Phosphoproteome and Transcriptome Analyses of ErbB Ligand-stimulated MCF-7 Cells

Cellular signal transduction pathways and gene expression are tightly regulated to accommodate changes in response to physiological environments. In the current study, molecules were identified that are activated as a result of intracellular signaling and immediately expressed as mRNA in MCF-7 breast cancer cells shortly after stimulation of ErbB receptor ligands, epidermal growth factor (EGF) or heregulin (HRG). For the identification of tyrosine-phosphorylated proteins and expressed genes, a SILAC (stable isotopic labeling using amino acids in cell culture) method and Affymetrix gene expression array system, respectively, were used. Unexpectedly, the overlapping of genes appeared in two experimental datasets was very low for HRG (43 hits in the proteome data, 1,655 in the transcriptome data, and 5 hits common to both datasets), while no overlapping gene was detected for EGF (15 hits in the proteome data, 211 hits in the transcriptome data, and no hits common to both datasets). The HRG overlapping genes included ERBB2, NEDD9, MAPK3, JUP and EPHA2. Biological pathway analysis indicated that HRG-stimulated molecular activation is significantly related to cancer pathways including bladder cancer, chronic myeloid leukemia and pancreatic cancer (p<0.05). The proteome datasets of EGF and HRG contain molecules that are related to Axon guidance, ErbB signaling and VEGF signaling at a high rate. Cells respond to external stimuli and immediately activate intracellular signal transduction pathways to adapt to the surrounding environment (1-3). These pathways consist of protein phosphorylation cascades followed by de novo gene expression. The purpose of gene expression is to supply and replenish necessary cellular proteins that impact on cell fate in terms of proliferation, differentiation or apoptosis. Therefore, if an existing protein is phosphorylated and its corresponding mRNA immediately expressed, such a protein should play a critical role in maintaining requisite activation pathways within the cell until cellular transition is made. When this is not the case, such phosphorylated proteins should be recycled through dephosphorylation under conditions where the signaling activity persists. Additionally, new stimulus-responsive gene expression products should possess functions that enable cells to adapt to altered physiological conditions (4). In this study, our aim was to identify protein molecules that are activated and immediately expressed as mRNA in MCF-7 breast cancer cells shortly after growth hormone stimulation. Epidermal growth factor (EGF) and heregulin (HRG) were used as growth hormone stimulants; both are known to be ErbB receptor ligands. EGF and HRG induce distinct signaling durations, transient and persistent, accompanied by different cellular effects in MCF-7 cells, comprising cell proliferation and differentiation, respectively (5, 6). For the determination of activated signaling proteins, a SILAC (stable isotopic labeling using amino acids in cell culture) method was employed and proteins modified by tyrosine phosphorylation were identified (7). This method allows us to identify dozens of known and unknown proteins that become phosphorylated following ligand stimulation at specific time points. For the identification of newly synthesized genes, the Affymetrix gene expression array system was used. Based on these two kinds of high throughput data, a series of genes that appeared in the proteome, transcriptome or both datasets were obtained together with their relationship to biological functions.