Role Of Estrogen Signaling Research Articles

Abstract P4-05-01: Diet, development and predisposition to breast cancer: The impact of sugar derived metabolites (AGEs) on pubertal mammary gland development

Abstract The mammary gland is one of the few organs that continues to develop postnatally through stages including puberty, pregnancy, lactation, and involution. The gland is composed of epithelial and stromal cells that include fibroblasts, adipocytes, endothelial cells, nerve cells, and macrophages. Terminal end bud (TEB) structures are found exclusively in the pubertal developmental stage. The formation of TEBs and side branching drives mammary gland epithelial cell invasion into the mammary fat pad, continuing until the entire fat pad is filled. Pubertal mammary gland morphogenesis integrates a balance of epithelial cell proliferation, differentiation, and apoptosis. Several studies have shown that the interaction between mammary epithelial and stromal cells is crucial for the proper postnatal development of the mammary ductal tree. Interestingly, studies have shown that processes important in mammary gland development are often deregulated during breast cancer tumorigenesis. Thus, understanding the complex signaling network as well as the interactions between the different cell types during mammary gland development will be vital for elucidating the mechanisms underlying breast cancer progression and metastasis. Glycation is the non-enzymatic glycosylation of sugar moieties to biological macromolecules such as protein and DNA which produces reactive metabolites known as advanced glycation end products (AGE's). AGE content in the Western Diet has consistently increased over the last 50 years due to increased consumption of sugar laden and cheap processed/manufactured foods which are high in reactive AGE metabolites. AGE containing food can lead to the accumulation of AGEs in the body overtime leading to pro-inflammatory and pro-oxidant effects when signaling through receptor for advanced glycation end products (RAGE). Leading too many complications associated with diseases including diabetes, Alzheimer's, heart disease and cancer. Preliminary data in our lab has shown that AGEs also have an effect on phosphorylation and signaling of estrogen receptor α (ERα), a key receptor and signaling pathway in the regulation of mammary gland development during puberty. This observation, together with the links between diet, mammary gland development and immune cell recruitment lead us to examine the biological effects of a diet high in AGEs on pubertal mammary gland development in mice. We observed a significant disruption of normal pubertal mammary gland development in mice fed a high AGE diet when compared to mice fed a control diet. Mice fed the high AGE diet showed increases in TEB number as well as width, length and area. We also observed an increase in ductal branching and a decrease in ductal extension. Future studies will assess the role of macrophage recruitment to the developing gland, specifically around the TEBs based on its reported role in normal TEB function. We also plan to assess ERa signaling in mice fed the high AGE diet based on the reported role of estrogen signaling in ductal elongation. Citation Format: Krisanits BA, Nogueira LM, Findlay VJ, Turner DP. Diet, development and predisposition to breast cancer: The impact of sugar derived metabolites (AGEs) on pubertal mammary gland development [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-05-01.

Membrane-Bound Estrogen Receptor Alpha Initiated Signaling is Dynamin Dependent in MCF-7 Cells

Although membrane-associated estrogen receptors (mERs) have been known to play important role in steroid induced signal transmission, we still know little about their function in the estrogen induced proliferation of breast cancer cells. In our current work we tried to separate membrane initiated estrogen receptor (ERα,) signaling from the overall estrogenic effect in MCF7 breast carcinoma cells. Re-analyzing expression data from multiple microarray experiments, we selected a set of key regulatory genes involved in proliferation regulation and estrogen signaling. Our quantitative real time-PCR results confirmed that the selective membrane receptor agonist, estrogen-BSA induces similarly pronounced expression changes regarding these genes as 17β-estradiol. Our light and electronmicroscopical studies revealed that the membrane-bound form of classical estrogen receptor alpha is internalized after ligand binding via dynamin dependent, caveola-mediated endocytosis. Inhibition of this internalization with dynamin inhibitor, dynasore practically abolished the regulatory effect of E2-BSA, suggesting that interaction and internalization with the scaffold protein is necessary for effective signaling. The physiological role of plasma membrane estrogen receptor alpha is intensively studied, yet there are still several aspects of it to be resolved. The dynamin-dependent, ligand mediated internalization of mERs seems to play an important role in estrogen signaling. Our results may serve as another example how membrane initiated and nuclear receptor signaling well-orchestrated is and form an integrated system.

Tyrosine Phosphorylation of the Pioneer Transcription Factor FoxA1 Promotes Activation of Estrogen Signaling

The pioneer transcription factor FoxA1 plays an important role in estrogen signaling by opening closed chromatin and promoting recruitment of the estrogen receptor to its target regions in DNA. In this study, we analyzed tyrosine phosphorylation of FoxA1 by the non-receptor-type tyrosine kinase c-Abl. c-Abl was shown to phosphorylate FoxA1 at multiple sites, especially in the N- and C-terminal regions. Tyr429 and Tyr464 were identified as the major phosphorylation sites in the FoxA1 C-terminal region. The phosphomimetic and nonphosphorylatable FoxA1 mutants were generated by glutamic acid and phenylalanine substitutions at these tyrosine residues, respectively. The phosphomimetic FoxA1 promoted the activation of estrogen signaling, whereas the nonphosphorylatable FoxA1 suppressed its activation. Stimulation with the epidermal growth factor, which activates c-Abl, enhanced the activation of estrogen signaling. In contrast, the c-Abl inhibitor imatinib reduced its activation. The phosphomimetic FoxA1 mutant showed a higher affinity toward histone H3 than the wild-type. These results suggest that c-Abl-mediated phosphorylation of FoxA1 promotes the activation of estrogen signaling by inducing its binding to histones. J. Cell. Biochem. 118: 1453-1461, 2017. © 2016 Wiley Periodicals, Inc.

Targeting Estrogen Receptor Signaling with Fulvestrant Enhances Immune and Chemotherapy-Mediated Cytotoxicity of Human Lung Cancer.

The conversion of tumor cells from an epithelial to a mesenchymal-like phenotype, via a process designated as the epithelial-mesenchymal transition (EMT), is known to mediate tumor resistance to a variety of cell death inducers, including cytotoxic effector immune cells. The goal of this study was to identify and potentially repurpose FDA-approved compounds capable of reducing mesenchymal features of human lung carcinoma cells, which could be used in combination with immunotherapies or chemotherapeutic strategies to improve clinical responses. In the current report, we have utilized a quantitative high-throughput screening (qHTS) of a pharmaceutical collection of more than 2,000 compounds to identify clinically approved drugs capable of augmenting the sensitivity of mesenchymal-like, lung cancer cells to immune- and chemotherapy-mediated lysis, both in vitro and in vivo RESULTS: The estrogen receptor antagonist fulvestrant was shown to reduce mesenchymal features of lung carcinoma cells, resulting in tumor sensitization to the cytotoxic effect of antigen-specific T cells, natural killer (NK) effector cells, and chemotherapy both in vivo and in vitro CONCLUSIONS: To our knowledge, this is the first report defining a potential role for estrogenic signaling in promoting tumor resistance to immune-mediated cytotoxicity and chemotherapy in lung cancer. Our data demonstrate a robust association between the acquisition of mesenchymal attributes, therapeutic resistance of lung carcinoma cells, and the expression of estrogen receptor 1 (ESR1), supporting further investigations on the role of estrogen signaling in lung cancer progression via the induction of EMT. Clin Cancer Res; 22(24); 6204-16. ©2016 AACR.

Abstract LB-079: Effect of diet-induced obesity on tobacco carcinogen-induced lung carcinogenesis in mice

Abstract Obesity has been shown to increase breast cancer risk by causing inflammation through altered estrogen signaling. Whether obesity plays a similar role in lung carcinogenesis is currently not known. However, the role of estrogen signaling in the development and progression of lung cancer is well-established and the inflammation-estrogen signaling axis may underlie the link between obesity and lung cancer risk as well. We used a preclinical animal model to determine the role of diet-induced obesity in promoting lung carcinogenesis and inflammation caused by tobacco carcinogen exposure [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone; NNK] and the ability of estrogen pathway and/or inflammatory inhibitors to reduce this effect. Female FVB/N mice were administered a high fat (HF; 60kcal% fat; N = 67) or low fat (LF; 10kcal% fat; N = 66) diet ad libitum starting 2 weeks before NNK exposure until sacrifice. NNK was administered for 4 weeks, followed by a 4 week holding period for preneoplasia development. Subsequently, placebo, the aromatase inhibitor anastrozole (0.1mg/kg; p.o. daily) and/or aspirin (25mg/kg; p.o. daily) treatment was administered for 13 weeks after which lung tumors and blood biomarkers were evaluated. Poisson regression was used to assess differences in tumor number and mixed-effects models for differences in tumor size. Overall, the LF diet resulted in more tumors per animal (HF mean number of tumors 8.8, range 1-16; LF mean number of tumors 11.5, range 5-20), but signficantly larger tumors were observed in the HF fed mice compared to LF fed mice in all four treatment groups (HF mean tumor size 0.60 mm2, range 0.05-12.24 mm2; LF mean tumor size 0.37 mm2, range 0.06-2.14 mm2) which correlated with increased tumor Ki67 expression. Anastrozole and aspirin as single agents were significantly more effective at decreasing tumor number in HF fed mice compared to LF fed mice; however, the combined treatment was equally effective in both dietary groups. Inflammatory cell count in bronchoalveolar lavage fluid, tumor infiltrating inflammatory cells and tumor aromatase, COX-2 and nuclear factor kappa-B expression were all increased in HF compared to LF fed mice. This increased inflammatory response may be responsible for the observed increase in tumor size in the HF fed mice. In addition, levels of circulating inflammatory cytokines such as IL-6, IL-10 and TNF-α were significantly down-modulated by anastrozole and also by combined treatment in LF fed mice but not in HF fed mice. These results suggest that while dietary fat may not affect lung cancer incidence, a HF diet may contribute to lung tumor progression potentially through dysregulation of the inflammation-estrogen signaling axis. Supported by R21 CA184611. Citation Format: Brenda Diergaarde, Beatriz Kanterewicz, Mary E. Rothstein, Autumn Gaither-Davis, Shira R. Abberbock, Brenda F. Kurland, Laura P. Stabile. Effect of diet-induced obesity on tobacco carcinogen-induced lung carcinogenesis in mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-079.

Estrogen and estrogen receptor alpha promotes malignancy and osteoblastic tumorigenesis in prostate cancer.

The role of estrogen signaling in regulating prostate tumorigenesis is relatively underexplored. Although, an increasing body of evidence has linked estrogen receptor beta (ERß) to prostate cancer, the function of estrogen receptor alpha (ERα) in prostate cancer is not very well studied. We have discovered a novel role of ERα in the pathogenesis of prostate tumors. Here, we show that prostate cancer cells express ERα and estrogen induces oncogenic properties in prostate cancer cells through ERα. Importantly, ERα knockdown in the human prostate cancer PacMetUT1 cells as well as pharmacological inhibition of ERα with ICI 182,780 inhibited osteoblastic lesion formation and lung metastasis in vivo. Co-culture of pre-osteoblasts with cancer cells showed a significant induction of osteogenic markers in the pre-osteoblasts, which was attenuated by knockdown of ERα in cancer cells suggesting that estrogen/ERα signaling promotes crosstalk between cancer and osteoblastic progenitors to stimulate osteoblastic tumorigenesis. These results suggest that ERα expression in prostate cancer cells is essential for osteoblastic lesion formation and lung metastasis. Thus, inhibition of ERα signaling in prostate cancer cells may be a novel therapeutic strategy to inhibit the osteoblastic lesion development as well as lung metastasis in patients with advanced prostate cancer.

Estrogen regulates luminal progenitor cell differentiation through H19 gene expression.

Although the role of estrogen signaling in breast cancer development has been extensively studied, the mechanisms that regulate the indispensable role of estrogen in normal mammary gland development have not been well studied. Because of the unavailability of culture system to maintain estrogen-receptor-positive (ERα+) cells in vitro, the molecular mechanisms that regulate estrogen/ERα signaling in the normal human breast are unknown. In the present study, we examined the effects of estrogen signaling on ERα+ human luminal progenitors using a modified matrigel assay and found that estrogen signaling increased the expansion potential of these progenitors. Furthermore, we found that blocking ERα attenuated luminal progenitor expansion and decreased the luminal colony-forming potential of these progenitors. Additionally, blocking ERα decreased H19 expression in the luminal progenitors and led to the development of smaller luminal colonies. We further showed that knocking down the H19 gene in the luminal progenitors significantly decreased the colony-forming potential of the luminal progenitors, and this phenotype could not be rescued by the addition of estrogen. Lastly, we explored the clinical relevance of the estrogen–H19 signaling axis in breast tumors and found that ERα+ tumors exhibited a higher expression of H19 as compared with ERα− tumors and that H19 expression showed a positive correlation with ERα expression in those tumors. Taken together, the present results indicate that the estrogen–ERα–H19 signaling axis plays a role in regulating the proliferation and differentiation potentials of the normal luminal progenitors and that this signaling network may also be important in the development of ER+ breast cancer tumors.

Fulvestrant-mediated inhibition of estrogen receptor signaling slows lung cancer progression.

Estrogens are key signaling molecules that regulate various physiological processes such as cell growth, development, and differentiation. They also play a major role in many pathological conditions, such as hormone-dependent cancer. The importance of inhibiting estrogen receptor signaling in diseases of estrogen target tissues, such as breast cancer, is well documented. However, the role of estrogen signaling in diseases of nontarget tissues, such as lung cancer, is not well characterized. The aim of the current study is to examine the expression of estrogen receptor β (ERβ) and the roles of estradiol (E2) and fulvestrant on the progression of lung cancer. Tissue microarray (TMA) and immunohistochemistry (IHC) analyses were used to detect the expression of aromatase, ERα, and ERβ in 198 patients. We performed analyses to determine if there was any correlation among these three proteins. A mouse model of urethane-induced lung adenocarcinoma was used in the study. Mice were divided into three treatment groups: blank control, E2 alone, and E2 + fulvestrant (ERβ antagonist). Western blot analysis and fluorescence quantitative PCR (FQ-PCR) were used to measure expression of ERβ protein and mRNA levels, respectively. ERβ, but not ERα, was overexpressed in NSCLC samples. Lung cancer progression in mice treated with E2 was significantly increased compared to either the control group or the E2 + fulvestrant group. Mice in the E2 treatment group had significantly increased expression of ERβ at both the mRNA and protein levels compared to mice treated with E2 + fulvestrant or control. Our data suggest that ERβ promotes lung cancer progression in mice and that this progression can be inhibited with fulvestrant. These findings may help elucidate the role of ERβ in lung cancer and suggest that estrogen receptor antagonists, such as fulvestrant, may be therapeutically beneficial for the treatment of the disease.

Abstract LB-275: Adult body size and physical activity in relation to risk of breast cancer defined by tumor androgen receptor status

Abstract Background: In contrast to the well-established role of estrogens in breast cancer risk and survival, the role of androgens in breast carcinogenesis is unclear. Previous studies have shown that, in postmenopausal women, higher body mass index (BMI) and being less physically active are associated with an increased risk of invasive breast cancer, and the associations generally are stronger for estrogen receptor (ER) and/or progesterone receptor (PR) positive tumors. Although the androgen pathway also may play a mechanistic role with these breast cancer risk factors, no prior studies have specifically evaluated these associations by tumor androgen receptor (AR) status. Observing differential associations by AR status would provide etiologic insights into the contribution of the androgen pathway in breast carcinogenesis. Methods: We evaluated the associations of adult body size and physical activity with risk of incident invasive breast cancer by AR status in the Nurses’ Health Study. We used validated questionnaires to assess height, weight, and physical activity. AR status was determined using immunohistochemistry on sections from tumor tissue microarrays (TMAs); ER and PR status were determined using TMAs or medical record pathology reports. We applied a constrained competing risk survival model to evaluate these associations by AR status while controlling for ER/PR status and established breast cancer risk factors. Results: A total of 2,198 cases (1,701 AR+; 497 AR- tumors) were documented during 26 years of follow-up of 96,966 eligible women. After adjusting for ER/PR status and other breast cancer risk factors, higher BMI was associated with an increased risk of both AR+ and AR- tumors. The multivariable hazard ratios (MV HRs) and 95% confidence intervals (95%CIs) for every 5 kg/m2 increase in BMI were 1.05 (0.99, 1.10) for AR+ and 1.10 (1.00, 1.22) for AR- tumors (p-value for heterogeneity=0.46). Further, women engaged in higher amounts of total physical activity including brisk walking were at a lower risk of developing breast cancer. The MV HRs (95%CIs) per approximately 5 hours of brisk walking per week were 0.87 (0.72, 1.05) for AR+ and 0.69 (0.46, 1.04) for AR- tumors (p-value for heterogeneity=0.32). We further examined these associations by the combinations of ER/PR/AR status and observed that only BMI associations differed significantly across these subtypes (p-value for heterogeneity=0.03). The MV HRs (95%CIs) for 5 kg/m2 increase in BMI were 1.15 (1.08, 1.23) for ER+PR+AR+, 1.23 (1.04, 1.45) for ER+PR+AR-, 0.88 (0.75, 1.03) for ER+PR-AR+, 1.08 (0.92, 1.28) for ER-PR-AR+, 1.19 (1.01, 1.39) for ER-PR-AR-, 0.92 (0.70,1.22) for ER+PR-AR-. Conclusion: In postmenopausal women, higher adult body size was associated with an increased risk of both AR+ and AR- tumors while physical activity, including brisk walking, was associated with a reduced risk of both subtypes. The strongest associations observed with the ER+PR+AR- and ER+PR+AR+ tumors (i.e., subtypes with the most active estrogen signaling) supports the important role of estrogen signaling in the postmenopausal BMI/breast cancer relationship. Further, the positive association observed between BMI and ER-PR-AR- tumors suggests pathways other than sex steroids (e.g., the insulin pathway) also might play an etiologic role and more studies are warranted to confirm and extend these findings. Citation Format: Xuehong Zhang, A. Heather Eliassen, Rulla Tamimi, Aditi Hazra, Andrew H. Beck, Myles Brown, Laura C. Collins, Bernard Rosner, Susan E. Hankinson. Adult body size and physical activity in relation to risk of breast cancer defined by tumor androgen receptor status. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-275. doi:10.1158/1538-7445.AM2014-LB-275

GPER mediated estradiol reduces miR-148a to promote HLA-G expression in breast cancer

Breast cancer is the most common malignant diseases in women. miR-148a plays an important role in regulation of cancer cell proliferation and cancer invasion and down-regulation of miR-148a has been reported in both estrogen receptor (ER) positive and triple-negative (TN) breast cancer. However, the regulation mechanism of miR-148a is unclear. The role of estrogen signaling, a signaling pathway is important in development and progression of breast cancer. Therefore, we speculated that E2 may regulate miR-148a through G-protein-coupled estrogen receptor-1 (GPER). To test our hypothesis, we checked the effects of E2 on miR-148a expression in ER positive breast cancer cell MCF-7 and TN cancer cell MDA-MB-231. Then we used GPER inhibitor G15 to investigate whether GPER is involved in regulation of E2 on miR-148a. Furthermore, we analyzed whether E2 affects the expression of HLA-G, which is a miR-148a target gene through GPER. The results showed that E2 induces the level of miR-148a in MCF-7 and MDA-MB-231 cells, GPER mediates the E2-induced increase in miR-148a expression in MCF-7 and MDA-MB-231 cells and E2-GPER regulates the expression of HLA-G by miR-148a. In conclusion, our findings offer important new insights into the ability of estrogenic GPER signaling to trigger HLA-G expression through inhibiting miR-148a that supports immune evasion in breast cancer.

ER-α36-Mediated Rapid Estrogen Signaling Positively Regulates ER-Positive Breast Cancer Stem/Progenitor Cells

The breast cancer stem cells (BCSC) play important roles in breast cancer occurrence, recurrence and metastasis. However, the role of estrogen signaling, a signaling pathway important in development and progression of breast cancer, in regulation of BCSC has not been well established. Previously, we identified and cloned a variant of estrogen receptor α, ER-α36, with a molecular weight of 36 kDa. ER-α36 lacks both transactivation domains AF-1 and AF-2 of the 66 kDa full-length ER-α (ER-α66) and mediates rapid estrogen signaling to promote proliferation of breast cancer cells. In this study, we aim to investigate the function and the underlying mechanism of ER-α36-mediated rapid estrogen signaling in growth regulation of the ER-positive breast cancer stem/progenitor cells. ER-positive breast cancer cells MCF7 and T47D as well as the variants with different levels of ER-α36 expression were used. The effects of estrogen on BCSC's abilities of growth, self-renewal, differentiation and tumor-seeding were examined using tumorsphere formation, flow cytometry, indirect immunofluorence staining and in vivo xenograft assays. The underlying mechanisms were also studied with Western-blot analysis. We found that 17-β-estradiol (E2β) treatment increased the population of ER-positive breast cancer stem/progenitor cells while failed to do so in the cells with knocked-down levels of ER-α36 expression. Cells with forced expression of recombinant ER-α36, however, responded strongly to E2β treatment by increasing growth in vitro and tumor-seeding efficiency in vivo. The rapid estrogen signaling via the AKT/GSK3β pathway is involved in estrogen-stimulated growth of ER-positive breast cancer stem/progenitor cells. We concluded that ER-α36-mediated rapid estrogen signaling plays an important role in regulation and maintenance of ER-positive breast cancer stem/progenitor cells.

Estrogen receptor-β expression and pharmacological targeting in bladder cancer

A role for estrogen signaling in urothelial carcinoma of the bladder (UCB) is suggested to be associated with more advanced disease with worse outcomes in women. Estrogen receptor β (ERβ) is the predominant receptor in bladder tissues. We aimed to ascertain whether ERβ correlates with clinicopathological predictors of aggressive bladder cancer and worse survival outcomes. ERβ was measured by immunohistochemistry in malignant and adjacent benign bladder tissues in patients (N=72) with UCB who underwent radical cystectomy. ERβ expression was tested for statistical association with clinicopathological variables and patient survival. ERβ expression was determined in bladder cancer cell lines, and the effects of the selective estrogen modulator tamoxifen and the ERβ agonist diarylpropionitrile on cell growth were determined. The ERβ level was significantly higher in malignant vs. benign urothelium (P<0.001) and was strongly associated with aggressive tumor histology characterized by lymphovascular (P=0.008) and perineural (P=0.006) invasion, and clinical histories of pelvic irradiation (P=0.005), hydronephrosis (P=0.022) and no intravesical chemotherapy (P=0.038). All patients with a high (>70%) percentage of ERβ positivity in tissue with >3-month follow-up developed recurrent disease (P=0.009). Higher ERβ level was predictive of worse recurrence-free and overall survival following cystectomy, after adjustment for tumor stage, and remained significantly associated with recurrence-free survival in the multivariable analysis including tumor stage, nodal stage and lymphovascular invasion. Activation of ERβ in bladder cancer cell lines led to significant increases in proliferation, while pharmacological inhibition with tamoxifen blocked cell growth. Our study supports a role for ERβ in aggressive UCB. Pharmacological targeting of ERβ warrants further investigation as a therapeutic strategy in UCB.

Functional Adaptation in Female Rats: The Role of Estrogen Signaling

BackgroundSex steroids have direct effects on the skeleton. Estrogen acts on the skeleton via the classical genomic estrogen receptors alpha and beta (ERα and ERβ), a membrane ER, and the non-genomic G-protein coupled estrogen receptor (GPER). GPER is distributed throughout the nervous system, but little is known about its effects on bone. In male rats, adaptation to loading is neuronally regulated, but this has not been studied in females.Methodology/Principal FindingsWe used the rat ulna end-loading model to induce an adaptive modeling response in ovariectomized (OVX) female Sprague-Dawley rats. Rats were treated with a placebo, estrogen (17β-estradiol), or G-1, a GPER-specific agonist. Fourteen days after OVX, rats underwent unilateral cyclic loading of the right ulna; half of the rats in each group had brachial plexus anesthesia (BPA) of the loaded limb before loading. Ten days after loading, serum estrogen concentrations, dorsal root ganglion (DRG) gene expression of ERα, ERβ, GPER, CGRPα, TRPV1, TRPV4 and TRPA1, and load-induced skeletal responses were quantified. We hypothesized that estrogen and G-1 treatment would influence skeletal responses to cyclic loading through a neuronal mechanism. We found that estrogen suppresses periosteal bone formation in female rats. This physiological effect is not GPER-mediated. We also found that absolute mechanosensitivity in female rats was decreased, when compared with male rats. Blocking of adaptive bone formation by BPA in Placebo OVX females was reduced.ConclusionsEstrogen acts to decrease periosteal bone formation in female rats in vivo. This effect is not GPER-mediated. Gender differences in absolute bone mechanosensitivity exist in young Sprague-Dawley rats with reduced mechanosensitivity in females, although underlying bone formation rate associated with growth likely influences this observation. In contrast to female and male rats, central neuronal signals had a diminished effect on adaptive bone formation in estrogen-deficient female rats.

P5-02-01: ErbB4 Ectodomain as a Biomarker and a Potential Therapeutic Target for Breast Cancer.

Abstract ErbB4 function is controversial in breast cancer and influenced by alternative splicing of the ERBB4 gene. Here we evaluated the cleavable ErbB4 JM-a isoform as a potential drug target and biomarker. To address ErbB4 cleavage in breast cancer tissues in vivo, extracellular and intracellular cleavage products of ErbB4 were assessed by ELISA and immunohistochemistry using matched serum and tissue samples from a series of 243 breast cancer patients. Elevated serum ectodomain level (≥40 ng/mL) and nuclear immunoreactivity were present in 21% and 52% of the patients, respectively. An elevated serum ErbB4 ectodomain concentration was not associated with tumor nuclear immunoreactivity, but was significantly associated with the premenopausal status at diagnosis (P = 0.04). Estradiol also enhanced ErbB4 cleavage and ErbB4 cleaving enzyme activity in vitro, supporting a role for estrogen signaling in regulation of ErbB4 cleavage. Selective targeting of the cleavable ErbB4 JM-a in human breast cancer cells with an ErbB4 isoform-specific mAb 1479 inhibited ErbB4 cleavage and reduced tumor formation in a mouse xenograft model. Consistent with blocking of ErbB4 cleavage, mutagenesis studies and the 3.4 Å X-ray crystal structure of a complex of the ErbB4 extracellular region and the 1479 Fab localized the binding site of mAb 1479 on ErbB4 to a region on subdomain IV encompassing JM-a-specific residues. These data demonstrate that ErbB4 is cleaved in a subset of breast cancer patients, and suggest that the mechanisms by which mAb 1479 suppresses breast cancer cell growth involves inhibition of ErbB4 cleavage. Serum ErbB4 ectodomain concentration could be used as a biomarker to monitor the activity of compounds, such as mAb 1479, that target ErbB4 cleavage. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-02-01.

Estrogen and Its Receptor Enhance Mechanobiological Effects in Compressed Bone Mesenchymal Stem Cells

Mechanical stimulation and estrogen have been proven to be two important factors in promoting mesenchymal stem cell activity, which is closely associated with bone formation, mass maintenance and remodeling. However, the superposition effects of mechanical stimulation and estrogen on stem cells remain unknown. It is also unclear if the estrogen receptor (ER) plays only a key role in estrogen signaling or if it is also involved in the mechanotransduction of stem cells. To investigate the role of estrogen and its receptors in the mechanobiological effects in bone mesenchymal stem cells (BMSCs), isolated mesenchymal stem cells from bone marrow were exposed to mechanical pressure under additional estrogen treatment or ER blockade. Cell proliferation was examined using an MTT assay and alkaline phosphatase (ALP) activity was determined by a modified enzyme kinetic method. Alignment of the cytoskeleton was observed by Coomassie brilliant blue staining and F-actin fluorescent staining. Cellular ultrastructure was observed under transmission electron microscope. Expression of ERα was investigated using Western blot analysis. Results indicated that mechanical pressure promoted cell proliferation, ALP activity, ERα expression and F-actin stress fiber formation. Overall, this effect was enhanced by the addition of estrogen and inhibited by ER blockade. We concluded that pressure stimulated proliferation and differentiation capability via F-actin transduction in BMSCs. The effects were enhanced by the addition of estrogen, and the ER plays an important role in regulating mechanobiological effects and the mechanotransduction processes of BMSCs.

Expression of Estrogen Receptor Alpha36 in the Hamster Ovary and Its Possible Regulation by Gonadotropins and Steroid Hormones.

In the ovary, estrogen signaling is of utmost importance that is carried out by the estrogen receptor, member of nuclear receptor superfamily. In reproductive females, estrogen plays an essential role in the reproductive cyclicity and other ovarian functions. Here we report the expression of estrogen receptor α36 (ERα36), a splice variant of the estrogen receptor α (ERα), in the hamster ovary throughout the estrous cycle, and its possible regulation by follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol-17β (E) and progesterone (P). Hamster ovaries were collected from Day 1 (estrus, D1) though Day 4 (proestrus, D4) morning and D4 afternoon, and the expression pattern of ERα36 protein was examined by immunofluorescence and Western immunoblotting. Immunoblot data indicated that ERα36 expression declined by D4:0900h through D4:1600h compared to other days of the estrous cycle. Immunofluorescence results revealed that ERα36 was expressed in the plasma membrane of both follicular and interstitial cells in the ovary with highest level of expression in the granulosa cells; however, the immunostaining declined by D4:0900h through D4:1600h. No staining was observed in the cell nuclei. To determine the role of individual reproductive hormones on ovarian ERα36 expression, hypophysectomized hamsters were treated with FSH, LH, E or P on postoperative day 10. Immunoblot data revealed a significant decline in ERα36 protein in hypophysectomized hamsters compared to intact hamster on D1. FSH-treatment upregulated the receptor protein level to that of D1 hamsters. LH-treatment showed a similar increase. In contrast, a combined treatment of FSH and LH failed to raise the receptor protein level to that of the either hormone alone. E or P treatment upregulated ERα36 protein level, but P effect was stronger. Nevertheless, the increase in ERα36 expression was much less than that was induced by FSH and LH. No increase was observed after a combined treatment of E and P. The data were validated by immunofluorescence localization. These results indicate that the ERα splice variant, ERα36, is translated into protein in ovarian follicular cells, but the receptor protein is mostly localized in the plasma membrane in contrast to the classic ERα, which is located in the nucleus. Further, the induction of ERα mRNA splicing and the translation of the truncated receptor mRNA seems to be regulated by gonadotropins, such FSH or LH. Ovarian steroids may have a different regulatory role on ERα36 expression. Therefore, ERα36 may play a unique regulatory role in estrogen signaling during ovarian follicular development. (poster)

Expression and Function of a Novel Variant of Estrogen Receptor–α36 in Murine Airways

Evidence suggests that estrogen signaling is involved in sex differences in the prevalence rates and control of asthma, but the expression patterns of estrogen receptor variants and estrogen function in the lung are not well established. We investigated the expression of major estrogen receptor variants occurring naturally and after the development of allergen-induced airway hyperreactivity in a murine model of allergic asthma, along with the role of estrogen signaling in small-airway ciliary motion and smooth muscle contraction. Female BALB/c mice were sensitized with ovalbumin, and estrogen receptor expression patterns were examined by immunofluorescence and Western blot analysis. Time-lapse video and photodiode-based displacement measurement systems were used to assess the effects of estrogen signaling on airway ciliary beat frequency and smooth muscle contraction. We found that a novel variant of estrogen receptor (ER)-α, ER-α36, is expressed in airway epithelial and smooth muscle cells. ER-α36 was predominately localized on the plasma membranes of airway cells. After sensitization to allergen, the expression levels of ER-α36 increased significantly (P < 0.01), whereas the expression of ER-β and ER-α66 did not significantly change. Estrogen treatment in vitro resulted in a rapid increase in airway cilia motion in a dose-dependent fashion, but did not exert any effect on airway smooth muscle contraction. We speculate that the up-regulation of estrogen receptor expression associated with allergen-induced airway hyperresponsiveness may constitute a protective mechanism to facilitate the clearance of mucus. The identification and localization of specific estrogen receptor subtypes in the lung could lead to newer therapeutic avenues aimed at addressing sex differences of asthma susceptibility.

Interaction of estrogen receptor‐α ligand binding domain with nuclear proteins of aging mouse brain

After the interaction of estrogen with the ligand binding domain (LBD) of mouse estrogen receptor-alpha (mERalpha) and hormone-responsive elements of target genes, many nuclear proteins are recruited to regulate the expression of specific genes. Because it is not known which brain proteins interact with LBD or whether these proteins vary with age and sex, we used pull-down assay and far Western blotting to detect five nuclear proteins of 160, 140, 87, 60, and 46 kD in the mouse brain. These interacting proteins were identified as PELP1, RIP140, PGC1alpha, BAF60, and ADA3, respectively. The level of PELP1, RIP140, PGC1alpha, and BAF60 decreased drastically in old compared with adult male mice, whereas the ADA3 level showed no significant change. PELP1, PGC1alpha, and BAF60 levels were lower in old male compared with female mice. Thus we report the identification and interaction of five nuclear proteins with mERalpha-LBD, indicating their role in estrogen signaling and brain functions during aging.

Cellular targets of estrogen signaling in regeneration of inner ear sensory epithelia

Estrogen signaling in auditory and vestibular sensory epithelia is a newly emerging focus propelled by the role of estrogen signaling in many other proliferative systems. Understanding the pathways with which estrogen interacts can provide a means to identify how estrogen may modulate proliferative signaling in inner ear sensory epithelia. Reviewed herein are two signaling families, EGF and TGFß. Both pathways are involved in regulating proliferation of supporting cells in mature vestibular sensory epithelia and have well characterized interactions with estrogen signaling in other systems. It is becoming increasingly clear that elucidating the complexity of signaling in regeneration will be necessary for development of therapeutics that can initiate regeneration and prevent progression to a pathogenic state.

Effects of Loss of Classical Estrogen Response Element Signaling on Bone in Male Mice

The role of estrogen signaling in the male skeleton via estrogen receptor (ER)-alpha is now well established. ERalpha can elicit responses through either classical estrogen response elements (ERE) pathways or nonclassical, non-ERE pathways. In the present study, we examined the effects of either the attenuation or loss of classical ERalpha signaling on the murine male skeleton. To accomplish this, we crossed male mice heterozygous for a knock-in mutation [nonclassical ERalpha knock-in (NERKI)], which abolishes the ERE-mediated pathway with female heterozygous ERalpha knockout mice (ERalpha+/-) and studied the F1 generation ERalpha+/+, ERalpha+/-, ERalpha+/NERKI, and ERalpha-/NERKI male progeny longitudinally using bone density and histomorphometry. The only ERalpha allele present in ERalpha-/NERKI mice is incapable of classical ERE-mediated signaling, whereas the heterozygous ERalpha+/NERKI mice have both one intact ERalpha and one NERKI allele. As compared with ERalpha+/+ littermates (n=10/genotype), male ERalpha+/NERKI and ERalpha-/NERKI mice displayed axial and appendicular skeletal osteopenia at 6, 12, 20, and 25 wk of age, as demonstrated by significant reductions in total bone mineral density (BMD) at representative sites (areal BMD by dual-energy x-ray absorptiometry at the lumbar vertebrae and femur and volumetric BMD by peripheral quantitative computed tomography at the tibia; P<0.05-0.001 vs. ERalpha+/+). The observed osteopenia in these mice was evident in both trabecular and cortical bone compartments. However, these decreases were more severe in mice lacking classical ERalpha signaling (ERalpha-/NERKI mice), compared with mice in which one wild-type ERalpha allele was present (ERalpha+/NERKI mice). Collectively, these data demonstrate that classical ERalpha signaling is crucial for the development of the murine male skeleton.