title: 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 (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 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 ERα-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 (ERα-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 ERα signaling.
authors.affiliation: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, M5T3L9; §Department of Clinical Biochemistry, University Health Network, Toronto, ON, Canada, M5T3L9; andrei.drabovich@utoronto.ca.
authors.forename: Andrei P
authors.identifier: 0000-0003-3049-7145
authors.initials: AP
authors.lastname: Drabovich
authors.affiliation: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, M5T3L9; ¶Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada, M5T3L9;
authors.forename: Maria P
authors.initials: MP
authors.lastname: Pavlou
authors.affiliation: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, M5T3L9; ¶Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada, M5T3L9;
authors.forename: Christina
authors.initials: C
authors.lastname: Schiza
authors.affiliation: From the ‡Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, M5T3L9; §Department of Clinical Biochemistry, University Health Network, Toronto, ON, Canada, M5T3L9; ¶Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada, M5T3L9; ‖Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, ON, Canada, M5T3L9.
authors.forename: Eleftherios P
authors.identifier: 0000-0002-1589-820X
authors.initials: EP
authors.lastname: Diamandis
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D018929:Cell Culture Techniques; D051776:Early Growth Response Protein 3 / Q000302:isolation & purification; D004958:Estradiol / Q000494:pharmacology*; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007553:Isotope Labeling; D061986:MCF-7 Cells; D060066:Protein Interaction Maps / Q000187:drug effects; D040901:Proteomics / Q000379:methods*; D012097:Repressor Proteins / Q000302:isolation & purification; D015290:Second Messenger Systems / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D013629:Tamoxifen / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: C079945:EGR3 protein, human; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; C491000:NAB2 protein, human; D012097:Repressor Proteins; D013629:Tamoxifen; D051776:Early Growth Response Protein 3; C016601:afimoxifene; D004958:Estradiol