ERα Isoform Research Articles

Estrogen receptors and extracellular matrix: the critical interplay in cancer development and progression.

Cancer remains a significant global health concern. Breast cancer is a multifaceted and prevalent disease influenced by several factors, among which estrogen receptors (ERs) and the extracellular matrix (ECM) play pivotal roles. ERs, encompassing ERα and ERβ, exert significant diversity on tumor behavior, cell signaling, invasion, and metastatic potential, thus guiding breast cancer prognosis. Understanding the multifunctional connections between ERs and ECM that mediate the dynamics of tumor microenvironment is vital for unraveling the complexity of breast cancer pathobiology and identifying novel therapeutic targets. This critical review delves into the intricate nature of ERs, emphasizing their structural isoforms and the consequential impact on breast cancer outcomes. A detailed examination of ER-mediated cell signaling pathways reveals how differential expression of ERα and ERβ isoforms influence breast cancer cell behavior. The functional ERs-matrix interactions emerge as a pivotal factor in modulating epigenetic mechanisms of breast cancer cells, orchestrating changes in cellular phenotype and expression patterns of matrix modulators. Specifically, ERα isoforms are shown to regulate ECM signaling cascades, while the effects of ECM components on ERα activity highlight a bidirectional regulatory axis. The diversity of ERβ isoforms is also highlighted, illustrating their distinct contribution to ECM-mediated cellular responses. This review underscores the complex interplay between ERα/β isoforms and the ECM, shedding light onto the potential therapeutic strategies targeting these interactions to improve breast cancer management.

Silmitasertib (CX-4945) Disrupts ERα/HSP90 Interaction and Drives Proteolysis through the Disruption of CK2β Function in Breast Cancer Cells.

Aberrant estrogen receptor (ERα) signaling mediates detrimental effects of tamoxifen including drug resistance and endometrial hyperplasia. ERα36, an alternative isoform of ERα, contributes to these effects. We have demonstrated that CK2 modulates ERα expression and function in breast cancer (BCa). Here, we assess if CX-4945 (CX), a clinical stage CK2 inhibitor, can disrupt ERα66 and ERα36 signaling in BCa. Using live cell imaging, we assessed the antiproliferative effects of CX in tamoxifen-sensitive and tamoxifen-resistant BCa cells in monolayer and/or spheroid cultures. CX-induced alterations in ERα66 and ERα36 mRNA and protein expression were assessed by RT-PCR and immunoblot. Co-immunoprecipitation was performed to determine the differential interaction of ERα isoforms with HSP90 and CK2 upon CX exposure. CX caused concentration-dependent decreases in proliferation in tamoxifen-sensitive MCF-7 and tamoxifen-resistant MCF-7 Tam1 cells and significantly repressed spheroid growth in 3D models. Additionally, CX caused dramatic decreases in endogenous or exogenously expressed ERα66 and ERα36 protein. Silencing of CK2β, the regulatory subunit of CK2, resulted in destabilization and decreased proliferation, similar to CX. Co-immunoprecipitation demonstrated that ERα66/36 show CK2 dependance for interaction with molecular chaperone HSP90. Our findings show that CK2 functions regulate the protein stability of ERα66 and ERα36 through a mechanism that is dependent on CK2β subunit and HSP90 chaperone function. CX may be a component of a novel therapeutic strategy that targets both tamoxifen-sensitive and tamoxifen-resistant BCa, providing an additional tool to treat ERα-positive BCa.

GENERATION AND CHARACTERIZATION OF POLYCLONAL ANTIBODIES SPECIFIC TO HUMAN ESTROGEN RECEPTOR ERα

Aim. The purpose of the study was to generate and characterize anti-hERα polyclonal antibodies for elucidation of functional relationships between isoforms of estrogen receptor ERα and isoforms of ribosomal protein S6 kinase — S6K1. Methods. cDNA cloning. Expression of recombinant proteins in bacterial system. Affinity purification of His-tag fused recombinant proteins using Ni-NTA chromatography from bacterial lysates. Generation of polyclonal sera by mice immunization. Western blot analysis and immunoprecipitation. Results. cDNA coding for full length hERα was cloned into expression vector pET28a in frame with His-tag sequence. Recombinant hERα-His protein was expressed in E.Coli and purified by Ni-NTA chromatography. Purified hERα-His was used as antigen for mice immunization and generation of polyclonal antibodies. Specificity of polyclonal antibodies was analyzed by Western blot and immunoprecipitation of hERα from MCf-7 cell lysates. Conclusions. Generated anti-hERα polyclonal antibodies are of conformational type since specifically recognized hERα only in immunoprecipitation but not in Western blot. Created polyclonal antibodies a suitable for detection and analysis of hERα protein complexes.

Laryngeal Cancer Cells Metabolize 25-Hydroxyvitamin D3 and Respond to 24R,25-dihydroxyvitamin D3 via a Mechanism Dependent on Estrogen Receptor Levels.

Studies have evaluated vitamin D3's therapeutic potential in estrogen-responsive cancers, with conflicting findings. We have shown that the proliferation of breast cancer cells is regulated by 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) depending on estrogen receptor alpha 66 (ERα66) expression, suggesting that this could also be the case for estrogen-sensitive laryngeal cancer cells. Accordingly, we examined levels of ERα isoforms in ERα66-positive UM-SCC-12 and ERα66-negative UM-SCC-11A cells and their response to 24R,25(OH)2D3. 24R,25(OH)2D3 stimulated proliferation, increased the expression of metastatic markers, and inhibited apoptosis in UM-SCC-12 cells while having the opposite effect in UM-SCC-11A cells. To evaluate if vitamin metabolites could act via autocrine/paracrine mechanisms, we assessed the expression, protein levels, and activity of vitamin D3 hydroxylases CYP24A1 and CYP27B1. Both cell types expressed both mRNAs; but the levels of the enzymes and their activities were differentially regulated by estrogen. ERα66-negative UM-SCC-11A cells produced more 24,25(OH)2D3 than UM-SCC-12 cells, but comparable levels of 1,25(OH)2D3 when treated with 25(OH)D3 These results suggest that the regulation of vitamin D3 metabolism in laryngeal cancer cells is modulated by ERα66 expression, and support a role for 24R,25(OH)2D3 as an autocrine/paracrine regulator of laryngeal cancer. The local metabolism of 25(OH)D3 should be considered when determining the potential of vitamin D3 in laryngeal cancer.

Estrogen receptor α isoforms generated by alternative use of cryptic exons.

Estrogen receptor α (ERα) regulates several physiological functions. In pathophysiological conditions, ERα is involved in the development and progression of estrogen-sensitive tumors. The ERα gene contains multiple 5'-untranslated exons and eight conventional coding exons and presents multiple isoforms generated by alternative promoter usage and alternative splicing. This gene also possesses non-conventional exons in the 3'- and intronic regions, and alternative use of cryptic exons contributes to further diversity of ERα mRNAs and proteins. Recently, the genomic organization of ERα genes and the splicing profiles of their transcripts were comparatively analyzed in humans, mice, and rats, and multiple ERα isoforms with distinct structures and functions were identified. These transcripts contain cryptic sequences that encode insertion-containing or truncated ERα proteins. In particular, alternative cryptic exons with in-frame stop codons yield transcripts encoding C-terminally-truncated ERα proteins. The C-terminally-truncated ERα isoforms lack part or all of the ligand-binding domain but possess an isoform-specific sequence. Some of these isoforms exhibit constitutive transactivation and resistance to estrogen receptor antagonists. Although numerous studies have reported conflicting results regarding their functions, the critical determinant for their gain-of-function has been identified structurally. Here we review recent progress in ERα variant research concerning the genomic organization of ERα genes, splicing profiles of ERα transcripts, and transactivation properties of ERα isoforms.

A review on phytoestrogens: Current status and future direction.

Phytoestrogens are plant secondary metabolite that is structurally and functionally similar to mammalian estrogens, which have been shown to have various health benefits in humans. Isoflavones, coumestans, and lignans are the three major bioactive classes of phytoestrogens. It has a complicated mechanism of action involving an interaction with the nuclear estrogen receptor isoforms ERα and ERβ, with estrogen agonist and estrogen antagonist effects. Depending on their concentration and bioavailability in various plant sources, phytoestrogens can act as estrogen agonist or antagonists. Menopausal vasomotor symptoms, breast cancer, cardiovascular disease, prostate cancer, menopausal symptoms, and osteoporosis/bone health have all been studied using phytoestrogens as an additional standard hormone supplemental remedy. The botanical sources, techniques of identification, classification, side effects, clinical implications, pharmacological and therapeutic effects of their proposed mode of action, safety issues, and future directions for phytoestrogens have all been highlighted in this review.

Intrauterine administration of autologous peripheral blood mononuclear cells regulates the endometrium estrogen and progesterone receptor expression: An RCT.

Repeated implantation failure (RIF) affects 15% of women of reproductive age. There is a high endometrial expression of both estrogen receptors and progesterone receptors (PRs) during the window of implantation in women with RIF. To evaluate the effects of intrauterine administration of human peripheral blood mononuclear cells (PBMC) on estrogen receptor α (ERα) and PRs expression in the endometrium of women with RIF during the implantation window. This randomized clinical trial study was conducted on 22 women with RIF history from January 2018 to August 2019 in Erfan hospital, Tehran, Iran. Participantswere divided into 2 groups (PBMC-treated group [n = 11] and control group [n = 11]). Endometrial tissue samples were collected at the implantation window time, during the mid-secretory phase (luteinizing hormone surge +7 days) of each menstrual cycle. The quantitative real-time polymerase chain reaction technique was used to measure the mRNA levels of ERα and PRs isoforms (PR-A and PR-B) in endometrial tissues. Furthermore, the protein expression of ERα and PRs was investigated using immunohistochemical staining. PBMC treatment significantly decreased the mRNA expression of endometrial ERα and PRs isoforms at the time of the implantation window (p 0.001). Moreover, the endometrial ERα and PRs protein localization were significantly lower in PBMC-treated women compared with controls (p = 0.01, and p 0.001 respectively). The intrauterine administration of PBMC decreased the endometrial ERα and PRs expression during the window of implantation in women with RIF. This local response to PBMC therapy could promote endometrial receptivity and embryo implantation.

Hypomorphism of a Novel Long ERα Isoform Causes Severe Reproductive Dysfunctions in Female Mice.

Estrogen receptor alpha (ERα)-mediated estrogen signaling plays a pivotal role in both reproductive and nonreproductive functions. Transcriptional regulation of the ERα gene is highly complex, with multiple transcript variants being differentially produced across the tissues. However, tissue-specific variation and physiological specificity of the ERα variants are not yet fully understood. In an attempt to generate a Cre-dependently restorable ERα-null mouse for functional genetic studies, we unexpectedly produced ERα hypomorphic mice with biased downregulation of a previously unappreciated long ERα isoform that is enriched in the female reproductive organs (uterus and ovaries) and the pituitary but minimally expressed in the brain. Female homozygous mutant mice were capable of pregnancy but displayed irregular estrus cycle and rarely kept newborn pups alive. No significant morphological and pathological changes in reproductive system or disruption of body weight homeostasis were seen in female homozygous mutant mice. Collectively, our results define a tissue-specific enriched long ERα isoform and its preferential role in female reproductive function rather than body weight homeostasis.

A review on phytoestrogens: Current status and future direction

Phytoestrogens are plant-based secondary metabolite that is structurally and functionally similar to mammalian estrogens that have been shown to have various health benefits for humans. Isoflavones, coumestans, and lignans are the three major bioactive classes of phytoestrogens. It has a complicated mechanism of action that involves interaction with the nuclear estrogen receptor isoforms ERα and ERβ, with estrogen-agonist and estrogen-antagonist actions. Phytoestrogens can act as estrogen agonists or antagonists depending on their concentration and bioavailability in various plant sources. Menopausal vasomotor symptoms, breast cancer, cardiovascular disease, prostate cancer, menopausal symptoms, osteoporosis/bone health have all been studied using phytoestrogens as an additional standard hormone additional remedy. The botanical sources, techniques of identification, classification, side effects, clinical impacts, pharmacological and therapeutic effects of their suggested modes of action, safety aspects, and future directions for phytoestrogens were all highlighted in this review study.

ERα-LBD, an isoform of estrogen receptor alpha, promotes breast cancer proliferation and endocrine resistance

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of an ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple-negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(−) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.

The Isoforms of Estrogen Receptor Alpha and Beta in Thyroid Cancer

The incidence of thyroid cancer was predominant in women, indicating that the sex hormone may have a role in thyroid cancer development. Generally, the sex hormone exerts its function by binding to the correspondent nuclear receptors. Therefore, aberrant of these receptors may be involved in the development of thyroid cancer. Estrogen receptor alpha (ERα) and beta (ERβ), two main estrogen receptors, have been reported to have an important role in the pathogenesis of thyroid cancer. When the ERα and ERβ genes undergo the alternative RNA splicing, some ERα and ERβ isoforms with incomplete functional domains may be formed. To date, several isoforms of ERα and ERβ have been identified. However, their expression and roles in thyroid cancer are far from clear. In this review, we summarized the expressions and roles of ERα and ERβ isoforms in thyroid cancer, aiming to provide the perspective of modulating the alternative RNA splicing of ERα and ERβ against thyroid cancer.

Abstract 2693: The vitamin D3 metabolite, 24R,25(OH)3D3 is differentially tumorigenic in ER+ and ER- laryngeal cancer cells in vitro

Abstract Due to the high vitamin D3 levels associated with improved prognosis (but not incidence) in post-menopausal breast cancer patients with ER+ tumors, vitamin D3 supplementation has been used as potential therapeutics. 24R,25(OH)2D3, an active metabolite of vitamin D3, is known to protect cartilage cells through a membrane-initiated, p53-mediated pathway, suggesting that 24R,25(OH)2D3 may affect cancer cells through a similar mechanism. Our lab has shown that the effect of 24R,25(OH)2D3 on breast cancer cell growth is highly dependent on the expression levels of ERα isoforms, specifically ERα66. Understanding the mechanisms by which 24R,25(OH)2D3 affects tumor growth in breast cancer will aid in determining its therapeutic potential in another estrogen responsive cancer, laryngeal cancer. The goal of this study was to examine the effects of 24R,25(OH)2D3 on proliferation, apoptosis, and epithelial to mesenchymal transition (EMT) in laryngeal cancer cell lines. To assess the effect of 24R,25(OH)2D3 on cell growth, UM-SCC-12 (ERα66+) and UM-SCC-11A (ERα66-) cell lines were cultured on 96 well plates until 80% confluence. Cells were serum starved for 24 hours pretreatment and then treated with full media containing either vehicle, or 24R,25(OH)2D3 (10-9M, 10-8M, or 10-7M) for 15 minutes, at which time the media were replaced with fresh media. After 20 hours, cells were harvested for proliferation by EDU assay. For other experiments cells were treated with either vehicle, or 24R,25(OH)2D3 (10-9M, 10-8M, or 10-7M) for 15 minutes and after 24 hours, cells were assessed for apoptosis (TUNEL, P53) and metastasis (OPG/RANKL). 12 hours after treatment, cells were harvested for PCR and examined for gene expression of apoptotic markers (BAX, BCL2) and various metastatic markers (SNAI1, ERBB2). 24R,25(OH)2D3 decreased proliferation in ERα66- cells (UM-SCC-11A) with the 10-7M dosage. It also increased apoptotic markers (TUNEL, P53, BAX/BCL2 expression). EMT and metastatic markers (ERBB2, SNAI1 expression) as well as bone metastatic markers (OPG/RANKL) were reduced. Conversely, in ERα66+ cells (UM-SCC-12) 24R,25(OH)2D3 had the opposite effect, increasing proliferation with 10-7M 24R,25(OH)2D3 treatment as well as decreasing DNA fragmentation (TUNEL). Treatment with 24R,25(OH)2D3 increased total P53 content and had no effect on EMT, BAX/BCL2 or other metastatic markers (SNAI1, ERBB2, OPG, RANKL). These data show that 24R,25(OH)2D3 is functionally active in laryngeal cancer tumor regulation. Contrary to results shown in breast cancer in which 24R,25(OH)2D3 was pro-tumorigenic in ERα66- cells, 24R,25(OH)2D3 was anti-tumorigenic in ERα66- laryngeal cancer cells. While this difference may be a result of a difference in other aspects of the two ERα66- cell lines, these results provide evidence of the therapeutic potential of 24R,25(OH)2D3 in laryngeal cancer and its ERα66 dependent effect. Citation Format: Cydney D. Dennis, David Joshua Cohen, Barbara D. Boyan, Zvi Schwartz. The vitamin D3 metabolite, 24R,25(OH)3D3 is differentially tumorigenic in ER+ and ER- laryngeal cancer cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2693.

Oestrogen Receptor Isoforms May Represent a Therapeutic Target in Oesophageal Adenocarcinoma.

Simple SummaryOesophageal adenocarcinoma is a lethal malignancy with limited treatment options. Recent studies have identified oestrogen receptors (ERs) in this cancer, which could represent a new target for therapy. In this study, we used laboratory models of oesophageal adenocarcinoma to look for the presence of variant forms of ERs. We also assessed the response to treatment with a drug that acts through these ERs. We found that variant forms of ERs do exist in this malignancy and that some of the variants appear to be important in order for the cells to respond to treatment. This could be due to interactions between different ERs, or between ERs and other molecules that are known to be important in cancer growth. Our findings are encouraging in that drugs that act through ERs might be useful for patients with oesophageal adenocarcinoma in the future.Oesophageal adenocarcinoma is a rapidly increasing problem in which treatment options are limited. Previous studies have shown that oesophageal adenocarcinoma cells and tissues express oestrogen receptors (ERs) and show growth suppression and apoptosis in response to ER modulator agents such as tamoxifen. ERs are known to be expressed in a number of isoforms that act together to regulate cell growth and cell death. In this study, we used western blotting to profile the expression of ERα and ERβ isoforms, and expression of the oncologically related molecules p53, HER2, and EGFR, in a panel of oesophageal adenocarcinoma cell lines. The cytotoxicity of tamoxifen in the cell lines was determined with Annexin V-FITC flow cytometry, and correlations between cytotoxicity and receptor expression were assessed using Spearman’s rank-order correlation. Oesophageal adenocarcinoma cell lines showed varying cytotoxicity in response to tamoxifen. The ER species ERα90, ERα50, and ERα46, as well as p53, were positively associated with a cytotoxic response. Conversely, ERα74, ERα70, and ERβ54 were associated with a lack of cytotoxic response. The ER species detected in oesophageal adenocarcinoma cells may work together to confer sensitivity to ER modulators in this disease, which could open up a new avenue for therapy in selected patients.

ERβ Isoforms Have Differential Clinical Significance in Breast Cancer Subtypes and Subgroups.

Simple SummaryERβ, an ER subtype first identified in 1996, is significantly expressed in ERα-negative breast cancer (BCa) and TNBC. Many studies investigated mostly ERβ1 protein expression in the entire cohort of BCa, and the results are inconsistent. In this study, we simultaneously investigated both ERβ mRNA and three ERβ 1, 2, and 5 protein isoforms in various subtypes and subgroups of BCa. Each ERβ isoform’s mRNA and protein expression seemingly plays a significant role in BCa subtypes and subgroups, and ERβ2 mRNA expression is risk factor for poor outcome. Studies in a large cohort of BCa are needed to explore the potential usefulness of ERβ as a prognostic and predictive marker and a therapeutic target in BCa. Furthermore, the standardization of a ERβ testing protocol may be required for ERβ testing to be utilized in a clinical setting.ERβ, an ER subtype first identified in 1996, is highly expressed in different types of BCa including ERα-negative BCa and TNBC. Many studies on ERβ expression investigated mostly on ERβ1 protein expression in ERα-positive and ERα-negative BCa combined. The results are conflicting. This may be due to the complexity of ERβ isoforms, subject heterogeneity, and various study designs targeting different ERβ isoforms and either ERβ protein or mRNA expression, as well as to the lack of a standardized testing protocol. Herein, we simultaneously investigated both mRNA and protein expression of ERβ isoforms 1, 2, and 5 in different BCa subtypes and clinical characteristics. Patient samples (138) and breast cancer cell lines (BCC) reflecting different types of BCa were tested for ERα and ERβ mRNA expression using quantitative real-time PCR, as well as for protein expression of ERα, ERβ1, ERβ2, and ERβ5 isoforms, PR, HER2/neu, Ki-67, CK 5/6, and p53 using immunohistochemistry. Associations of ERβ isoform expression with clinical characteristics and overall survival (OS) were analyzed. ERβ1, 2, and 5 isoforms are differentially expressed in different BCa subtypes including ERα-negative and TNBC. Each ERβ isoform seemingly plays a distinct role and is associated with clinical tumor characteristics and patient outcomes. ERβ isoform expression is significantly associated with >15% Ki-67 positivity and poor prognostic markers, and it predicts poorer OS, mostly in the subgroups. High ERβ2 and 5 isoform expression in ERα-negative BCa and TNBC is predictive of poor OS. Further investigation of ERβ isoforms in a larger cohort of BCa subgroups is needed to evaluate the role of ERβ for the potential usefulness of ERβ as a prognostic and predictive marker and for therapeutic use. The inconsistent outcomes of ERβ isoform mRNA or protein expression in many studies suggest that the standardization of ERβ testing would facilitate the use of ERβ in a clinical setting.

Impact of pubertal onset on region-specific Esr2 expression.

In female rats, pubertal onset is associated with maturation of the medial prefrontal cortex (mPFC) and mPFC-mediated behaviours. These behavioural and anatomical changes are likely a result of the effects of oestrogens at the nuclear oestrogen receptor (ER)β, which is expressed at higher levels than the ERα isoform in the adult mPFC. Researchers have previously quantified ERβ protein and Esr2 RNA in rodents during early postnatal development and adulthood, although an adolescent-specific trajectory of this receptor in the mPFC has not been documented. Given that Esr2 expression can fluctuate in the presence or absence of oestrogens, puberty and the subsequent rise in gonadal hormones could influence levels of ERβ in the adolescent brain. To further explore this, we used RNAscope® technology to quantify the amount of Esr2 mRNA in pre-pubertal adolescent, recently post-pubertal adolescent and adult female rats. We show that Esr2 expression decreases significantly in the mPFC, striatum and motor cortex between pre-pubertal adolescence and adulthood. In the mPFC, this decrease occurs rapidly at pubertal onset, with no significant decrease in Esr2 levels between the recently post-pubertal and adult cohort. By contrast, the striatum and motor cortex had no significant differences in the amount of Esr2 mRNA between pre- and post-pubertal females. Insofar as the amount of Esr2 expression is proportional to functional ERβ, these results suggest ERβ decreases in a region-specific pattern in response to pubertal onset and highlight a role for this receptor in the maturational events that occur in the female rat mPFC at puberty.

ERα36-GPER1 Collaboration Inhibits TLR4/NFκB-Induced Pro-Inflammatory Activity in Breast Cancer Cells.

Inflammation is important for the initiation and progression of breast cancer. We have previously reported that in monocytes, estrogen regulates TLR4/NFκB-mediated inflammation via the interaction of the Erα isoform ERα36 with GPER1. We therefore investigated whether a similar mechanism is present in breast cancer epithelial cells, and the effect of ERα36 expression on the classic 66 kD ERα isoform (ERα66) functions. We report that estrogen inhibits LPS-induced NFκB activity and the expression of downstream molecules TNFα and IL-6. In the absence of ERα66, ERα36 and GPER1 are both indispensable for this effect. In the presence of ERα66, ERα36 or GPER1 knock-down partially inhibits NFκB-mediated inflammation. In both cases, ERα36 overexpression enhances the inhibitory effect of estrogen on inflammation. We also verify that ERα36 and GPER1 physically interact, especially after LPS treatment, and that GPER1 interacts directly with NFκB. When both ERα66 and ERα36 are expressed, the latter acts as an inhibitor of ERα66 via its binding to estrogen response elements. We also report that the activation of ERα36 leads to the inhibition of breast cancer cell proliferation. Our data support that ERα36 is an inhibitory estrogen receptor that, in collaboration with GPER1, inhibits NFκB-mediated inflammation and ERα66 actions in breast cancer cells.

The Relative Expression of ERα Isoforms ERα66 and ERα36 Controls the Cellular Response to 24R,25-Dihydroxyvitamin D3 in Breast Cancer.

Vitamin D3 and its metabolites have antitumorigenic properties in vitro and in vivo; however, clinical trials and retrospective studies on the effectiveness of vitamin D3 oral supplementation against cancer have been inconclusive. One reason for this may be that clinical trials ignore the complex vitamin D metabolome and the many active vitamin D3 metabolites present in the body. Recent work by our lab showed that 24R,25(OH)2D3, a vitamin D3 metabolite that is active in chondrocyte proliferation and differentiation, has antitumorigenic properties in estrogen receptor alpha-66 (ERα66)-positive (ER+) breast cancer, but not in ERα66-negative (ER-) breast cancer. Here we show that 24R,25(OH)2D3 is protumorigenic in an in vivo mouse model (NOD.Cg-PrkdcscidIl2rgtm1Wjl /SzJ (NSG) mice) of ER- breast cancer, causing greater tumor growth than in mice treated with vehicle alone. In vitro results indicate that the effect of 24R,25(OH)2D3 is via a membrane-associated mechanism involving ERs and phospholipase D. 24R,25(OH)2D3 increased proliferation and reduced apoptosis in ERα66-negative HCC38 breast cancer cells, and stimulated expression of metastatic markers. Overexpressing ESRI, which encodes ERα66, ERα46, and ERα36, reduced the proapoptotic response of ERα66- cells to 24R,25(OH)2D3, possibly by upregulating ERα66. Silencing ESR1 in ERα66+ cells increased apoptosis. This suggests 24R,25(OH)2D3 is differentially tumorigenic in cancers with different ERα isoform profiles. Antiapoptotic actions of 24R,25(OH)2D3 require ERα36 and proapoptotic actions require ERα66. IMPLICATIONS: These results suggest that 24R,25(OH)2D3, which is a major circulating metabolite of vitamin D, is functionally active in breast cancer and that the regulatory properties of 24R,25(OH)2D3 are dependent upon the relative expression of ERα66 and ERα36.

Estrogen Receptors in Nonfunctioning Pituitary Neuroendocrine Tumors: Review on Expression and Gonadotroph Functions.

Estrogen (17β-estradiol or E2) is a crucial regulator of the synthesis and secretion of pituitary reproductive hormones luteinizing hormone, follicle-stimulating hormone, and prolactin. In this review, we summarize the role of estrogen receptors in nonfunctioning pituitary neuroendocrine tumors (NF-Pitnets), focusing on immunoexpression and gonadotroph cell proliferation and apoptosis. Gonadotroph tumors are the most common subtype of NF-Pitnets. Two major estrogen receptor (ER) isoforms expressed in the pituitary are estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Overall, estrogen actions are mostly exerted through the ERα isoform on the pituitary. The G protein–coupled estrogen receptor (GPER) located at the plasma membrane may contribute to nongenomic effects of estrogen. Nuclear immunoreactivity for ERα and ERβ was highest among gonadotroph and null cell tumors. Silent corticotroph tumors are the least immunoreactive for both receptors. A significantly elevated ERα expression was observed in macroadenomas compared with microadenomas. ERα and ERβ may act in opposite directions to regulate the Slug-E-cadherin pathway and to affect invasiveness of NF-Pitnets. In the cellular pathway, ERs regulate estrogen-induced proliferation and differentiation and impact several signaling pathways including the MAPK and PI3K/Akt pathway. Estrogen was the first-discovered inducer of pituitary tumor transforming gene 1 that was abundantly expressed in NF-Pitnets. ERα can be a potential biomarker for predicting tumor size and invasiveness as well as therapeutic target for NF-Pitnets. Selective estrogen receptor modulators or antiestrogen may represent as an alternative choice for the treatment of NF-Pitnets.

A Role for Estrogen Receptor alpha36 in Cancer Progression.

Estrogen receptor α (ERα) functions as a ligand dependent transcription factor that directly binds specific estrogen responsive elements, thus regulating the transcription of estrogen sensitive genes. ERα has also been shown to be associated with the plasma membrane (membrane associated ERα, mERα), concentrated in lipid rafts, plasma membrane microdomains with a distinct lipid composition, where it transduces membrane-initiated estrogen-dependent activation of the mitogen-activated protein (MAP) kinase signaling pathway. Two isoforms of ERα have been described: the “traditional” ERα66 (66 kDa) and a lower molecular weight variant: the ERα46 (46 kDa). More recently, a novel ERα variant with a molecular mass of 36 kDa (ERα36) has been discovered. Notably, ERα36 has been found expressed in different human tumor cells, including both ER- positive and ER- negative breast cancer cells. Estrogen signaling at the cell membrane via ERα36 appears as capable of activating multiple pathways of importance for cancer aggressiveness and metastatic potential. The presence of serum autoantibodies reacting with mERα (anti-ERα Abs) in a large percentage of patients with breast cancer has recently been reported by our group. These anti-ERα Abs seem to act as estrogen agonists rapidly triggering MAP kinase pathway activation thus inducing tumor cell proliferation and overcoming cell resistance to anti-estrogen drug tamoxifen. In this review, we describe the involvement of ERα36 in different tumors. We also report the potential pathogenetic activity of anti-ERα Abs and their implication in drug resistance.

Exploring the transcriptional impact of ERα in the B cell response of systemic lupus erythematosus

Abstract Systemic lupus erythematosus (SLE) is a heterogeneous, autoimmune disease. Being female is a significant, baseline risk factor for developing lupus. The mechanisms responsible for enhanced risk remain unknown. The nuclear hormone receptor and key transcription factor, estrogen receptor alpha (ERα), has a number of immune gene targets; yet, understanding of its regulation of the immune response is incomplete. Lupus prone mice, expressing a truncated functional ERα knockout, survived longer and had reduced renal disease. However, a complete knockout of ERα was not protective. Our goal is to identify the molecular mechanisms utilized by ERα in regulating the immune response in lupus B cells. We identified ERα associated genes and determined expression differences in B cells isolated from female African American (AA) lupus patients. RNA-seq analysis indicates that 60% of ERα target genes were deferentially expressed and 82% of these genes were upregulated in patients compared to controls. To understand ERα function, full-length ERα and an isoform, ERα46 (lacking the AF-1 activation domain), were transfected into an EBV transformed SLE B cell line and the expression of ERα gene targets, including inflammatory cytokines (IL1β and IL6) examined. Transfection results varied by gene and suggest the interaction between ERα isoforms is complex. The effect of epigenetic modifying agents on ERα protein expression in transfected SLE B cells was also studied. Evidence suggests that an increase in DNA methylation leads to increased protein expression of ERα isoforms. The regulatory role of ERα in lupus B cells is complex, involves epigenetic changes and interplay between isoforms. Results support a role for ERα in the pathogenesis of SLE.