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␣)-Steroid hormone receptors are ubiquitous nuclear receptors with key roles in control of reproduction, fetal development, metabolism, homeostasis, immune response and cognitive functions (1). Estrogen receptor-alpha (ER␣) 1 belongs to the family of transcription factors which control cell growth and differentiation and regulate the expression of proto-oncogenes. ER␣ action is exerted through four distinct pathways: direct ligand-dependent transcription through binding to estrogen response elements (EREs), tethered mode through its binding to other transcription factors which interact with their DNA response elements, nongenomic pathway mediated through membrane or cytoplasmic ER␣ and rapid signaling by protein kinases, and finally, estrogen-independent pathway through growth factor signaling and ER␣ phosphorylation (2). Although the nongenomic pathways are rapidly exerted in a matter of minutes, ERE-mediated genomic pathways are relatively slow, with protein expression being deployed over the course of hours. Because some ER␣ target genes include transcription factors and regulatory proteins, a secondary cascade of gene expression is triggered following the initial stimulation of ER␣.Given the fundamental importance of estrogen signaling and its involvement in breast cancer progression, numerous approaches have been undertaken to discover primary ER␣ target genes and their secondary messengers. Global profiling of estradiol-stimulated ER␣-positive cells by ChIP-sequencing and DNA microarrays revealed thousands of EREs (3-6) and hundreds of estrogen-regulated mRNA transcripts

Moonlighting Proteins and Cardiopathy in the Spatial Response of MCF‐7 Breast Cancer Cells to Tamoxifen

“…Although a restoration of the ERα complex probably linked to the half‐life of OHT was recently observed at the latest time point of 24 h by interactome dynamics studies, a longer stimulation up to 36 h was adopted by others for the investigation of late‐response proteins, in agreement with results on late‐response genes at 24 h by transcriptomic approaches …”

Preclinical evaluation of a TEX101 protein ELISA test for the differential diagnosis of male infertility

“…The total protein was measured by bicinchoninic acid (BCA) assay, and 5 and 10 μg of total protein of spermatozoa lysate and SP, respectively, were mixed with 50 mM ammonium bicarbonate. SRM assays were developed using previously described selectivity and reproducibility criteria [20–22]. Two hundred fmoles of the heavy isotope-labeled TEX101 peptide AGTETAILATK (present in both membrane/secreted isoform Q9BY14-1 and intracellular isoform Q9BY14-2) and 500 fmoles of heavy isotope-labeled peptide QIQTSSSQTSPEEAMGTPR (present exclusively in the intracellular isoform Q9BY14-2) were spiked before trypsin digestion.…”

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays

“…The development of SRM assays and the analysis of clinical samples were performed using a TSQ Quantiva TM triple quadrupole mass spectrometer (Thermo Scientific), as previously described (26). Polarity was set to positive, ion transfer tube temperature was 300°C, and the CID argon pressure was 1.5 mTorr.…”