Abstract
Most breast cancers arise from luminal epithelial cells, and 25–30% of these tumors overexpress the ErbB2/HER2 receptor that correlates with disease progression and poor prognosis. The mechanisms of ErbB2 signaling and the effects of its overexpression are not fully understood. Herein, stable isotope labeling by amino acids in cell culture (SILAC), expression profiling, and phosphopeptide enrichment of a relevant, non-transformed, and immortalized human mammary luminal epithelial cell model were used to profile ErbB2-dependent differences in protein expression and phosphorylation events triggered via EGF receptor (EGF treatment) and ErbB3 (HRG1β treatment) in the context of ErbB2 overexpression. Bioinformatics analysis was used to infer changes in cellular processes and signaling events. We demonstrate the complexity of the responses to oncogene expression and growth factor signaling, and we identify protein changes relevant to ErbB2-dependent altered cellular phenotype, in particular cell cycle progression and hyper-proliferation, reduced adhesion, and enhanced motility. Moreover, we define a novel mechanism by which ErbB signaling suppresses basal interferon signaling that would promote the survival and proliferation of mammary luminal epithelial cells. Numerous novel sites of growth factor-regulated phosphorylation were identified that were enhanced by ErbB2 overexpression, and we putatively link these to altered cell behavior and also highlight the importance of performing parallel protein expression profiling alongside phosphoproteomic analysis.
Highlights
The expression and activity of the ErbB/HER family of receptor tyrosine kinases are frequently deregulated in human
Epithelial, or stroma-derived growth factors bind with different affinities and specificities to the ErbB receptor family, including EGF, amphiregulin, and TGF␣ (EGFR-specific); betacellulin and epiregulin [4]; and the neuregulin/heregulin (HRG) family [5]
We have identified proteomic changes associated with ErbB2 overexpression in a relevant human mammary luminal epithelial cell (HMLEC) model and characterized in parallel the early phosphorylation events triggered by ErbB receptor-specific ligands in the context of ErbB2 overexpression
Summary
The expression and activity of the ErbB/HER family of receptor tyrosine kinases are frequently deregulated in human. Signaling through the ErbB family (EGFR, ErbB2, ErbB3, and ErbB4) is initiated by ligand-induced receptor homo- and heterodimerization with subsequent activation of intrinsic tyrosine kinase activity and receptor phosphorylation. This creates docking sites for adaptor proteins and enzymes to initiate signal transduction leading to altered gene and protein expression and modulation of cellular phenotypes [3]. ErbB2 is an orphan receptor but preferentially dimerizes with the other family members to potentiate signaling, whereas ErbB3 lacks intrinsic kinase activity and is reliant upon heterodimerization for signal transduction [5, 6].
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