS) and a validation set (n = 666 comprising 534 pure DCIS and 132 cases wherein DCIS and invasive breast carcinoma were co-existent) were assessed. Seven different scoring methods were applied to the training set to identify the most optimal reproducible method associated with strongest prognostic value. Among different methods, TILs touching ducts' basement membrane or away from it by one lymphocyte cell thickness provided the strongest significant association with outcome and highest concordance rate [inter-cluster correlation coefficient = 0.95]. Assessment of periductal TILs at increasing distances from DCIS (0.2 , 0.5 , and 1 mm) as well as percent of stromal TILs were practically challenging and showed lower concordance rates than touching TILs. TILs hotspots and lymphoid follicles did not show prognostic significance. Within the pure DCIS validation set, dense TILs were associated with younger age, symptomatic presentation, larger size, higher nuclear grade, comedo necrosis and estrogen receptor negativity as well as shorter recurrence-free interval (p = 0.002). In multivariate survival analysis, dense TILs were independent predictor of shorter recurrence-free interval (p = 0.002) in patients treated with breast conservation. DCIS associated with invasive carcinoma showed denser TILs than pure DCIS (p = 9.0 × 10−13). Dense TILs is an independent prognostic variable in DCIS. Touching TILs provides a reproducible method for their assessment that can potentially be used to guide management

Impairments in the reproductive axis of female mice lacking estrogen receptor β in GnRH neurons.

The effect of estrogen on the differentiation and maintenance of reproductive tissues is mediated by two nuclear estrogen receptors (ERs), ERα, and ERβ. Lack of functional ERα and ERβ genes in vivo significantly affects reproductive function; however, the target tissues and signaling pathways in the hypothalamus are not clearly defined. Here, we describe the generation and reproductive characterization of a complete-ERβ KO (CERβKO) and a GnRH neuron-specific ERβKO (GERβKO) mouse models. Both ERβKO mouse models displayed a delay in vaginal opening and first estrus. Hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression levels in both ERβKO mice were similar to control mice; however female CERβKO and GERβKO mice had lower basal and surge serum gonadotropin levels. Although a GnRH stimulation test in both female ERβKO models showed preserved gonadotropic function in the same animals, a kisspeptin stimulation test revealed an attenuated response by GnRH neurons, suggesting a role for ERβ in normal GnRH neuron function. No alteration in estrogen-negative feedback was observed in either ERβKO mouse models after ovariectomy and estrogen replacement. Further, abnormal development of ovarian follicles with low serum estradiol levels and impairment of fertility were observed in both ERβKO mouse models. In male ERβKO mice, no differences in the timing of pubertal onset or serum luteinizing hormone and follicle-stimulating hormone levels were observed as compared with controls. Taken together, these data provide in vivo evidence for a role of ERβ in GnRH neurons in modulating puberty and reproduction, specifically through kisspeptin responsiveness in the female hypothalamic-pituitary-gonadal axis.

27-hydroxycholesterol decreases cell proliferation in colon cancer cell lines

Colorectal cancer (CRC) is the third most diagnosed cancer in the western world, affecting 1 out of approximately 22 people in their lifetime. Several epidemiological studies suggest a positive association between high plasma cholesterol levels and colorectal cancer. However, the molecular mechanisms by which cholesterol may alter the risk of colorectal cancer (CRC) are ill-defined as the cholesterol lowering drugs statins do not appear to decrease a patient's risk of developing colorectal cancer. Cholesterol is metabolized to active derivatives including cholesterol oxidization products (COP), known as oxysterols, which have been shown to alter cellular proliferation. These metabolites and not cholesterol per se, may therefore affect the risk of developing colorectal cancer. The cholesterol metabolite or the oxysterol 27-hydroxycholesterol (27-OHC) is the most abundant oxysterol in the plasma and has been shown to be involved in the pathogenesis of several cancers including breast and prostate cancer. However, the role of 27-OHC in colorectal cancer has not been investigated. We treated Caco2 and SW620, two well characterized colon cancer cells with low, physiological and high concentrations of 27-OHC, and found that 27-OHC reduces cellular proliferation in these cells. We also found that the effects of 27-OHC on cell proliferation are not due to cellular cytotoxicity or apoptotic cellular death. Additionally, 27-OHC-induced reduction in cell proliferation is independent of actions on its target nuclear receptors, liver-X-receptors (LXR) and estrogen receptors (ER) activation. Instead, our study demonstrates that 27-OHC significantly decreases AKT activation, a major protein kinase involved in the pathogenesis of cancer as it regulates cell cycle progression, protein synthesis, and cellular survival. Our data shows that treatment with 27-OHC substantially decreases the activation of AKT by reducing levels of its active form, p-AKT, in Caco2 cells but not SW620 cells. All-together, our results show for the first time that the cholesterol metabolite 27-OHC reduces cell proliferation in colorectal cancer cells.

Abstract 5243: Prognostic value of nuclear and non-nuclear estrogen receptor expression by immunohistochemistry with phosphor-integrated dots in hormone receptor-positive early breast cancer

Abstract Background: Estrogen receptor α (ERα) is associated to cell proliferation and survival through genomic (nuclear) and non-genomic (non-nuclear) signaling pathway. Endocrine therapy is the most effective therapies for patients with hormone receptor(HR) positive breast cancer. Recent progress shows that both nuclear and non-nuclear crosstalk was considered to contribute to endocrine resistance. Clinically, ERα is predominantly observed using immunohistochemistry(IHC) as a nuclear protein while unable to detect nuclear and non-nuclear ERα separately. Our group recently created new fluorescent nanoparticles called phosphor-integrated dots (PIDs). PIDs has extremely brightness so that it could be double stained with hematoxylin and quantitated without influence. In addition, the localization of biomarker protein can be visualized and analyzed on cell as PIDs score by this method. Methods: 65 HR+/HER2- breast cancer patients from 2001 to 2003 who treated with postoperative endocrine therapy were selected in this study. Expression levels of total ERα, nuclear-ERα, non-nuclear-ERα, progesterone receptor (PR) was examined by IHC with PIDs. Nuclear-ERα was recognized by double staining using hematoxylin. IHC with DAB was carried out to analyze the expression levels of ER and PR and H score was automatically calculated. In addition, PIDs score of non-nuclear-ERα can be calculated through PIDs score of total ERα and nuclear-ERα by PID analyzer. Optimal cutoff value of PIDs score of each receptor to predict survival were derived and independently tested. The correlation among every clinical characteristic and pathological feature was examined. Result: PIDs score of total ERα(χ2=5.17, p=0.023) and nuclear-ERα is negative correlated with relapse (χ2=6.46, p=0.011), ratio of non-nuclear-ERα to total ERα is positive correlated with relapse (χ2=5.82, p=0.016).The cutoff value as 37.46 of nuclear-ERα PIDs score was statistically proved significant for predicting disease-free survival(DFS) (log-rank p=0.020).We also found that cutoff value as 0.32 which represents ratio of non-nuclear-ERα PIDs score to total ERα PIDs score was statistically proved significant for predicting DFS (log-rank p=0.022). H score of ER, PR and PIDs score of non-nuclear-ERα, PR has no predictive value for recurrence. Conclusion: Our result demonstrates that nuclear-ERα PIDs score obtain a higher prognostic value than that of total-ERα PIDs score and H score detected by DAB-IHC. Ratio of non-nuclear-ERα to total ERα may play a role of prognostic factor in HR positive breast cancer. Citation Format: Zhaorong Guo, Hiroshi Tada, Narufumi Kitamura, Yoh Hamada, Shin-ichi Hayashi, Noriaki Ohuchi, Kosuke Gonda, Takanori Ishida. Prognostic value of nuclear and non-nuclear estrogen receptor expression by immunohistochemistry with phosphor-integrated dots in hormone receptor-positive early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5243.