Dataset Information

Dynamics of protein expression reveals primary targets and secondary messengers of estrogen receptor signaling in MCF-7 breast cancer cells

ABSTRACT: Estrogen receptor-mediated proliferation of breast cancer cells is facilitated through expression of multiple primary target genes, products of which induce the secondary response to stimulation. To differentiate between the primary and secondary target genes of estrogen receptor signaling, we measured dynamics of protein expression induced by estradiol in MCF-7 breast cancer cells. Measurement of the global proteomic effects of estradiol by SILAC resulted in identification of 104 estradiol-regulated proteins, with only 41 corresponding genes having estrogen response elements (EREs). Selected reaction monitoring assays were used to validate 32 proteins and measure the dynamics of their expression within 72 hours upon estradiol stimulation and in the presence of 4-hydroxytamoxifen, thus confirming estrogen receptor-mediated signaling. Dynamics of protein expression revealed immediate early response proteins including TFF1 and CDK1 and delayed response proteins including CDK2 and NAB2. Presence or absence of EREs in the corresponding genes indicated early or delayed expression of proteins identified by SILAC. Finally, we measured accurate dynamics of estradiol-induced protein expression in the sub-network of primary and secondary targets of estrogen receptor. Interestingly, since NAB2 protein is also a repressor of EGR3-induced transcription, siRNA-mediated silencing of NAB2 resulted in the over-expression of some EGR3-induced proteins, such as ITGA2. To conclude, quantitative proteomics revealed secondary targets of estrogen receptor signaling potentially overlooked by genome-wide profiling of EREs and provided dynamics of protein expression in the network of transcription factors with a negative feedback loop.

INSTRUMENT(S): LTQ Orbitrap XL

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Mammary Gland Epithelial Cell

DISEASE(S): Breast Cancer

SUBMITTER: Andrei Drabovich

LAB HEAD: Andrei Drabovich

PROVIDER: PXD002797 | Pride | 2016-04-14

REPOSITORIES: Pride

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Publications

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:Using a chromatin immunoprecipitation-paired end diTag cloning and sequencing strategy, we mapped estrogen receptor alpha (ERalpha) binding sites in MCF-7 breast cancer cells. We identified 1,234 high confidence binding clusters of which 94% are projected to be bona fide ERalpha binding regions. Only 5% of the mapped estrogen receptor binding sites are located within 5 kb upstream of the transcriptional start sites of adjacent genes, regions containing the proximal promoters, whereas vast majority of the sites are mapped to intronic or distal locations (>5 kb from 5' and 3' ends of adjacent transcript), suggesting transcriptional regulatory mechanisms over significant physical distances. Of all the identified sites, 71% harbored putative full estrogen response elements (EREs), 25% bore ERE half sites, and only 4% had no recognizable ERE sequences. Genes in the vicinity of ERalpha binding sites were enriched for regulation by estradiol in MCF-7 cells, and their expression profiles in patient samples segregate ERalpha-positive from ERalpha-negative breast tumors. The expression dynamics of the genes adjacent to ERalpha binding sites suggest a direct induction of gene expression through binding to ERE-like sequences, whereas transcriptional repression by ERalpha appears to be through indirect mechanisms. Our analysis also indicates a number of candidate transcription factor binding sites adjacent to occupied EREs at frequencies much greater than by chance, including the previously reported FOXA1 sites, and demonstrate the potential involvement of one such putative adjacent factor, Sp1, in the global regulation of ERalpha target genes. Unexpectedly, we found that only 22%-24% of the bona fide human ERalpha binding sites were overlapping conserved regions in whole genome vertebrate alignments, which suggest limited conservation of functional binding sites. Taken together, this genome-scale analysis suggests complex but definable rules governing ERalpha binding and gene regulation. Experiment Overall Design: We used oligonucleotide expression microarrays (Affymetrix GeneChip U133 Plus 2.0) to identify estradiol (E2)-responsive genes in the estrogen-receptor positive breast cancer cell line, MCF7. MCF7 cells were grown to 30-50% confluency and exposed to 10 nM E2 (or vehicle only) at 12, 24, and 48 hours. Each timepoint was performed in triplicate (ie, biological replicates). Total RNA was isolated from cells using the Qiagen RNeasy kit, and 5 micrograms of total RNA was amplified, labeled and hybridized to the array according to the manufacturerâs protocols.

Project description:Estrogen receptor Î± (ERÎ±) is an important biomarker of breast cancer severity and a common therapeutic target. Recent studies have demonstrated that in addition to its role in promoting proliferation, ERÎ± also protects tumors against metastatic transformation. Current therapeutics antagonize ERÎ± and interfere with both beneficial and detrimental signaling pathways stimulated by ERÎ±. The goal of this study is to uncover the dynamics of coding and non-coding RNA (microRNA) expression in response to estrogen stimulation and identify potential therapeutic targets that more specifically inhibit ERÎ±-stimulated growth and survival pathways without interfering with its protective features. To achieve this, we exposed MCF7 cells (an estrogen receptor positive model cell line for breast cancer) to estrogen and prepared a time course of paired mRNA and miRNA sequencing libraries at ten time points throughout the first 24 hours of the response to estrogen. From these data, we identified three primary expression trendsâ??transient, induced, and repressedâ??that were each enriched for genes with distinct cellular functions. Integrative analysis of paired mRNA and microRNA temporal expression profiles identified miR-503 as the strongest candidate master regulator of the estrogen response, in part through suppression of ZNF217â??an oncogene that is frequently amplified in cancer. We confirmed experimentally that miR-503 directly targets ZNF217 and that over-expression of miR-503 suppresses breast cancer cell proliferation. Overall, these data indicate that miR-503 acts as a potent estrogen-induced tumor suppressor microRNA that opposes cellular proliferation and has promise as a therapeutic for breast cancer. More generally, our work provides a systems-level framework for identifying functional interactions that shape the temporal dynamics of gene expression. Quantification of miRNAs in MCF7 cells responding to estrogen following a period of estrogen starvation. Three independent biological replicates (30 samples: 3 replicates x 10 time points) of MCF7 cells were exposed to 10nM Estradiol for 0, 1, 2, 3, 4, 5, 6, 8, 12 , or 24 hours, and total RNA was extracted from the samples. Total RNA was used to generate paired RNA and miRNA sequencing. The miRNA libraries were prepared using the Bioo Scientific NextFLEX v2 library preparation kit.

Project description:Estrogen receptor Î± (ERÎ±) is an important biomarker of breast cancer severity and a common therapeutic target. Recent studies have demonstrated that in addition to its role in promoting proliferation, ERÎ± also protects tumors against metastatic transformation. Current therapeutics antagonize ERÎ± and interfere with both beneficial and detrimental signaling pathways stimulated by ERÎ±. The goal of this study is to uncover the dynamics of coding and non-coding RNA (microRNA) expression in response to estrogen stimulation and identify potential therapeutic targets that more specifically inhibit ERÎ±-stimulated growth and survival pathways without interfering with its protective features. To achieve this, we exposed MCF7 cells (an estrogen receptor positive model cell line for breast cancer) to estrogen and prepared a time course of paired mRNA and miRNA sequencing libraries at ten time points throughout the first 24 hours of the response to estrogen. From these data, we identified three primary expression trendsâ??transient, induced, and repressedâ??that were each enriched for genes with distinct cellular functions. Integrative analysis of paired mRNA and microRNA temporal expression profiles identified miR-503 as the strongest candidate master regulator of the estrogen response, in part through suppression of ZNF217â??an oncogene that is frequently amplified in cancer. We confirmed experimentally that miR-503 directly targets ZNF217 and that over-expression of miR-503 suppresses breast cancer cell proliferation. Overall, these data indicate that miR-503 acts as a potent estrogen-induced tumor suppressor microRNA that opposes cellular proliferation and has promise as a therapeutic for breast cancer. More generally, our work provides a systems-level framework for identifying functional interactions that shape the temporal dynamics of gene expression. Quantification of mRNAs in MCF7 cells responding to estrogen following a period of estrogen starvation. Three independent biological replicates (30 samples: 3 replicates x 10 time points) of MCF7 cells were exposed to 10nM Estradiol for 0, 1, 2, 3, 4, 5, 6, 8, 12 , or 24 hours, and total RNA was extracted from the samples. Total RNA was used to generate paired RNA and miRNA sequencing. RNA libraries were prepared using an Illumina TruSeq stranded mRNA library preparation kit.

Dataset Information

Dynamics of protein expression reveals primary targets and secondary messengers of estrogen receptor 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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