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Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells.

ABSTRACT: Estrogen receptor alpha (ER?)-mediated proliferation of breast cancer cells is facilitated through expression of multiple primary target genes, products of which induce a secondary response to stimulation. To differentiate between the primary and secondary target proteins of ER? signaling, we measured dynamics of protein expression induced by 17?-estradiol in MCF-7 breast cancer cells. Measurement of the global proteomic effects of estradiol by stable isotope labeling by amino acids in cell culture (SILAC) resulted in identification of 103 estrogen-regulated proteins, with only 40 of the corresponding genes having estrogen response elements. Selected reaction monitoring (SRM) assays were used to validate the differential expression of 19 proteins and measure the dynamics of their expression within 72 h after estradiol stimulation, and in the absence or presence of 4-hydroxytamoxifen, to confirm ER?-mediated signaling. Dynamics of protein expression unambiguously revealed early and delayed response proteins and well correlated with presence or absence of estrogen response elements in the corresponding genes. Finally, we quantified dynamics of protein expression in a rarely studied network of transcription factors with a negative feedback loop (ER?-EGR3-NAB2). Because NAB2 protein is a repressor of EGR3-induced transcription, siRNA-mediated silencing of NAB2 resulted in the enhanced expression of the EGR3-induced protein ITGA2. To conclude, we provided a high-quality proteomic resource to supplement genomic and transcriptomic studies of ER? signaling.

SUBMITTER: Drabovich AP

PROVIDER: S-EPMC5083091 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells.

Drabovich Andrei P AP Pavlou Maria P MP Schiza Christina C Diamandis Eleftherios P EP

Molecular & cellular proteomics : MCP 20160411 6

Estrogen receptor alpha (ERα)-mediated proliferation of breast cancer cells is facilitated through expression of multiple primary target genes, products of which induce a secondary response to stimulation. To differentiate between the primary and secondary target proteins of ERα signaling, we measured dynamics of protein expression induced by 17β-estradiol in MCF-7 breast cancer cells. Measurement of the global proteomic effects of estradiol by stable isotope labeling by amino acids in cell cult ...[more]

PMID: 27067054

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Project description:BackgroundThe orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) is a member of the nuclear receptor superfamily. It was identified through a search for genes encoding proteins related to estrogen receptor alpha (ERalpha). An endogenous ligand has not been found. Novel ERRalpha antagonists that are highly specific for binding to the ligand binding domain (LBD) of ERRalpha have been recently reported. Research suggests that ERRalpha may be a novel drug target to treat breast cancer and/or metabolic disorders and this has led to an effort to characterize the mechanisms of action of N-[(2Z)-3-(4,5-dihydro-1,3-thiazol-2-yl)-1,3-thiazolidin-2-yl idene]-5H dibenzo[a,d][7]annulen-5-amine, a novel ERRalpha specific antagonist.Methodology/principal findingsWe demonstrate this ERRalpha ligand inhibits ERRalpha transcriptional activity in MCF-7 cells by luciferase assay but does not affect mRNA levels measured by real-time RT-PCR. Also, ERalpha (ESR1) mRNA levels were not affected upon treatment with the ERRalpha antagonist, but other ERRalpha (ESRRA) target genes such as pS2 (TFF1), osteopontin (SPP1), and aromatase (CYP19A1) mRNA levels decreased. In vitro, the ERRalpha antagonist prevents the constitutive interaction between ERRalpha and nuclear receptor coactivators. Furthermore, we use Western blots to demonstrate ERRalpha protein degradation via the ubiquitin proteasome pathway is increased by the ERRalpha-subtype specific antagonist. We demonstrate by chromatin immunoprecipitation (ChIP) that the interaction between ACADM, ESRRA, and TFF1 endogenous gene promoters and ERRalpha protein is decreased when cells are treated with the ligand. Knocking-down ERRalpha (shRNA) led to similar genomic effects seen when MCF-7 cells were treated with our ERRalpha antagonist.Conclusions/significanceWe report the mechanism of action of a novel ERRalpha specific antagonist that inhibits transcriptional activity of ERRalpha, disrupts the constitutive interaction between ERRalpha and nuclear coactivators, and induces proteasome-dependent ERRalpha protein degradation. Additionally, we confirmed that knocking-down ERRalpha lead to similar genomic effects demonstrated in vitro when treated with the ERRalpha specific antagonist.

Project description:UBC9 encodes a protein that conjugates small ubiquitin-related modifier (SUMO) to target proteins thereby changing their functions. Recently, it was noted that UBC9 expression and activity play a role in breast tumorigenesis and response to anticancer drugs. However, the underlying mechanism is poorly understood. To investigate the transcriptional regulation of the UBC9 gene, we identified and characterized its promoter and cis-elements. Promoter activity was tested using luciferase reporter assays. The binding of transcription factors to the promoter was detected by chromatin immunoprecipitation (ChIP), and their functional role was confirmed by siRNA knockdown. UBC9 mRNA and protein levels were measured by quantitative reverse transcription PCR and Western blot analysis, respectively. An increased expression of UBC9 mRNA and protein was found in MCF-7 breast cancer cells treated with 17?-estradiol (E2). Analysis of various deletion mutants revealed a 137 bp fragment upstream of the transcription initiation site to be sufficient for reporter gene transcription. Mutations of putative estrogen receptor ? (ER-?) (one imperfect estrogen response element, ERE) and/or nuclear factor Y (NF-Y) binding sites (two CCAAT boxes) markedly reduced promoter activity. Similar results were obtained in ER-negative MDA-MB-231 cells except that the ERE mutation did not affect promoter activity. Additionally, promoter activity was stimulated upon E2 treatment and overexpression of ER-? or NF-YA in MCF-7 cells. ChIP confirmed direct binding of both transcription factors to the UBC9 promoter in vivo. Furthermore, UBC9 expression was diminished by ER-? and NF-Y siRNAs on the mRNA and protein levels. In conclusion, we identified the proximal UBC9 promoter and provided evidence that ER-? and NF-Y regulate UBC9 expression on the transcriptional level in response to E2 in MCF-7 cells. These findings may contribute to a better understanding of the regulation of UBC9 in ER-positive breast cancer and be useful for the development of cancer therapies targeting UBC9.

Project description:While breast cancer patients with tumors that express estrogen receptor α (ER) generally respond well to hormone therapies that block ER activity, a significant number of patients relapse. Approximately 30% of these recurrences harbor activating mutations in the ligand binding domain (LBD) of ER, which have been shown to confer ligand-independent function. However, much is still unclear regarding the effect of mutant ER beyond its estrogen independence. To investigate the molecular effects of mutant ER, we developed multiple isogenic ER-mutant cell lines for the most common LBD mutations, Y537S and D538G. These mutations induced differential expression of thousands of genes, the majority of which were mutant allele specific and were not observed upon estrogen treatment of wild-type (WT) cells. These mutant-specific genes showed consistent differential expression across ER-mutant lines developed in other laboratories. WT cells with long-term estrogen exposure only exhibited some of these transcriptional changes, suggesting that mutant ER causes novel regulatory effects that are not simply due to constant activity. While ER mutations exhibited minor effects on ER genomic binding, with the exception of ligand independence, ER mutations conferred substantial differences in chromatin accessibility. Mutant ER was bound to approximately a quarter of mutant-enriched accessible regions that were enriched for other DNA binding factors, including FOXA1, CTCF, and OCT1. Overall, our findings indicate that mutant ER causes several consistent effects on gene expression, both indirectly and through constant activity. SIGNIFICANCE: This study demonstrates the multiple roles of mutant ER in breast cancer progression, including constant ER activity and secondary regulatory effects on gene expression and chromatin accessibility. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/539/F1.large.jpg.See related commentary by Hermida-Prado and Jeselsohn, p. 537 See related article by Williams and colleagues, p. 732.

Dataset Information

Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells.

Publications

Dynamics of Protein Expression Reveals Primary Targets and Secondary Messengers of Estrogen Receptor Alpha Signaling in MCF-7 Breast Cancer Cells.

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