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.     

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.     

miR-142-3p is a tumor suppressor that inhibits estrogen receptor expression in ER-positive breast cancer

Estrogen receptors (ERs) are involved in the development of many types of malignant tumors, in particular, breast cancer. Among others, ERs affect cell growth, proliferation, and differentiation. The microRNA (miRNA) miR-142-3p has been shown to inhibit carcinogenesis by regulating various cellular processes, including cell cycle progression, cell migration, apoptosis, and invasion. It does so via targeting molecules involved in a range of signaling pathways. We surgically collected 20 ER-positive breast cancer samples, each with matched adjacent normal breast tissue, and measured the expression of miR-142-3p via quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics methods, luciferase reporter assay, qRT-PCR, and western blot analysis were used to assess whether miR-142-3p could target ESR1, which encodes the estrogen receptor, in ER-positive breast cancer cells and patient samples. We also restored miRNA expression and performed cell viability, cytotoxicity, and colony formation assays. Western blot analysis and qRT-PCR were used to study the expression of apoptosis and stemness markers. We found that miR-142-3p is downregulated in ER-positive breast cancers. Restoration of miR-142-3p expression in ER-positive breast cancer cells reduced cell viability, induced apoptosis via the intrinsic pathway and decreased both colony formation and the expression of stem cell markers. Bioinformatic analysis predicted miR-142-3p could bind to 3′-untranslated region ESR1 messenger RNA (mRNA). Consistently, we demonstrated that miR-142-3p reduced luciferase activity in ER-positive breast cancer cells, and decreased ESR1 expression in both mRNA and protein levels. The results revealed miR-142-3p and ESR1 expression correlated negatively in ER-positive breast cancer samples. The results suggest miR-142-3p acts as a tumor suppressor via multiple mechanisms. Thus, restoration of miR-142-3p expression, for example, via miRNA replacement therapy, may represent an effective strategy for the treatment of ER-positive breast cancer patients.     

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。     

CAPER,a novel regulator of human breast cancer progression

CAPER is an estrogen receptor (ER) co-activator that was recently shown to be involved in human breast cancer pathogenesis. Indeed, we reported increased expression of CAPER in human breast cancer specimens. We demonstrated that CAPER was undetectable or expressed at relatively low levels in normal breast tissue and assumed a cytoplasmic distribution. In contrast, CAPER was expressed at higher levels in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) specimens, where it assumed a predominantly nuclear distribution. However, the functional role of CAPER in human breast cancer initiation and progression remained unknown. Here, we used a lentiviral-mediated gene silencing approach to reduce the expression of CAPER in the ER-positive human breast cancer cell line MCF-7. The proliferation and tumorigenicity of MCF-7 cells stably expressing control or human CAPER shRNAs was then determined via both in vitro and in vivo experiments. Knockdown of CAPER expression significantly reduced the proliferation of MCF-7 cells in vitro. Importantly, nude mice injected with MCF-7 cells harboring CAPER shRNAs developed smaller tumors than mice injected with MCF-7 cells harboring control shRNAs. Mechanistically, tumors derived from mice injected with MCF-7 cells harboring CAPER shRNAs displayed reduced expression of the cell cycle regulators PCNA, MCM7, and cyclin D1, and the protein synthesis marker 4EBP1. In conclusion, knockdown of CAPER expression markedly reduced human breast cancer cell proliferation in both in vitro and in vivo settings. Mechanistically, knockdown of CAPER abrogated the activity of proliferative and protein synthesis pathways.     

Electromagnetic fields alter the expression of estrogen receptor cofactors in breast cancer cells

Breast cancer is the most common malignancy of women in Western societies. The increasing exposure to electromagnetic fields has been suspected to contribute to the rising incidence of breast cancer in industrialized countries. The majority of breast tumors is treated with the partial antiestrogen tamoxifen. Most tumors become resistant to tamoxifen in the course of treatment resulting in treatment failure. Electromagnetic fields reduce the efficacy of tamoxifen similar to tamoxifen resistance. In this study we investigated the mechanism by which electromagnetic fields influence the sensitivity to tamoxifen. In cells exposed to 1.2 microT of a 50 Hz electromagnetic field gene expression of cofactors of the estrogen receptors was compared to sham exposed cells. Using a gene array technology several cofactors were found to be differentially expressed. The expression of the coactivators, SRC-1 and AIB1, and of two corepressors, N-Cor and SMRT, was quantified by RT-PCR. Both coactivators were expressed more strongly in the exposed cells while the expression of two corepressors decreased. The RNA analysis was confirmed by Western blots. The contradirectional changes in gene expression of coactivators and corepressors by electromagnetic fields results in a lower sensitivity to tamoxifen. Electromagnetic fields may contribute to the induction of tamoxifen resistance in vivo.     

Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka’s nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E₂/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E₂/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E₂ signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E₂. Because E₂ stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E₂ to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.     

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.     

Proteomics profiling identify CAPS as a potential predictive marker of tamoxifen resistance in estrogen receptor positive breast cancer

Background

Despite the success of tamoxifen since its introduction, about one-third of patients with estrogen (ER) and/or progesterone receptor (PgR) - positive breast cancer (BC) do not benefit from therapy. Here, we aim to identify molecular mechanisms and protein biomarkers involved in tamoxifen resistance.

Results

Using iTRAQ and Immobilized pH gradient-isoelectric focusing (IPG-IEF) mass spectrometry based proteomics we compared tumors from 12 patients with early relapses (<2 years) and 12 responsive to therapy (relapse-free > 7 years). A panel of 13 proteins (TCEAL4, AZGP1, S100A10, ALDH6A1, AHNAK, FBP1, S100A4, HSP90AB1, PDXK, GFPT1, RAB21, MX1, CAPS) from the 3101 identified proteins, potentially separate relapse from non-relapse BC patients. The proteins in the panel are involved in processes such as calcium (Ca²⁺) signaling, metabolism, epithelial mesenchymal transition (EMT), metastasis and invasion. Validation of the highest expressed proteins in the relapse group identify high tumor levels of CAPS as predictive of tamoxifen response in a patient cohort receiving tamoxifen as only adjuvant therapy.

Conclusions

This data implicate CAPS in tamoxifen resistance and as a potential predictive marker.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9080-y) contains supplementary material, which is available to authorized users.     

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.     

Epigenetic regulation of estrogen signaling in breast cancer

Estrogen signaling is mediated by ERα and ERβ in hormone dependent, breast cancer (BC). Over the last decade the implication of epigenetic pathways in BC tumorigenesis has emerged: cancer-related epigenetic modifications are implicated in both gene expression regulation, and chromosomal instability. In this review, the epigenetic-mediated estrogen signaling, controlling both ER level and ER-targeted gene expression in BC, are discussed: (1) ER silencing is frequently observed in BC and is often associated with epigenetic regulations while chemical epigenetic modulators restore ER expression and increase response to treatment;(2) ER-targeted gene expression is tightly regulated by co-recruitment of ER and both coactivators/corepressors including HATs, HDACs, HMTs, Dnmts and Polycomb proteins.     

Paradoxial changes in the expression of estrogen receptor alpha in breast cancer multicellular spheroids

Multicellular spheroids are excellent models for the analysis of cancer behavior. Just like small avascular tumors, they present a marked zonal heterogeneity which influences gene expression and thus, growth and response to chemotherapy. In the present paper, we sought to analyze the effects of three-dimensional culture in the expression and distribution of estrogen receptor alpha. Using MCF-7 breast cancer cells, we found that multicellular spheroids in estrogen-containing medium presented a paradoxical regulation of estrogen receptor alpha, with a decrease in protein expression and a marked increase in mRNA steady-state levels. Immunohistochemistry showed that only sparse cells in the periphery of the spheroid expressed estrogen receptor, in sharp contrast with progresterone receptor, which was more extensively expressed and HIF-alpha, which was expressed in the central core of the spheroid. This could mean that both hypoxia and ERA activation by estrogen participate in the expression heterogeneity of this hormone receptor in breast cancer These results are important to considerate in the analysis and interpretation of immunohistochemistry of ERA and downstream targets in samples of solid tumors.     

Nuclear estrogen receptor targeted photodynamic therapy: selective uptake and killing of MCF-7 breast cancer cells by a C17alpha-alkynylestradiol-porphyrin conjugate

We hypothesized that over-expression of estrogen receptor (ER) in hormone-sensitive breast cancer could be harnessed synergistically with the tumor-migrating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17-alpha-alkynylestradiol and chlorin e6-dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ER alpha. In a cellular uptake assay with ER-positive MCF-7 and ER-negative MDA-MB 231 human breast cancer cell-lines, we observed that one such conjugate (E17-POR, XIV) was selectively taken up in a dose-dependent and saturable manner by MCF-7 cells, but not by MDA-MB 231 cells. Furthermore, MCF-7 cells, but not MDA-MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17-POR. Therefore, the combination approach, including drug and process modalities has the potential to be applied clinically for hormone-sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo-induced selective destruction of tumor cells and/or adjuvant therapy in post-surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor.     

Estrogen activates raf-1 kinase and induces expression of Egr-1 in MCF-7 breast cancer cells

We have demonstrated that in normal and b/b rat red blood cells (RBCs) hsp70-like protein (heat shock protein 70-like) is localized in the cytosol and it is exported via exosomes during in vivo reticulocytes maturation. As we have presumed, in the mutant (b/b) rat, hsp70-like protein transfers from cytosol to the RBC membrane. In the normal rat RBCs this happens when those cells are submitted to heat stress conditions. Our study indicates that the presence of hsp70-like protein in the b/b rat RBC plasma membrane is consistent with a primary defect and is not a consequence of life long stress, i.e. hypoxia.     

17beta-estradiol (E2) induces cdc25A gene expression in breast cancer cells by genomic and non-genomic pathways

Cdc25A is a potent tyrosine phosphatase that catalyzes specific dephosphorylation of cyclin/cyclin-dependent kinase (cdk) complexes to regulate G1 to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17beta-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs that bind NF-Y, and E2F sites that bind DP/E2F1 proteins. Studies with inhibitors and dominant negative expression plasmids show that E2 activates cdc25A expression through activation of genomic ERalpha/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of cdc25A in breast cancer cells.     

A novel gene STYK1/NOK is upregulated in estrogen receptor-alpha negative estrogen receptor-beta positive breast cancer cells following estrogen treatment

The human STYK1/NOK protein is approximately 30–35% similar to mouse fibroblast growth factor receptor 3 and a kinase homologue in D. melanogaster in the tyrosine protein kinase region. STYK1/NOK was identified as being up regulated in MDA-MB-231, an estrogen receptor-alpha negative breast cancer cell line, following 12 h of estrogen treatment at 1 × 10⁻⁹ M. On further investigation of STYK1/NOK in estrogen treated cell line MDA-MB-231, STYK1/NOK was up regulated at 6 h post treatment when compared to untreated cells. We also investigated the expression levels of STYK1/NOK in other breast cancer cell lines MCF-7, MDA-MB-231, BT-549, and MDA-MB-435S using QRT-PCR. In addition, the analysis of message accumulation was increased with other synthetic estrogen response modifiers. We propose that the regulation of STYK1/NOK is achieved independent of ERα and suggests further investigation to the relevance of this kinase in breast cancer progression.