NDF induces expression of a novel 46 kD protein in estrogen receptor positive breast cancer cells

Most human breast tumors start as estrogen-dependent, but during the course of the disease become refractory to hormone therapy. The transition of breast tumors from estrogen dependent to independent behavior may be regulated by autocrine and/or paracrine growth factor(s) that are independent of the estrogen receptor (ER). We have investigated the role(s) of NDF (neu-differentiation factor) in the biology of estrogen positive breast cancer cells by using MCF-7 cells as a model system. Treatment of MCF-7 cells with human recombinant NDF-β2 (NDF) inhibited the ER expression by 70% and this was associated with growth stimulation in an estrogen-independent manner. To explore the mechanism(s) of action of NDF in MCF-7 cells, we examined the expression of NDF-inducible gene products. We report here that NDF stimulated the levels of expression of a 46 kD protein (p46) (in addition to few minor proteins) in ER positive breast cancer cells including MCF-7, T-47-D, and ZR-75-R cells but not in ER negative breast cancer cells including MDA-231, SK-BP-3, and MDA-468 cells. This effect of NDF was due to induction in the rate of synthesis of new p46. The observed NDF-mediated induction of p46 expression was specific as there was no such effect by epidermal growth factor or 17-β-estradiol, and inclusion of actinomycin D partially inhibited the p46 induction elicited by NDF. NDF-inducible stimulation of p46 expression was an early event (2–6 h) which preceded the period of down-regulation of ER expression by NDF. These results support the existence of NDF-responsive specific cellular pathway(s) that may regulate ER, and these interactions could play a role(s) in hormone-independence of ER positive breast cancer cells. © 1996 Wiley-Liss, Inc.     

Synthesis and evaluation of raloxifene derivatives as a selective estrogen receptor down-regulator

Estrogen receptors (ERs) play a major role in the growth of human breast cancer cells. A selective estrogen receptor down-regulator (SERD) that acts as not only an inhibitor of ligand binding, but also induces the down-regulation of ER, would be useful for the treatment for ER-positive breast cancer. We previously reported that tamoxifen derivatives, which have a long alkyl chain, had the ability to down-regulate ERα. With the aim of expanding range of the currently available SERDs, we designed and synthesized raloxifene derivatives, which had various lengths of the long alkyl chains, and evaluated their SERD activities. All compounds were able to bind ERα, and RC10, which has a decyl group on the amine moiety of raloxifene, was shown to be the most potent compound. Our findings suggest that the ligand core was replaceable, and that the alkyl length was important for controlling SERD activity. Moreover, RC10 showed antagonistic activity and its potency was superior to that of 4,4′-(heptane-4,4-diyl)bis(2-methylphenol) (18), a competitive antagonist of ER without SERD activity. These results provide information that will be useful for the development of promising SERDs candidates.     

DNA methylation in breast cancers: Differences based on estrogen receptor status and recurrence

DNA methylation plays a role in the etiology of primary breast cancers. We analyzed paired primary and second breast tumors to elucidate the role of methylation in recurrence. Methylation profiles from paired primary and second breast tumors of 23 women were assessed using the HumanMethylation450 BeadChip. Twelve women had estrogen receptor positive (ERpos) primary and second tumors, five had estrogen receptor negative (ERneg) primary and second tumors, and six had an ERpos primary tumor but an ERneg second tumor. Stratifying tumors by occurrence revealed that the greater methylation previously associated with ERpos tumors, is more pronounced in primary tumors than in second tumors. Further, ERneg second tumors are more methylated than ERpos second tumors among women who had ERpos primary tumors. Pathway analyses using gene lists generated from comparisons of methylation in ERpos primary tumors from the paired sets with ERpos tumors from six women without recurrences, identified differences between groups based on the ER status of the second tumor. Hypermethylated genes of significantly enriched pathways were differentially associated with survival. DNA methylation profiles of ERpos primary breast tumors support the development and use of tumor methylation profiles for stratifying women with breast cancer both for prognosis and therapy.     

雌激素受体信号通路在乳腺癌发生和治疗中的作用

雌激素受体信号通路在调控乳腺细胞增殖和凋亡等生理机能中发挥重要功能,该通路出现调控异常时可导致乳腺癌发生。雌激素受体在乳腺癌发生中的作用机制包括核受体介导的基因组信号通路和膜受体介导的非基因组信号通路以及二者的相互作用。基于雌激素受体信号通路及其关键信号分子的靶向治疗是开展乳腺癌治疗的重要策略与有效途径。对雌激素受体结构以及雌激素受体信号通路在乳腺癌发生和治疗中的作用作一综述。     

An estrogen receptor (ER)‐related signature in predicting prognosis of ER‐positive breast cancer following endocrine treatment

Quite a few estrogen receptor (ER)‐positive breast cancer patients receiving endocrine therapy are at risk of disease recurrence and death. ER‐related genes are involved in the progression and chemoresistance of breast cancer. In this study, we identified an ER‐related gene signature that can predict the prognosis of ER‐positive breast cancer patient receiving endocrine therapy. We collected RNA expression profiling from Gene Expression Omnibus database. An ER‐related signature was developed to separate patients into high‐risk and low‐risk groups. Patients in the low‐risk group had significantly better survival than those in the high‐risk group. ROC analysis indicated that this signature exhibited good diagnostic efficiency for the 1‐, 3‐ and 5‐year disease‐relapse events. Moreover, multivariate Cox regression analysis demonstrated that the ER‐related signature was an independent risk factor when adjusting for several clinical signatures. The prognostic value of this signature was validated in the validation sets. In addition, a nomogram was built and the calibration plots analysis indicated the good performance of this nomogram. In conclusion, combining with ER status, our results demonstrated that the ER‐related prognostic signature is a promising method for predicting the prognosis of ER‐positive breast cancer patients receiving endocrine therapy.     

Estradiol and a selective estrogen receptor modulator affect steroid hormone receptor messenger RNA levels and turnover in explant cultures of sheep endometrium

Estrogens upregulate estrogen receptor (ER) and progesterone receptor (PR) gene expression in endometrium immediately before ovulation to prepare it for nurturing embryos. Most in vitro model systems have lost the ability to upregulate expression of the ER gene in response to estradiol (E2) or the ability to express the ER gene at all. Here, we used explant cultures from control and E2-treated ewes and assessed expression of four genes (ER, PR, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and cyclophilin [CYC] genes) that are upregulated by E2 in vivo on Northern blots. In cultures from control and E2-treated ewes, ER and PR messenger ribonucleic acid (mRNA) levels dropped significantly during 24 h of culture in the absence of E2. Glyceraldehyde 3-phosphate dehydrogenase mRNA levels increased 300% in explants from control ewes to match the higher levels in the endometrium of the E2-treated ewe (in vivo and in explant culture). The only effect of E2 in the explant cultures was to prevent the decrease in PR mRNA. The new selective ER modulator, EM-800 (EM), decreased ER and PR mRNA levels in explants from control ewes but upregulated GAPDH and CYC mRNA levels. The EM treatment in vitro mimicked that of E2 by increasing the half-life of ER mRNA in endometrial explants. These data illustrate distinct, gene-specific effects of the explant culture process, E2, and EM on the expression of endometrial genes.     

The nuclear receptor coactivator PGC-1alpha exhibits modes of interaction with the estrogen receptor distinct from those of SRC-1

Estrogen receptor (ER) function is mediated by multi-domain co-regulator proteins. A fluorescently labelled fragment of the human PGC-1alpha co-regulator (residues 91-408) bearing the two motifs most strongly implicated in interactions with nuclear receptors (NR box2 and NR box3), was used to characterize in vitro binding of PGC-1alpha to ER. Anisotropy measurements revealed that the affinity of this PGC-1alpha fragment for human ERalpha and beta was fairly strong in the presence of estradiol (approximately 5 nM), and that unlike a similar fragment of SRC-1 (570-780), PGC-191-408 exhibited ligand-independent interactions with ER, particularly with ERbeta (Kd approximately 30 nM). Competition experiments of the complex between ERalpha and fluorescently labelled PGC-1 91-408 with unlabelled SRC-1 570-780 showed that PGC-1 91-408 was an efficient competitor of SRC-1 570-780, while the inverse was not true, underscoring their distinct modes of binding. The anisotropy data provide strong evidence for a ternary complex between ERalpha, SRC-1 570-780 and PGC-1 91-408. GST-pull-down experiments with deletion mutants of ERalpha revealed that the constitutive binding of PGC-1 91-408 requires the presence of the linker domain between the DNA binding and ligand binding domains (DBD and LBD). Homology modeling studies of the different regions of full length PGC-1alpha confirmed the lack of compact tertiary structure of the N-terminal region bearing the NR box motifs, and suggested a slightly different mode of interaction compared to the NR box motifs of SRC-1. They also provided reasonable structural models for the coiled-coil dimerization motif at residues 633-675, as well as the C-terminal putative RNA binding domain, raising important questions concerning the stoichiometry of its complex with the nuclear receptors.     

Estrogen receptor alpha/beta isoforms, but not betacx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells

The actions of 17beta-estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor-alpha (ERalpha) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERbeta, along with multiple estrogen receptor (ER) splice variants such as ERbetacx, an ERbeta splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERalpha, ERbeta, or ERbetacx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH-tamoxifen (4HT) treated Hs578T ERalpha and ERbeta cells revealed distinct ligand and receptor-dependent patterns of gene regulation, while the induction of ERbetacx did not alter gene expression patterns. E2 stimulation of Hs578T ERbeta cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERalpha or ERbeta cells to 4HT. Expression of ERbetacx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2-stimulated Hs578T ERbeta cells entering S-phase, along with a 17% increase in G0/G1 cell-cycle arrest. We demonstrate here that ERalpha and ERbeta regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform-specific changes in cell proliferation. Hs578T ER expressing cell-lines provide a unique BC model system, permitting the comparison of ERalpha, ERbeta, and ERbetacx actions in the same cell-line.     

二恶英对雌激素受体的干扰作用

核受体相关的肿瘤,如乳腺癌和前列腺癌的发生、发展主要依赖于性激素的分泌。环境内分泌干扰物如Ⅲ二恶英,可作为雌激素受体的诱导配体,直接或间接地与转录因子相互作用,从而干扰基因的转录。此外,二恶英还能使雌激素受体发生泛素化修饰,促进它的降解,并使雌激素依赖性的基因转录受到抑制,成为内分泌干扰因子。本文综述了二恶英的类雌激素效应、致癌性以及激素信号干扰的最新研究进展。     

雌激素受体在垂体中的作用

垂体和性腺是生殖轴重要组成部分,两者之间的协同与制衡是动物维持正常生长发育的保证。性腺产生的雌激素可以反馈调控垂体神经内分泌活动。近年来,随着基因芯片和差异表达谱分析技术的发展,垂体内雌激素受体介导的基因调控网络不断取得进展,垂体生殖生理功能备受关注。通过综述雌激素及其受体在促性腺激素、催乳素、生长激素合成分泌中的调控作用,以及雌激素对垂体生长发育的影响,探讨雌激素受体通过垂体影响生殖过程,希望能为进一步研究雌激素及其受体的生殖生理作用开拓思路。     

Differential expression of estrogen receptor alpha (ERalpha) protein in MCF-7 breast cancer cells chronically exposed to TCDD

Estrogens play a key role in the development and evolution of breast cancer tumors. Estrogen receptor alpha (ERalpha) mediates many of the biological activities of estrogens, and its expression is associated with low invasiveness and good prognosis. Recent epidemiological reports suggest that long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is implicated in the increased incidence of breast cancer in exposed women. TCDD interferes with the expression of some ERalpha-dependent genes and inhibits estradiol (E2)- dependent growth of breast cancer cells in vitro. However, E2-dependent xenographs of MCF-7 human breast cancer cells resumed growth after a 2-week exposure to TCDD. The mechanisms involved in the resumption of cell growth are not completely understood. In this study, we show that short term-exposure (16 days) to 1 nM TCDD results in the suppression of ERalpha protein expression, while chronic exposure for more than 1 year (LTDX cells) results in the partial re-expression of the receptor. Immunocytochemistry studies showed that re-expression of ERalpha in LTDX cells occurred in some of the cells. Analysis by Western immunoblots indicated that four out of five LTDX clones expressed ERalpha at levels comparable to those in unexposed MCF-7 cells. Removal of TCDD treatment for 16 days restored the expression of ERalpha in the ERalpha-negative clonal cells. These results suggest that MCF-7 cells chronically exposed to TCDD contain at least two cell subpopulations that may respond differently to the ERalpha-mediated effects of TCDD.     

Analysis of 3D models of octopus estrogen receptor with estradiol: evidence for steric clashes that prevent estrogen binding

Relatives of the vertebrate estrogen receptor (ER) are found in Aplysia californica, Octopus vulgaris, Thais clavigera, and Marisa cornuarietis. Unlike vertebrate ERs, invertebrate ERs are constitutively active and do not bind estradiol. To investigate the molecular basis of the absence of estrogen binding, we constructed a 3D model of the putative steroid-binding domain on octopus ER. Our 3D model indicates that binding of estradiol to octopus ER is prevented by steric clashes between estradiol and amino acids in the steroid-binding pocket. In this respect, octopus ER resembles vertebrate estrogen-related receptors (ERR), which have a ligand-binding pocket that cannot accommodate estradiol. Like ERR, octopus ER also may have the activation function 2 domain (AF2) in a configuration that can bind to coactivators in the absence of estrogens, which would explain constitutive activity of octopus ER.     

雌激素受体与脑肿瘤

雌激素通过其受体对脑结构与功能具有重要的调节作用,如调节脑结构的性别差异、生殖行为、神经可塑性与学习记忆等。脑肿瘤是一种危害严重的肿瘤,研究表明肿瘤细胞表达的雌激素受体的种类、水平与脑肿瘤的生长过程相关。雌激素受体可通过直接作用或与部分其它受体相互作用,调控相关基因的转录,进而调节肿瘤的生长、增殖、转移、扩散以及凋亡等生理、生化过程。本文就雌激素受体在肿瘤细胞的表达水平、表达种类以及雌激素受体参与的信号通路做一简要综述。