High content analysis of estrogen receptor activity: a mechanism-driven approach to compound testing
ABSTRACT: Analysis of the genome-wide response of the ER:PRL-HeLa cell line to treatment with estrogen receptor ligands estradiol, 4H-tamoxifen and bisphenol-A. Overall design: Total RNA obtained from ER:PRL-HeLa cells treated for 4 hours with estradiol, 4H-tamoxifen or bisphenol -A is compared to vehicle treated controls
Project description:Analysis of the genome-wide response of the ER:PRL-HeLa cell line to treatment with estrogen receptor ligands estradiol, 4H-tamoxifen and bisphenol-A. Total RNA obtained from ER:PRL-HeLa cells treated for 4 hours with estradiol, 4H-tamoxifen or bisphenol -A is compared to vehicle treated controls
Project description:The aim of this study is to understand the role of tamoxifen in the transcription regulation of estrogen receptor positive breast cancer cells using GRO-seq experiment. ER positive MCF-7 cells was depleted with lipid hormone in stripped culture media for 4 days, and stimulated with 17-β-estradiol (100nM), 4-OH-tamoxifen (1μM), and the combination of both for 40min. Each treatment was generated in duplicates. Then nuclei was isolated and used as the starting material for GRO-seq experiments. Sequencing data was analyzed and differential gene expression analysis was performed. A rapid induction of gene expression by 17-β-estradiol was observed as previous studies reported. While also a number of genes were also up-regulated by 4-OH-tamoxifen treatment which is considered as a ER antagnist. The data strongly indicate that as a recpressor of estrogen receptor induced transcription, tamoxifen could also perform as a partial agnist at the same time. Overall design: Transcriptome profiling of hormone depleted MCF-7 cells treated with vehicle (control), 17-β-estradiol (100nM), 4-OH-tamoxifen (1μM) and combination of both for 40min. 2 replicates for each
Project description:Tamoxifen is an anti-estrogen drug used in the treatment of Estrogen Receptor (ER) positive breast cancer. Effects and side effects of tamoxifen is the sum of tamoxifen and all its metabolites. Using concentrations that mimic the clinical situation we examined effects of 4OHtam, 4OHNDtam and NDtam on global gene expression in 17beta-estradiol (E2) treated MCF-7 cells.
Project description:To obtain an integrated view of gene regulation in response to environmental and endogenous estrogens on a genome-wide scale, we performed ChIP-seq, to identify estrogen receptor 1 (ER) binding sites, and RNA-seq in endometrial cancer cells exposed to bisphenol A (BPA; found in plastics), genistein (GEN; found in soybean), or 17β-estradiol (E2; an endogenous estrogen). GEN and BPA treatment induces thousands of ER binding sites and >50 gene expression changes, representing a subset of E2‑induced gene regulation changes. Genes affected by E2 were highly enriched for ribosome-associated proteins; however, GEN and BPA failed to regulate most ribosome-associated proteins and instead enriched for transporters of carboxylic acids. Treatment-dependent changes in gene expression were associated with treatment-dependent ER binding sites, with the exception that many genes up-regulated by E2 harbored a BPA-induced ER binding site, but failed to show any expression change after BPA treatment. GEN and BPA exhibited a similar relationship to E2 in the breast cancer line T-47D, where cell type specificity played a much larger role than treatment specificity. Overall, both environmental estrogens clearly regulate gene expression through ER on a genome-wide scale, although with lower potency resulting in less ER binding sites and less gene expression changes compared to the endogenous estrogen, E2. RNA-seq of human cancer cell lines treated with estradiol, bisphenol A, genistein or DMSO (control)
Project description:Treatment with the breast cancer drug tamoxifen confers a risk of developing uterine tumors or other endometrial pathologies. Tamoxifen is a selective estrogen receptor modulator, which demonstrates tissue-specific activity although the mechanisms remain poorly understood. Both estradiol and tamoxifen act as estrogen agonists on the human uterus, and therefore have the potential to promote carcinogenicity. Estradiol and tamoxifen elicit cellular responses via the estrogen receptors (ER), which are involved in multiple signalling pathways. The effects at the molecular level are further influenced by the differential recruitment of co-factors and the presence of specific promoter motifs in target genes. In this study, ER positive (+) Ishikawa cells are used as a model to investigate the overall effect of treatment with either 17b-estradiol or 4-hydroxytamoxifen on the gene expression profiles. Ishikawa cells were serum-starved for 72 hours prior to treatment with 10-8M 17b-estradiol (E2) or 10-6M 4-hydroxytamoxifen (tam) for 24 or 48 hours. Cells were collected for total RNA extraction, and the quality and quantity of the RNA was determined spectrophotometrically. cDNA was prepared from treated (tam or E2) and control (vehicle only) Ishikawa cells, and was both forward and reverse labelled using Cy-3 dUTP/ Cy-5 dUTP and hybridised to oligo microarray slides representing >19,000 human genes (MRC HGMP-RFC). The data were analysed using GenePix Pro 3.0 software (Axon instruments) and statistical analyses applied to select significant gene changes (p<0.05).
Project description:Extracts from the rhizome of Cimicifuga racemosa (black cohosh) are increasingly popular as herbal alternative to hormone replacement therapy (HRT) for the alleviation of postmenopausal disorders. However, the molecular mode of action and the active principles are presently not clear. Previously published data have been largely contradictory. We, therefore, investigated the effects of a lipophilic Cimicifuga rhizome extract on the ER+ breast cancer MCF-7 cells at transcriptional level in comparision to 17beta-estradiol and the ER antagonist tamoxifen. With the extract 431 genes were regulated more than 1.5 fold. The overall expression pattern differed from those of 17β-estradiol or the estrogen receptor antagonist tamoxifen. We observed an enrichment of genes in an anti-proliferative and apoptosis-sensitizing manner, together with an increase of mRNAs coding for gene products involved in several stress response pathways. Regulated genes of these functional groups were highly overrepresented among all regulated genes. Various transcripts coding for oxidoreductases were induced, as for example the cytochrome P450 family members 1A1 and 1B1. In addition, some transcripts associated with antitumor but also tumor-promoting activity were regulated. Experiment Overall Design: MCF-7 cells were treated for 24 h with a lipophilic (dichloromethane) Cimicifuga rhizome extract, 17beta-estradiol, tamoxifen and the solvent control (DMSO 0,1%) in duplicate
Project description:Human estrogen-responsive breast cancer cell line MCF-7 wt were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (Ct-ER-beta and Nt-ER-beta) as previously described. MCF7 wt and beta clone cells were cultured in steroid-free medium for 5 days and then were treated with 10nM of 17-beta-estradiol, or vehicle (ETOH). RNA was extracted after 2h, 4h and 8h of stimulation with 17-ß-estradiol 10 nM (+E2) or ethanol vehicle . Total RNA extracted by Ct-ER-beta and Nt-ER-beta cells were pooled (TAP-ER-beta). For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 18 hours on Illumina HumanHT-12 v3.0 BeadChips and after scanning, data analysis was performed.
Project description:Products derived from roots of Leuzea carthamoides DC. (maral root) are being promoted as anti-aging and adaptogenic. The phytoecdysteroids are considered as active principles with numerous beneficial effects, but little is known about the pharmacological properties of Leuzea extracts. We, therefore, investigated the effects of a lipophilic Leuzea root extract on ER+ breast cancer MCF-7 cells at transcriptional level in comparison to 17beta-estradiol and the ER antagonist tamoxifen. With the extract 241 genes were regulated more than 1.5 fold. We observed gene regulation in an anti-proliferative and pro-apoptotic manner. Additionally, expression of several enzymes with oxidoreductase activity was induced including a very strong increase of the phase I enzyme CYP1A1, a possible link to the AhR pathway, which might explain other expression results, e.g. the correlated regulation of about 20 genes by Leuzea compared to 17beta-estradiol. Experiment Overall Design: MCF-7 cells were treated for 24 h with a lipophilic (dichloromethane) Leuzea root extract, 17beta-estradiol, tamoxifen and the solvent control (0,1% DMSO) in duplicate
Project description:The beneficial effect of the selective estrogen receptor (ER) modulator tamoxifen in the treatment and prevention of breast cancer is assumed to be through its ability to antagonize the stimulatory actions of estrogen, although tamoxifen can also have some estrogen-like agonist effects. Here, we report that, in addition to these mixed agonist/antagonist actions, tamoxifen can also selectively regulate a unique set of >60 genes, which are minimally regulated by estradiol (E2) or raloxifene in ERalpha-positive MCF-7 human breast cancer cells. This gene regulation by tamoxifen is mediated by ERalpha and reversed by E2 or ICI 182,780. Introduction of ERbeta into MCF-7 cells reverses tamoxifen action on approximately 75% of these genes. To examine whether these genes might serve as markers of tamoxifen sensitivity and/or the development of resistance, their expression level was examined in breast cancers of women who had received adjuvant therapy with tamoxifen. High expression of two of the tamoxifen-stimulated genes, YWHAZ/14-3-3z and LOC441453, was found to correlate significantly with disease recurrence following tamoxifen treatment in women with ER-positive cancers and hence seem to be markers of a poor prognosis. Our data indicate a new dimension in tamoxifen action, involving gene expression regulation that is tamoxifen preferential, and identify genes that might serve as markers of tumor responsiveness or resistance to tamoxifen therapy. This may have a potential effect on the choice of tamoxifen versus aromatase inhibitors as adjuvant endocrine therapy.
Project description:Progesterone and estrogen are important drivers of breast cancer proliferation. Herein, we probed estrogen receptor-α (ER) and progesterone receptor (PR) cross-talk in breast cancer models. Stable expression of PR-B in PR-low/ER+ MCF7 cells increased cellular sensitivity to estradiol and insulin-like growth factor 1 (IGF1), as measured in growth assays performed in the absence of exogenous progestin; similar results were obtained in PR-null/ER+ T47D cells stably expressing PR-B. Genome-wide microarray analyses revealed that unliganded PR-B induced robust expression of a subset of estradiol-responsive ER target genes, including cathepsin-D (CTSD). Estradiol-treated MCF7 cells stably expressing PR-B exhibited enhanced ER Ser167 phosphorylation and recruitment of ER, PR and the proline-, glutamate- and leucine-rich protein 1 (PELP1) to an estrogen response element in the CTSD distal promoter; this complex co-immunoprecipitated with IGF1 receptor (IGFR1) in whole-cell lysates. Importantly, ER/PR/PELP1 complexes were also detected in human breast cancer samples. Inhibition of IGF1R or phosphoinositide 3-kinase blocked PR-B-dependent CTSD mRNA upregulation in response to estradiol. Similarly, inhibition of IGF1R or PR significantly reduced ER recruitment to the CTSD promoter. Stable knockdown of endogenous PR or onapristone treatment of multiple unmodified breast cancer cell lines blocked estradiol-mediated CTSD induction, inhibited growth in soft agar and partially restored tamoxifen sensitivity of resistant cells. Further, combination treatment of breast cancer cells with both onapristone and IGF1R tyrosine kinase inhibitor AEW541 was more effective than either agent alone. In summary, unliganded PR-B enhanced proliferative responses to estradiol and IGF1 via scaffolding of ER-α/PELP1/IGF1R-containing complexes. Our data provide a strong rationale for targeting PR in combination with ER and IGF1R in patients with luminal breast cancer. Overall design: The study contains 4 different sample groups measured in triplicate, for a total of 12 individual samples (12 arrays). From parental MCF7 human breast cancer cell lines, we created two stable clones expressing either (1) an empty vector (pSG5) or (2) the wild type progesterone receptor isoform B (pSG5-PR-B). These two cell lines were treated with either (1) vehicle control (ethanol) or (2) estradiol 10e-7 M (E2) for 6 hours before total RNA harvest. Thus, the experiment contains two cell lines, and two treatments (4 sample groups) treated and analyzed in triplicate (12 microarrays). Standard Illumina HT-12v4 chip controls were used during hybridization.