Estrogen Receptor-α Signaling Research Articles

WITHDRAWN: Genetic polymorphisms and promoter methylation of miR-9-3 in the modulation of breast cancer risk in a Chinese population

<h2>Abstract</h2><h3>Background</h3> <b>:</b> Genome-wide association study has discovered some susceptibility loci of breast cancer (BC), such as 1q32.1, 5q14.3, and 15q26.1. miR-9-3, located on 15q26.1, was down-regulated in BC, involving in estrogen receptor-α signaling pathway and p53-related apoptotic pathway. We aimed to investigate the role of genetic polymorphisms and promoter methylation of miR-9-3 in the modulation of BC risk. <h3>Materials and Methods</h3> <b>:</b> The rs4335754 and rs4932217 in the promoter of miR-9-3 were genotyped using the Taqman assay. The relative expression levels of miR-9-3 and <i>CDH1</i> were measured by qRT-PCR. miR-9-3 promoter methylation was examined by bisulfate genomic sequencing and transcriptional activity was evaluated by dual-luciferase reporter assay. <h3>Results</h3> <b>:</b> The rs4335754 GG, AG/GG genotypes, and G allele were associated with a reduced risk of BC (GG vs. AA: adjusted OR = 0.46, 95 % CI, 0.26-0.79, <i>p</i> = 0.004; AG/GG vs. AA: adjusted OR = 0.72, 95 % CI, 0.56-0.92, <i>p</i> = 0.009; G vs. A: adjusted OR = 0.73, 95 % CI, 0.60-0.89, <i>p</i> = 0.002, respectively). The rs4335754 AG/GG carriers displayed high levels of miR-9-3 and the rs4335754 G allele increased promoter activity. Moreover, miR-9-3 promoter hypermethylation can also reduce the expression of miR-9-3. Ectopic expression of miR-9-3 inhibited cell proliferation through targeting <i>CDH1.</i> <h3>Conclusion</h3> <b>:</b> These findings indicate that rs4335754 as well as CpGs methylation in the promoter of miR-9-3 altered its expression, and ultimately affected individual's susceptibility for the development of BC.

Endometriosis-associated Extraovarian Malignancies: A Challenging Question for the Clinician and the Pathologist.

Endometriosis is an estrogen-dependent disease, which affects 10% of women in the reproductive age. Malignant transformation is an uncommon event, which affects approximately 0.7-2.5% of women, and, when it occurs, it involves ovarian and extraovarian sites in 75% and 25% of the cases, respectively. Endometriosis correlates with presentation of clear cell and endometrioid carcinoma of the ovary. Activation of phosphatidylinositol 3-kinase (PIK3) - protein kinase B (AKT) - mammalian target of rapamycin (mTOR) pathway, aberrant chromatin remodeling due to AT-rich interactive domain-containing protein 1A (ARID1A) mutation and inactivation of estrogen receptor-α signaling seem to play a major role in the carcinogenesis. To date, little data are available regarding endometriosis-associated extraovarian malignancies. The aim of the present study was to review the clinical, pathological and prognostic features of endometriosis-related neoplasms arising from extraovarian sites, with particular focus on intestinal malignancies, urinary tract malignancies and tumors arising from surgical scars.

Neurofibromin Is an Estrogen Receptor-α Transcriptional Co-repressor in Breast Cancer

We report that neurofibromin, a tumor suppressor and Ras-GAP (GTPase-activating protein), is also an estrogen receptor-α (ER) transcriptional co-repressor through leucine/isoleucine-rich motifs that are functionally independent of GAP activity. GAP activity, in turn, does not affect ER binding. Consequently, neurofibromin depletion causes estradiol hypersensitivity and tamoxifen agonism, explaining the poor prognosis associated with neurofibromin loss in endocrine therapy-treated ER+ breast cancer. Neurofibromin-deficient ER+ breast cancer cells initially retain sensitivity to selective ER degraders (SERDs). However, Ras activation does play a role in acquired SERD resistance, which can be reversed upon MEK inhibitor addition, and SERD/MEK inhibitor combinations induce tumor regression. Thus, neurofibromin is a dual repressor for both Ras and ER signaling, and co-targeting may treat neurofibromin-deficient ER+ breast tumors.

Estrogen receptor-α signaling maintains immunometabolic function in males and is obligatory for exercise-induced amelioration of nonalcoholic fatty liver

The role of estrogen receptor-α (ERα) signaling in immunometabolic function is established in females. However, its necessity in males, while appreciated, requires further study. Accordingly, we first determined whether lower metabolic function in male mice compared with females is related to reduced ERα expression. ERα protein expression in metabolically active tissues was lower in males than in females, and this lower expression was associated with worse glucose tolerance. Second, we determined whether ERα is required for optimal immunometabolic function in male mice consuming a chow diet. Despite lower expression of ERα in males, its genetic ablation (KO) caused an insulin-resistant phenotype characterized by enhanced adiposity, glucose intolerance, hepatic steatosis, and metaflammation in adipose tissue and liver. Last, we determined whether ERα is essential for exercise-induced metabolic adaptations. Twelve-week-old wild-type (WT) and ERα KO mice either remained sedentary (SED) or were given access to running wheels (WR) for 10 wk while fed an obesogenic diet. Body weight and fat mass were lower in WR mice regardless of genotype. Daily exercise obliterated immune cell infiltration and inflammatory gene transcripts in adipose tissue in both genotypes. In the liver, however, wheel running suppressed hepatic steatosis and inflammatory gene transcripts in WT but not in KO mice. In conclusion, the present findings indicate that ERα is required for optimal immunometabolic function in male mice despite their reduced ERα protein expression in metabolically active tissues. Furthermore, for the first time, we show that ERα signaling appears to be obligatory for exercise-induced prevention of hepatic steatosis.

Abstract GS2-02: Direct regulation of estrogen receptor-α (ER) transcriptional activity by NF1

Abstract Inactivating germline mutations in the NF1 gene (encoding neurofibromin) cause neurofibromatosis type 1. In addition to peripheral nervous system tumors, NF1 patients are at higher risk for other cancers, including breast cancer. Tumor exome-sequencing studies demonstrate that approximately 20% of all human cancers have somatic NF1 mutations. NF1 has been best known for its ability to inactivate Ras as a GAP (GTPase Activating Protein). However, this function is served by a small GAP domain in a very large protein. Recurrent missense mutations inactivating the GAP activity are infrequent. In contrast, it is common to detect frameshift (FS) and nonsense (NS) NF1 mutations, which can create an NF1-null state deleting not only GAP, but also, potentially, undefined NF1 functions whose loss could also drive tumorigenesis. As we reported at SABCS previously, in 600+ patients treated by tamoxifen adjuvant monotherapy, we found that FS/NS NF1 mutations independently correlate with relapse risk (HR=2.6, p=0.03). To explore this finding, we silenced NF1 in preclinical models of ER+ breast cancer, which markedly enhanced ER transcriptional activities, causing estradiol (E2) hypersensitivity and converted tamoxifen into an agonist (in vitro and in vivo). Most important, these activities depend on ER, but not on NF1's GAP activity. These findings readily explain the poor patient outcomes associated with NS/FS NF1 mutations, and reveal a previously unrecognized function for NF1 in ER regulation. In the presence of an agonist, liganded ER repels co-repressors and recruits co-activators, while the reverse is true with an antagonist such as tamoxifen. Many co-regulators contain leucine/isoleucine rich motifs, which bind directly to the ligand-binding domain (LBD) in ER. NF1 has several of these motifs that are much more highly conserved in species with a functional ER pathway, and some of these are mutated in cancers (e.g., in our patient cohort). Furthermore, we found that NF1 canbind directly to ER, and that this binding is mediated between the ER LBD and the NF1 leucine-rich regions. Like a classic co-repressor, wildtype NF1 (but not mutants lacking GAP activity or the Leu-rich motif) binds to ER, and is recruited by ER to the ERE in the presence of tamoxifen, but not E2. Further preclinical treatment studies indicate that while NF1-deficient ER+ breast cancer should not be treated by tamoxifen or AIs, fulvestrant remains effective. Furthermore, when fulvestrant is combined with dabrafinib and trametinib to inhibit Ras effectors Raf and MEK, apoptosis is induced in vitro, and tumor regression is observed in vivo. In conclusion, we have demonstrated that NF1 is a dual negative regulator at the intersection of two potent oncogenic signaling pathways, Ras and ER, and that NF1-deficient ER+ breast cancer patients may be more effectively treated by co-targeting the Ras and ER signaling. These patients, up to 10% of those with advanced ER+ breast cancer, can be readily identified for treatment by ctDNA analysis. A clinical trial is under development. Citation Format: Chang EC, Zheng Z, Philip L, Burcu C, Lei J, Singh P, Anurag M, Chan D, Li JD, Du XP, Shafaee MN, Banks K, Sacker S, Song W, Nguyen T, Cao J, Chen X, Haricharan S, Kavuri M, Kim B-J, Zhang B, Gutmann DH, Lanman RB, Foulds C, Ellis M. Direct regulation of estrogen receptor-α (ER) transcriptional activity by NF1 [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS2-02.

Estrogen mediated expression of nucleophosmin 1 in human endometrial carcinoma clinical stages through estrogen receptor-α signaling.

BackgroundEndometrial carcinoma is one of the most common gynecologic malignancies. Estrogen plays a critical role in its pathogenesis, but the underlying mechanism is not clear. Nucleophosmin 1 (NPM1), a multifunctional protein involved in many cellular activities, has been implicated in the tumorigenesis processes. However, the role of NPM1 in endometrial carcinogenesis remains to be elucidated. The present study was aimed to elucidate the role of NPM1 in different clinical stages of human endometrial carcinoma and the underlying mechanism of NPM1 action.MethodsThe distribution and expression of NPM1 in normal endometrium, FIGO stages I to IV endometrial carcinoma tissues was analyzed using immunohistochemistry, RT-qPCR and Western blotting. The association between NPM1 expression and estrogen and estrogen receptor signaling was investigated in primary-cultured FIGO stage I endometrial adenocarcinoma cells.ResultsA strong positive correlation between NPM1 level and the clinical stage and histological grade of endometrial carcinomas was observed. Expression of NPM1 was up-regulated by estrogen in primary-cultured human endometrial adenocarcinoma cells. Furthermore, estrogen increased NPM1 level via estrogen receptor-α (ERα) signaling, nor estrogen receptor-β signaling.ConclusionsExpression of NPM1 was gradually increased with the increase of clinical stages of endometrial carcinomas. Overexpression of NPM1 may play a role in the effects of estrogen on the malignant progression of endometrioid adenocarcinoma via ERα signaling. These findings may extend our understanding of the oncogenesis of steroid hormone-related cancers and have significance for the diagnosis and treatment of this carcinoma.

Estrogen receptor-α signaling and localization regulates autophagy and unfolded protein response activation in ER+ breast cancer.

Antiestrogen therapy is commonly used to treat estrogen receptor (ER)+ breast cancers but acquired and de novo resistance limits their overall curative potential. An endoplasmic reticulum stress pathway, the unfolded protein response, and autophagy are both implicated in the development of antiestrogen therapy resistance in estrogen receptor-α (ER) positive breast cancer. Thus, we recently investigated how ERα can regulate autophagy and the unfolded protein response (Cook et al., FASEBJ, 2014). We showed that inhibiting ERα signaling stimulates autophagosome formation and flux. Moreover, we showed that ERα knockdown inhibited the unfolded protein response (UPR) signaling components. Here we support and extend this recent report showing additional data on ERα localization and provide a schematic of the overall signaling implicated by our results. Differential activation of UPR and autophagy highlight the pivotal role of ERα in regulating pro-survival signaling in breast cancer through UPR and autophagy. Furthermore, these data suggest new approaches to successful targeting ERα and preventing the regulation of key pro-survival signaling that confers resistance to endocrine therapies.

Timing and completion of puberty in female mice depend on estrogen receptor α-signaling in kisspeptin neurons

Puberty onset is initiated by activation of neurons that secrete gonadotropin-releasing hormone (GnRH). The timing and progression of puberty may depend upon temporal coordination of two opposing central mechanisms--a restraint of GnRH secretion before puberty onset, followed by enhanced stimulation of GnRH release to complete reproductive maturation during puberty. Neuronal estrogen receptor α (ERα) has been implicated in both controls; however, the underlying neural circuits are not well understood. Here we test whether these mechanisms are mediated by neurons that express kisspeptin, a neuropeptide that modulates GnRH neurosecretion. Strikingly, conditional ablation of ERα in kisspeptin neurons results in a dramatic advancement of puberty onset in female mice. Furthermore, subsequent pubertal maturation is arrested in these animals, as they fail to acquire normal ovulatory cyclicity. We show that the temporal coordination of juvenile restraint and subsequent pubertal activation is likely mediated by ERα in two separate kisspeptin neuronal populations in the hypothalamus.

Response of adult mouse uterus to early disruption of estrogen receptor-α signaling is influenced by Krüppel-like factor 9

Inappropriate early exposure of the hormone-responsive uterus to estrogenic compounds is associated with increased risk for adult reproductive diseases including endometrial cancers. While the dysregulation of estrogen receptor-alpha (ESR1) signaling is well acknowledged to mediate early events in tumor initiation, mechanisms contributing to sustained ESR1 activity later in life and leading to induction of oncogenic pathways remain poorly understood. We had shown previously that the transcription factor Krüppel-like factor 9 (KLF9) represses ESR1 expression and activity in Ishikawa endometrial glandular epithelial cells. We hypothesized that KLF9 functions as a tumor suppressor, and that loss of its expression enhances ESR1 signaling. Here, we evaluated the contribution of KLF9 to early perturbations in uterine ESR1 signaling pathways elicited by the administration of synthetic estrogen diethylstilbestrol (DES) to wild-type (WT) and Klf9 null (KO) mice on postnatal days (PNDs) 1-5. Uterine tissues collected at PND84 were subjected to histological, immunological, and molecular analyses. Compared with WT mice, KO mice demonstrated larger endometrial glands and lower endometrial gland numbers; DES exposure exacerbated these differences. Loss of KLF9 expression resulted in increased glandular ESR1 immunoreactivity with DES, without effects on serum estradiol levels. Quantitative RT-PCR analyses indicated altered expression of uterine genes commonly dysregulated in endometrial cancers (Akt1, Mmp9, Slpi, and Tgfbeta1) and of those involved in growth regulation (Fos, Myc, Tert, and Syk), with loss of Klf9, alone or in concert with DES. Our data support a molecular network between KLF9 and ESR1 in the uterus, and suggest that silencing of KLF9 may contribute to endometrial dysfunctions initiated by aberrant estrogen action.

Estrogen-Dependent and -Independent Estrogen Receptor-α Signaling Separately Regulate Male Fertility

Estrogen receptor-alpha (ERalpha) plays a critical role in male reproductive tract development and fertility. To determine whether estrogen-dependent and -independent ERalpha mechanisms are involved in male fertility, we examined male estrogen nonresponsive ERalpha knock-in mice. These animals have a point mutation (G525L) in the ligand-binding domain of ERalpha that significantly reduces interaction with, and response to, endogenous estrogens but does not affect growth factor activation of ligand-independent ERalpha pathways. Surprisingly, we found that ligand-independent ERalpha signaling is essential for concentrating epididymal sperm via regulation of efferent ductule fluid reabsorption. In contrast, estrogen-dependent ERalpha signaling is required for germ cell viability, most likely through support of Sertoli cell function. By treating estrogen nonresponsive ERalpha knock-in (ENERKI) mice with the ERalpha selective synthetic agonist propyl pyrazole triol, which is able to bind and activate G525L ERalpha in vivo, we discovered male fertility required neonatal estrogen-mediated ERalpha signaling. Thus, our work indicates both estrogen-dependent and -independent pathways play separable roles in male murine reproductive tract development and that the role of ERalpha in human infertility should be examined more closely.

Immunomodulation of FOXP3+ Regulatory T Cells by the Aromatase Inhibitor Letrozole in Breast Cancer Patients

We have shown previously that tumor infiltration by FOXP3+ regulatory T cells (Treg) is associated with increased relapse and shorter survival of patients with both in situ and invasive breast cancer. Because estrogen regulates Treg numbers in mice and promotes the proliferation of human Tregs, we hypothesized that blocking estrogen receptor-alpha signaling would abrogate Tregs and be associated with response to hormonal therapy and increased survival. FOXP3+ Tregs were quantified in tumor samples collected at baseline by incisional biopsy and after 6 months at definitive surgery in 83 elderly breast cancer patients (T2-4 N0-1) enrolled in a randomized phase II trial based on 6 months of primary letrozole (2.5 mg/d) or 6 months of letrozole plus oral "metronomic" cyclophosphamide (50 mg/d). Treg number ranged from 0 to 380 (median, 30) before treatment and from 0 to 300 (median, 8) after treatment. There was a significant reduction in Tregs in letrozole and letrozole-cyclophosphamide patients (P < 0.0001 and P < 0.002, respectively) after treatment. Treg number at residual histology was inversely related with response (P < 0.03 and P = 0.50, respectively) and a greater Treg reduction was observed in responding patients (P < 0.03). This study suggests that aromatase inhibitors may have an indirect antitumor mechanism of action through reducing Tregs in breast tumors and may be of use in estrogen receptor-alpha-negative tumors in combination with immunotherapy approaches.

Estrogen Receptor-α Signaling in Growth of the Ventral Prostate: Comparison of Neonatal Growth and Postcastration Regrowth

A role for estrogen receptor (ER)-alpha in branching morphogenesis in the ventral prostate (VP) has previously been demonstrated; in the VP of ERalpha(-/-) mice, there are fewer side branches than in wild-type littermates. In the present study, we show that in the postnatal VP, fibroblast growth factor 10 (FGF10) is expressed in wild-type mice but not in ERalpha(-/-) mice, and because branching involves proliferation pathways also used in malignant growth, we investigated whether branching during regrowth of the VP after castration involves ERalpha and FGF10. ERalpha was not detectable in the prostates of sham-operated or castrated mice but was expressed in the prostatic epithelium between d 3 and 5 after testosterone replacement. Blocking either ERalpha or ERbeta with ICI 182,780 had no detectable effects on epithelial cell proliferation during regrowth by testosterone. The ERalpha agonist, propylpyrazoletriol, did not induce regrowth by itself, but exposure to propylpyrazoletriol on d 3-5 of testosterone replacement resulted in cyclin D1-positive cells in the ductal epithelium, invasion of FGF10-positive immune cells in the regrowing prostate, and budding 14 d later. Testosterone replacement alone did not induce cyclin D1, FGF10, or bud formation. These results indicate that stimulation of ERalpha is essential for ductal branching during postnatal prostate growth. During regrowth after castration, there is a window in time when selective stimulation of ERalpha can also induce ductal branching. The FGF10 for this growth comes from the immune system, not from the prostatic mesenchyme.

P-Cadherin Is Up-Regulated by the Antiestrogen ICI 182,780 and Promotes Invasion of Human Breast Cancer Cells

P-cadherin expression in breast carcinomas has been associated with tumors of high histologic grade and lacking estrogen receptor-alpha, suggesting a link between these proteins. In the MCF-7/AZ breast cancer cell line, blocking estrogen receptor-alpha signaling with the antiestrogen ICI 182,780 induced an increase of P-cadherin, which coincided with induction of in vitro invasion. Retroviral transduction of MCF-7/AZ cells, as well as HEK 293T cells, showed the proinvasive activity of P-cadherin, which requires the juxtamembrane domain of its cytoplasmic tail. This study establishes a direct link between P-cadherin expression and the lack of estrogen receptor-alpha signaling in breast cancer cells and suggests a role for P-cadherin in invasion, through its interaction with proteins bound to the juxtamembrane domain.