Extranuclear ERα Research Articles

Macrophage-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes atherosclerosis in male mice

Hypercholesterolemia and vascular inflammation are key interconnected contributors to the pathogenesis of atherosclerosis. How hypercholesterolemia initiates vascular inflammation is poorly understood. Here we show in male mice that hypercholesterolemia-driven endothelial activation, monocyte recruitment and atherosclerotic lesion formation are promoted by a crosstalk between macrophages and endothelial cells mediated by the cholesterol metabolite 27-hydroxycholesterol (27HC). The pro-atherogenic actions of macrophage-derived 27HC require endothelial estrogen receptor alpha (ERα) and disassociation of the cytoplasmic scaffolding protein septin 11 from ERα, leading to extranuclear ERα- and septin 11-dependent activation of NF-κB. Furthermore, pharmacologic inhibition of cyp27a1, which generates 27HC, affords atheroprotection by reducing endothelial activation and monocyte recruitment. These findings demonstrate cell-to-cell communication by 27HC, and identify a major causal linkage between the hypercholesterolemia and vascular inflammation that partner to promote atherosclerosis. Interventions interrupting this linkage may provide the means to blunt vascular inflammation without impairing host defense to combat the risk of atherosclerotic cardiovascular disease that remains despite lipid-lowering therapies.

Both Nuclear and Membrane Estrogen Receptor Alpha Impact the Expression of Estrogen Receptors and Plasticity Markers in the Mouse Hypothalamus and Hippocampus.

Estrogens via estrogen receptor alpha (ERα) genomic and nongenomic signaling can influence plasticity processes in numerous brain regions. Using mice that express nuclear only ERα (NOER) or membrane only ERα (MOER), this study examined the effect of receptor compartmentalization on the paraventricular nucleus of the hypothalamus (PVN) and the hippocampus. The absence of nuclear and membrane ERα expression impacted females but not males in these two brain areas. In the PVN, quantitative immunohistochemistry showed that the absence of nuclear ERα increased nuclear ERβ. Moreover, in the hippocampus CA1, immuno-electron microscopy revealed that the absence of either nuclear or membrane ERα decreased extranuclear ERα and pTrkB in synapses. In contrast, in the dentate gyrus, the absence of nuclear ERα increased pTrkB in synapses, whereas the absence of membrane ERα decreased pTrkB in axons. However, the absence of membrane only ERα decreased the sprouting of mossy fibers in CA3 as reflected by changes in zinc transporter immunolabeling. Altogether these findings support the idea that both membrane and nuclear ERα contribute overlapping and unique actions of estrogen that are tissue- and cellular-specific.

Segregation of nuclear and membrane-initiated actions of estrogen receptor using genetically modified animals and pharmacological tools

Estrogen receptor alpha (ERα) and beta (ERβ) are members of the nuclear receptor superfamily, playing widespread functions in reproductive and non-reproductive tissues. Beside the canonical function of ERs as nuclear receptors, in this review, we summarize our current understanding of extra-nuclear, membrane-initiated functions of ERs with a specific focus on ERα. Over the last decade, in vivo evidence has accumulated to demonstrate the physiological relevance of this ERα membrane-initiated-signaling from mouse models to selective pharmacological tools. Finally, we discuss the perspectives and future challenges opened by the integration of extra-nuclear ERα signaling in physiology and pathology of estrogens.

Automated Quantification of Extranuclear ERα using Phosphor-integrated Dots for Predicting Endocrine Therapy Resistance in HR+/HER2- Breast Cancer.

In addition to genomic signaling, Estrogen receptor alpha (ERα) is associated with cell proliferation and survival through extranuclear signaling contributing to endocrine therapy (ET) resistance. However, the relationship between extranuclear ERα and ET resistance has not been extensively studied. We sought to measure extranuclear ERα expression by immunohistochemistry using phosphor-integrated dots (IHC-PIDs) and to assess its predictive value for ET resistance. After quantitative detection of ERα by IHC-PIDs in vitro, we developed “the nearest-neighbor method” to calculate the extranuclear ERα. Furthermore, tissue sections from 65 patients with HR+/HER2- BC were examined by IHC-PIDs, and the total ERα, nuclear ERα, extranuclear ERα PIDs score, and ratio of extranuclear-to-nuclear ERα (ENR) were measured using the novel method. We demonstrate that quantification of ERα using IHC-PIDs exhibited strong correlations to real-time qRT-PCR (r2 = 0.94) and flow cytometry (r2 = 0.98). High ERα ENR was significantly associated with poor overall survival (p = 0.048) and disease-free survival (DFS) (p = 0.007). Multivariate analysis revealed that the ERα ENR was an independent prognostic factor for DFS [hazard ratio, 3.8; 95% CI, 1.4–11.8; p = 0.006]. Our automated measurement has high accuracy to localize and assess extranuclear ERα. A high ERα ENR in HR+/HER2− BC indicates decreased likelihood of benefiting from ET.

Males Require Estrogen Signaling Too: Sexual Dimorphism in the Regulation of Glucose Homeostasis by Nuclear ERα.

Females are more protected against insulin resistance and cardiovascular disease compared with males of the same age or BMI, and this relative protection is diminished in postmenopausal women, attributable to a loss in estrogen signaling (1–4). Evidence from genetic animal models further points to the beneficial effects of estrogen signaling via estrogen receptor-α (ERα) in regard to glucose homeostasis in health and metabolic diseases (5). Mice harboring whole-body deletion of ERα (ERαKO) reveal that estrogen signaling through this receptor regulates glucose homeostasis in part by modulating hepatic insulin sensitivity (6) and glucose uptake in the skeletal muscle and adipose tissue (7). Moreover, ERα signaling has been demonstrated to promote pancreatic insulin-producing β-cell function, survival, and proliferation, as well as protection from development of diabetes in mice of both sexes (8–10). Thus, estrogen is generally thought to positively regulate glucose homeostasis primarily through ERα, which is expressed in both male and female tissues, but the respective importance of nuclear and membrane ERα pools in the regulation of glucose homeostasis is not clear. ERα is predominantly characterized as a nuclear receptor and thus noted for exclusively regulating transcription of target genes. However, extranuclear ERα action or membrane compartment–initiated estrogen signaling is now widely accepted to activate different pathways occurring in the cytoplasm and nucleus. For example, membrane localization of ERα (which accounts for ∼5–10% of the ERα pool depending on cell type) facilitates membrane-initiated signaling events important for reproduction and vascular physiology (11) as well as β-cell function and survival (12). Although it is clear that ERα positively regulates glucose homeostasis, the distinct and overlapping contributions of the extranuclear and nuclear pool of ERα remain unknown. Therefore, mouse models specifically engineered to have only nuclear or membrane ERα action are necessary to delineate specific ERα functions related to …

Selective activation of estrogen receptors, ERα and GPER-1, rapidly decreases food intake in female rats

Many of estradiol's behavioral effects are mediated, at least partially, via extra-nuclear estradiol signaling. Here, we investigated whether two estrogen receptor (ER) agonists, targeting ERα and G protein-coupled ER-1 (GPER-1), can promote rapid anorexigenic effects. Food intake was measured in ovariectomized (OVX) rats at 1, 2, 4, and 22 h following subcutaneous (s.c.) injection of an ERα agonist (PPT; 0–200 μg/kg), a GPER-1 agonist (G-1; 0–1600 μg/kg), and a GPER-1 antagonist (G-36; 0–80 μg/kg). To investigate possible cross-talk between ERα and GPER-1, we examined whether GPER-1 blockade affects the anorexigenic effect of PPT. Feeding was monitored in OVX rats that received s.c. injections of vehicle or 40 μg/kg G-36 followed 30 min later by s.c. injections of vehicle or 200 μg/kg PPT. Selective activation of ERα and GPER-1 alone decreased food intake within 1 h of drug treatment, and feeding remained suppressed for 22 h following PPT treatment and 4 h following G-1 treatment. Acute administration of G-36 alone did not suppress feeding at any time point. Blockade of GPER-1 attenuated PPT's rapid (within 1 h) anorexigenic effect, but did not modulate PPT's ability to suppress food intake at 2, 4 and 22 h. These findings demonstrate that selective activation of ERα produces a rapid (within 1 h) decrease in food intake that is best explained by a non-genomic signaling pathway and thus implicates the involvement of extra-nuclear ERα. Our findings also provide evidence that activation of GPER-1 is both sufficient to suppress feeding and necessary for PPT's rapid anorexigenic effect.

Estrogen receptor-alpha isoforms are the main estrogen receptors expressed in non-small cell lung carcinoma

The expression profile of estrogen receptors (ER) in Non-Small Cell Lung Carcinoma (NSCLC) remains contradictory. Here we investigated protein and transcriptome expression of ERα wild type and variants. Tissue Micro-Arrays of 200 cases of NSCLC (paired tumor/non-tumor) were assayed by immunohistochemistry using a panel of ERα antibodies targeting different epitopes (HC20, 6F11, 1D5, ERα36 and ERα17p). ERβ epitopes were also examined for comparison. In parallel we conducted a probe-set mapping (Affymetrix HGU133 plus 2 chip) meta-analysis of 12 NSCLC tumor public transcriptomic studies (1418 cases) and 39 NSCLC cell lines. Finally, we have investigated early transcriptional effects of 17β-estradiol, 17β-estradiol-BSA, tamoxifen and their combination in two NSCLC cell lines (A549, H520). ERα transcript and protein detection in NSCLC specimens and cell lines suggests that extranuclear ERα variants, like ERα36, prevail, while wild-type ERα66 is minimally expressed. In non-tumor lung, the wild-type ERα66 is quasi-absent. The combined evaluation of ERα isoform staining intensity and subcellular localization with sex, can discriminate NSCLC subtypes and normal lung. Overall ERα transcription decreases in NSCLC. ERα expression is sex-related in non-tumor tissue, but in NSCLC it is exclusively correlating with tumor histologic subtype. ERα isoform protein expression is higher than ERβ. ERα isoforms are functional and display specific early transcriptional effects following steroid treatment. In conclusion, our data show a wide extranuclear ERα-variant expression in normal lung and NSCLC that is not reported by routine pathology ER evaluation criteria, limited in the nuclear wild type receptor.

Retraction Note to: Extranuclear ER\u03b1 is associated with regression of T47D PKC\u03b1-overexpressing, tamoxifen-resistant breast cancer

Prior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCα)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCα preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17β-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCα tumor regression requires extranuclear ERα and interaction with the extracellular matrix. T47D:A18/PKCα cells were grown in vitro using two-dimensional (2D) cell culture, three-dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERα) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERα with caveolin-1. We report that although T47D:A18/PKCα cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERα rapidly translocates to extranuclear sites during T47D:A18/PKCα tumor regression in response to both raloxifene and E2, whereas ERα is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERα to the nucleus. T47D:A18/neo tumors that do not overexpress PKCα maintain ERα in the nucleus during tamoxifen-mediated regression. An association between ERα and caveolin-1 increases in tumors regressing in response to E2. Extranuclear ERα plays a role in the regression of PKCα-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERα in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCα-expressing tumors encountered in the clinic.

Exon 7 splicing variant of estrogen receptor α is associated with pathological invasiveness in smoking-independent lung adenocarcinoma.

Patients with smoking-independent lung cancer mainly consist of females, yet the molecular background of this epidemiological feature, other than epidermal growth factor receptor (EGFR) mutation, remains unclear. Several studies have revealed the association between female hormone-associated factors and the prognosis of lung cancer, however the data remain inconsistent. The present study focused on the expression of estrogen receptor (ER)α in order to elucidate this association in smoking-independent lung cancer. Immunohistochemistry staining (IHC) of aromatase, ERα and ERβ was performed against formalin-treated tissues from 38 patients who had never-smoked who underwent complete surgical resection between 2012 and 2013. Among them, adequate RNA of the tumor and adjacent normal lung cancer was extracted from 31 matching deep frozen samples. Considering the IHC results, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure the expression level of 2 different exons of ERα, exon 6 and exon 7, which are part of the ligand binding domain of ERα, using the Taqman gene expression assay. Extra-nuclear expression of ERα using IHC demonstrated a statistically significant association with pathological invasiveness. RT-qPCR results exhibited a decreased expression of ERα exon 7 in invasive tumor tissues, compared with their adjacent normal tissues. This is consistent with the findings of previous in vitro studies indicating that extra-nuclear ERα were exon 7 splicing variants. No difference was observed in ERα exon 7 expression between normal and tumor tissues in non-invasive lung cancer tissues. When considering the EGFR mutation status, EGFR wild-type lung cancers exhibited decreased ERα exon 7 expression levels compared with EGFR mutated lung cancers. Extra-nuclear expression of ERα, which may represent exon 7 splicing variants of ERα, showed statistical association with pathological invasiveness in smoking-independent lung cancer. The post-translational splicing mechanism of ERα may be involved in the acquired invasiveness of smoking independent lung cancer.

Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice

Steroid hormone receptors including estrogen receptors (ER) classically function as ligand-regulated transcription factors. However, estrogens also elicit cellular effects through binding to extra-nuclear ER (ERα, ERβ, and G protein-coupled ER or GPER) that are coupled to kinases. How extra-nuclear ER actions impact cardiac ischemia-reperfusion (I/R) injury is unknown. We treated ovariectomized wild-type female mice with estradiol or an estrogen-dendrimer conjugate (EDC), which selectively activates extra-nuclear ER, or vehicle interventions for two weeks. I/R injury was then evaluated in isolated Langendorff perfused hearts. Two weeks of treatment with estradiol significantly decreased infarct size and improved post-ischemic contractile function. Similarly, EDC treatment significantly decreased infarct size and increased post-ischemic functional recovery compared to vehicle-treated hearts. EDC also caused an increase in myocardial protein S-nitrosylation, consistent with previous studies showing a role for this post-translational modification in cardioprotection. In further support of a role for S-nitrosylation, inhibition of nitric oxide synthase, but not soluble guanylyl cyclase blocked the EDC mediated protection. The administration of ICI182,780, which is an agonist of G-protein coupled estrogen receptor (GPER) and an antagonist of ERα and ERβ, did not result in protection; however, ICI182,780 significantly blocked EDC-mediated cardioprotection, indicating participation of ERα and/or ERβ. In studies determining the specific ER subtype and cellular target involved, EDC decreased infarct size and improved functional recovery in mice lacking ERα in cardiomyocytes. In contrast, protection was lost in mice deficient in endothelial cell ERα. Thus, extra-nuclear ERα activation in endothelium reduces cardiac I/R injury in mice, and this likely entails increased protein S-nitrosylation. Since EDC does not stimulate uterine growth, in the clinical setting EDC-like compounds may provide myocardial protection without undesired uterotrophic and cancer-promoting effects.

Neuroglobin, a pro-survival player in estrogen receptor α-positive cancer cells.

Recently, we reported that human neuroglobin (NGB) is a new player in the signal transduction pathways that lead to 17β-estradiol (E2)-induced neuron survival. Indeed, E2 induces in neuron mitochondria the enhancement of NGB level, which in turn impairs the activation of a pro-apoptotic cascade. Nowadays, the existence of a similar pathway activated by E2 in non-neuronal cells is completely unknown. Here, the role of E2-induced NGB upregulation in tumor cells is reported. E2 induced the upregulation of NGB in a dose- and time-dependent manner in MCF-7, HepG2, SK-N-BE, and HeLa cells transfected with estrogen receptor α (ERα), whereas E2 was unable to modulate the NGB expression in the ERα-devoid HeLa cells. Both transcriptional and extranuclear ERα signals were required for the E2-dependent upregulation of NGB in MCF-7 and HepG2 cell lines. E2 stimulation modified NGB intracellular localization, inducing a significant reduction of NGB in the nucleus with a parallel increase of NGB in the mitochondria in both HepG2 and MCF-7 cells. Remarkably, E2 pretreatment did not counteract the H2O2-induced caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage, as well as Bcl-2 overexpression in MCF-7 and HepG2 cells in which NGB was stably silenced by using shRNA lentiviral particles, highlighting the pivotal role of NGB in E2-induced antiapoptotic pathways in cancer cells. Present results indicate that the E2-induced NGB upregulation in cancer cells could represent a defense mechanism of E2-related cancers rendering them insensitive to oxidative stress. As a whole, these data open new avenues to develop therapeutic strategies against E2-related cancers.

Differential expression patterns and clinical significance of estrogen receptor-α and β in papillary thyroid carcinoma

BackgroundThe incidence of papillary thyroid cancer (PTC) is markedly higher in women than men during the reproductive years. In vitro studies have suggested that estrogen may play an important role in the development and progression of PTC through estrogen receptors (ERs). This study aimed to investigate the expression patterns of the two main ER subtypes, α and β1 (wild-type ERβ), in PTC tissue and their clinical significance.MethodsImmunohistochemical staining of thyroid tissue sections was performed to detect ER expression in female patients with PTC (n = 89) and nodular thyroid goiter (NTG; n = 30) using the Elivision™ plus two-step system. The relationships between ER subtype expression and clinicopathological/biological factors were further analyzed.ResultsThe positive percentage and expression levels of ERα were significantly higher in female PTC patients of reproductive age (18–45 years old; n = 50) than age-matched female NTG patients (n = 30), while ERβ1 exhibited the opposite pattern. There was no difference in ERα or ERβ1 expression between female PTC patients of reproductive age and those of advanced reproductive age (>45 years old; n = 39). In the female PTC patients of reproductive age, ERα expression level was positively correlated with that of Ki-67, while ERβ1 was negatively correlated with mutant P53. Furthermore, more patients with exclusively nuclear ERα expression had extrathyroidal extension (ETE) as compared with those with extranuclear ERα localization. VEGF expression was significantly decreased in female PTC patients of reproductive age with only nuclear ERβ1 expression when compared with those with extranuclear ERβ1 localization. In PTC patients of advanced reproductive age, neither ERα nor ERβ1 expression showed any correlation with that of Ki-67, mutant P53, VEGF, tumor size, TNM stage, ETE, or lymph node metastases.ConclusionsThe differential expression patterns of the two ER subtypes between PTC and NTG indicate that ERα may be a useful immunohistochemical marker for differential diagnosis of PTC. The associations of ER subtype expression with Ki-67, mutant P53, VEGF expression and ETE in female PTC patients of reproductive age suggest that estrogen-activated ERα may mediate stimulatory effects on PTC growth and progression whereas ERβ1 has some inhibitory actions.

Significance of ERα, HER2, and CAV1 expression and molecular subtype classification to canine mammary gland tumor.

Canine mammary gland tumor (CMT) and human breast cancer (HBC) share many similarities regarding their risk factors, histological features, and behavior. Despite the increasing evidence of molecular marker expression as a prognostic indicator for HBC, few studies have applied this approach to CMT. The aim of the present study is to evaluate the significance of the expression of estrogen receptor-alpha (ERα), human epidermal growth factor receptor 2 (HER2), and caveolin-1 (CAV1) to the behavior and the clinical outcome of CMT. Additionally, the correlation between subtype classification (luminal A, luminal B, HER2-overexpressing, basal-like, and normal-like) and tumor behavior prognosis were assessed. Canine mammary gland tissues were immunohistochemically stained for ERα, HER2, and CAV1 and evaluated and classified into 5 subtypes on the basis of immunoreactivity. Although there were no statistically significant differences in the molecular marker immunoreactivity of different subtypes, the degree of positive staining for ERα, extranuclear ERα, HER2, and CAV1 showed significant correlations (P < 0.05) with the behavior and prognosis of the tumor. The current study indicates the prognostic value of immunohistochemical staining status of ERα, HER2, and CAV1 for CMT. In addition, some trends were seen in subtype classification on the prognosis of the tumor, implying that, although further analysis is needed, there is potential clinical application of 5-subtype classification for CMT.

Xenoestrogens Alter Estrogen Receptor (ER) α Intracellular Levels

17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.

RETRACTED ARTICLE: Extranuclear ERα is associated with regression of T47D PKCα-overexpressing, tamoxifen-resistant breast cancer

BackgroundPrior to the introduction of tamoxifen, high dose estradiol was used to treat breast cancer patients with similar efficacy as tamoxifen, albeit with some undesirable side effects. There is renewed interest to utilize estradiol to treat endocrine resistant breast cancers, especially since findings from several preclinical models and clinical trials indicate that estradiol may be a rational second-line therapy in patients exhibiting resistance to tamoxifen and/or aromatase inhibitors. We and others reported that breast cancer patients bearing protein kinase C alpha (PKCα)- expressing tumors exhibit endocrine resistance and tumor aggressiveness. Our T47D:A18/PKCα preclinical model is tamoxifen-resistant, hormone-independent, yet is inhibited by 17β-estradiol (E2) in vivo. We previously reported that E2-induced T47D:A18/PKCα tumor regression requires extranuclear ERα and interaction with the extracellular matrix.MethodsT47D:A18/PKCα cells were grown in vitro using two-dimensional (2D) cell culture, three-dimensional (3D) Matrigel and in vivo by establishing xenografts in athymic mice. Immunofluoresence confocal microscopy and co-localization were applied to determine estrogen receptor alpha (ERα) subcellular localization. Co-immunoprecipitation and western blot were used to examine interaction of ERα with caveolin-1.ResultsWe report that although T47D:A18/PKCα cells are cross-resistant to raloxifene in cell culture and in Matrigel, raloxifene induces regression of tamoxifen-resistant tumors. ERα rapidly translocates to extranuclear sites during T47D:A18/PKCα tumor regression in response to both raloxifene and E2, whereas ERα is primarily localized in the nucleus in proliferating tumors. E2 treatment induced complete tumor regression whereas cessation of raloxifene treatment resulted in tumor regrowth accompanied by re-localization of ERα to the nucleus. T47D:A18/neo tumors that do not overexpress PKCα maintain ERα in the nucleus during tamoxifen-mediated regression. An association between ERα and caveolin-1 increases in tumors regressing in response to E2.ConclusionsExtranuclear ERα plays a role in the regression of PKCα-overexpressing tamoxifen-resistant tumors. These studies underline the unique role of extranuclear ERα in E2- and raloxifene-induced tumor regression that may have implications for treatment of endocrine-resistant PKCα-expressing tumors encountered in the clinic.

Distribution and Posttranslational Modification of Synaptic ERα in the Adult Female Rat Hippocampus

Acute 17β-estradiol (E2) signaling in the brain is mediated by extranuclear estrogen receptors. Here we used biochemical methods to investigate the distribution, posttranslational modification, and E2 regulation of estrogen receptor-α (ERα) in synaptosomal fractions isolated by differential centrifugation from the adult female rat hippocampus. We find that ERα is concentrated presynaptically and is highly enriched with synaptic vesicles. Immunoisolation of vesicles using vesicle subtype-specific markers showed that ERα is associated with both glutamate and γ-aminobutyric acid-containing neurotransmitter vesicles as well as with some large dense core vesicles. Experiments using broad spectrum and residue-specific phosphatases indicated that a portion of ERα in synaptosomal fractions is phosphorylated at serine/threonine residues leading to a mobility shift in SDS-PAGE and creating a double band on Western blots. The phosphorylated form of ERα runs in the upper of the two bands and is particularly concentrated with synaptic vesicles. Finally, we used E2 with or without the acyl protein thioesterase 1 inhibitor, Palmostatin B, to show that 20 min of E2 treatment of hippocampal slices depletes ERα from the synaptosomal membrane by depalmitoylation. We found no evidence that E2 regulates phosphorylation of synaptosomal ERα on this time scale. These studies begin to fill the gap between detailed molecular characterization of extranuclear ERα in previous in vitro studies and acute E2 modulation of hippocampal synapses in the adult brain.

Estrogen receptor signaling as a target for novel breast cancer therapeutics

In breast cancer (BC) epithelial cells, the mitogenic action of estradiol is transduced through binding to two receptors, ERα and ERβ, which act as transcription factors. Anti-estrogens (AEs) and aromatase inhibitors (AIs) are used clinically to arrest the estrogen-dependent growth of BC. In the case of AE or AI resistance, Herceptin or lapatinib may be used to inhibit growth factors. Estrogen effects are mediated not only through nuclear ERs but also through cytoplasmic/membrane ERs and G-protein-coupled ERs. These estrogen-binding systems associate with various proteins that direct cell cycle signaling, proliferation and survival. The partners of nuclear ER include SRC1-3, HDACs and ERβ itself as well as newly identified proteins, such as E6-AP, LKB1, PELP1, PAX-2 and FOXA1. The partners of extra-nuclear ERα include PI3K and the tyrosine kinase Src. These various factors are all potential targets for therapeutic intervention. In addition, BC proliferation is enhanced by insulin and EGF, which stimulate signaling through the MAPK and PI3K/AKT pathways by activation of the IGF-1R and EGFR axes, respectively. These pathways are tightly interconnected with ER-activated signaling, and membrane ERα forms complexes with Src and PI3K. Chemokine-mediated signaling also modulates the estrogen response. Inhibiting these pathways with specific inhibitors or activating some of the pathways by gene manipulation may be therapeutically valuable for arresting BC cell cycle progression and for inducing apoptosis to antagonize hormone-resistance. Here, we review some newly identified putatively targetable ER partners and highlight the need to develop tumor-targeting drug carrier systems affecting both the tumor cells and the tumor environment.

Abstract 1210: Tetraspanin CD63 interacts with extranuclear estrogen receptor alpha and regulates non-genomic signaling pathways in breast cancer

Abstract Tetraspanin is a four transmembrane protein family whose members are regarded as molecular linkers. They also serve as scaffold proteins for many signaling processes. CD63 is a member of the tetraspanin family that serves as a lysosomal protein. Estrogen receptor alpha (ERα) plays critical roles in breast carcinogenesis. Extranuclear ERα could initiate rapid estrogen responses by forming protein complexes with many signaling molecules. Here, we report a novel protein complex formed between tetraspanin CD63 and extranuclear ERα. Immunoprecepitation (IP) experiments in MCF-7 and BT474 cells showed that CD63 interacts with ERα at endogenous levels. IP experiments using extracts from different cellular compartments (nucleus, cytoplasm and plasma membrane) showed that the interaction between CD63 and ERα is mainly located on the plasma membrane. Moreover, using co-immunofluorescence (IF) staining, we found CD63 co-localized with ERα on plasma membrane and/or in the cytoplasm. To further explore the effects of CD63 on ERα signaling pathways, we stably knocked down CD63 by shRNA in MCF-7 and BT474 cells. Interestingly, the expression of ERα was increased with lower expression of CD63. We further examined ERα from extracts of nucleus, cytoplasm, and membrane by western blots, and found that ERα in cytoplasm and/or membrane was increased by knocking down CD63 whereas the ERα level in the nucleus was not changed. IF staining showed similar results. Furthermore, P-MAPK level was up-regulated in both MCF-7 and BT474 cell lines with suppressed expression of CD63. These data suggest that the tetraspanin CD63 may facilitate the transport of ERα between plasma membrane and/or cytoplasm and nucleus, an important new function. CD63 may play a key role on organizing and regulating non-genomic ERα signaling pathways in breast cancer. 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 1210. doi:1538-7445.AM2012-1210

Abstract 2301: E2-induced T47D:A18/PKCα tumor regression is accompanied by translocation of ERα to extranuclear sites in association with caveolin-1

Abstract Background: Acquired resistance to tamoxifen (TAM) is a serious clinical problem in the treatment of breast cancer patients. Our laboratory developed and characterized a TAM-resistant and 17 beta-estradiol (E2) independent preclinical model where PKCα was stably transfected into T47D:A18 cells compared with the E2-dependent T47D:A18/neo cells (Chisamore et al, 2001). We and others have shown that PKCα overexpression is associated with TAM resistance in cell lines and in the clinic (Tonetti et al, 2003, Assender et al, 2007). Additionally the T47D:A18/PKCα model exhibits E2-induced tumor regression in vivo or when grown in matrigel (3D matrix) but not on 2D plastic. Recently we have demonstrated that tumor regression involves participation of estrogen receptor alpha (ERα), Fas/FasL and the extracellular matrix (Zhang et al, 2009). Interestingly the membrane impermeable E2-BSA conjugate mediates growth inhibition of T47D:A18/PKCα colonies in matrigel similar to E2 suggesting a potential role of extranuclear ERα. On the basis of our preliminary data we investigated whether extranuclear ERα signaling pathway might play a critical role in E2-induced tumor regression. Materials and methods: Four micrometer thick sections were prepared from paraffin embedded T47D:A18/PKCα tumor tissues from untreated (control) and E2-treated mice and T47D:A18/neo tumors. Fluorescence tagged secondary antibodies were used for immunofluorescence staining. Co-immunoprecipitation (coIP) was done with control and E2 treatment group tumor extracts. Results: Immunofluorescence images of T47D:A18/PKCα tumor sections from untreated control mice showed that the ERα was mainly localized in the nucleus. Interestingly, treatment with E2 resulted in significant translocation of ERα from the nucleus to the cytoplasm and the plasma membrane of tumor cells. However in T47D:A18/neo tumors the majority of ERα was found in the nucleus suggesting the involvement of ERE mediated classical pathway in tumor growth. Confocal microscopy clearly indicates the colocalization of ERα with caveolin-1 in E2-treated T47D:18/PKCα tumor sections. Furthermore coIP performed with T47D:A18/PKCα tumor extracts revealed greater association of ERα with caveolin-1 in E2-treated tumors compared with control tumors. Discussion: We present evidence that E2-induced regression of our clinically relevant PKCα-overexpressing tumor model is accompanied by translocation of ERα to extranuclear sites in association with caveolin. This result suggests that E2-induced tumor regression is likely to be mediated by a membrane-associated signaling pathway. Therapies that stimulate the extranuclear ERα signaling pathway in PKCα overexpressing tumors may be an attractive approach to treat endocrine resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2301. doi:10.1158/1538-7445.AM2011-2301

Hippocampal Synthesis of Sex Steroids and Corticosteroids: Essential for Modulation of Synaptic Plasticity

Sex steroids play essential roles in the modulation of synaptic plasticity and neuroprotection in the hippocampus. Accumulating evidence shows that hippocampal neurons synthesize both estrogen and androgen. Recently, we also revealed the hippocampal synthesis of corticosteroids. The accurate concentrations of these hippocampus-synthesized steroids are determined by liquid chromatography–tandem mass-spectrometry in combination with novel derivatization. The hippocampal levels of 17β-estradiol (E2), testosterone (T), dihydrotestosterone (DHT), and corticosterone (CORT), are 5–15 nM, and these levels are sufficient to modulate synaptic plasticity. Hippocampal E2 modulates memory-related synaptic plasticity not only slowly/genomically but also rapidly/non-genomically. Slow actions of E2 occur via classical nuclear receptors (ERα or ERβ), while rapid E2 actions occur via synapse-localized or extranuclear ERα or ERβ. Nanomolar concentrations of E2 change rapidly the density and morphology of spines in hippocampal neurons. ERα, but not ERβ, drives this enhancement/suppression of spinogenesis in adult animals. Nanomolar concentrations of androgens (T and DHT) and CORT also increase the spine density. Kinase networks are involved downstream of ERα and androgen receptor. Newly developed Spiso-3D mathematical analysis is useful to distinguish these complex effects by sex steroids and kinases. Significant advance has been achieved in investigations of rapid modulation by E2 of the long-term depression or the long-term potentiation.