Effects of HER2 overexpression on cell signaling networks governing proliferation and migration

Abstract

Although human epidermal growth factor receptor 2 (HER2) overexpression is implicated in tumor progression for a variety of cancer types, how it dysregulates signaling networks governing cell behavioral functions is poorly understood. To address this problem, we use quantitative mass spectrometry to analyze dynamic effects of HER2 overexpression on phosphotyrosine signaling in human mammary epithelial cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). Data generated from this analysis reveal that EGF stimulation of HER2‐overexpressing cells activates multiple signaling pathways to stimulate migration, whereas HRG stimulation of these cells results in amplification of a specific subset of the migration signaling network. Self‐organizing map analysis of the phosphoproteomic data set permitted elucidation of network modules differentially regulated in HER2‐overexpressing cells in comparison with parental cells for EGF and HRG treatment. Partial least‐squares regression analysis of the same data set identified quantitative combinations of signals within the networks that strongly correlate with cell proliferation and migration measured under the same battery of conditions. Combining these modeling approaches enabled association of epidermal growth factor receptor family dimerization to activation of specific phosphorylation sites, which appear to most critically regulate proliferation and/or migration. ### Synopsis HER2, also known as Erb‐B2, belongs to the epidermal growth factor receptor (EGFR) family of highly regulated receptor tyrosine kinases (RTKs) composed of human epidermal growth factor receptors 1, 2, 3, and 4 (EGFR, HER2, HER3, and HER4). Mutation and dysregulation of EGFR family members has been correlated with cancer development, and progression and overexpression of HER2 has been found in association with a variety of tumor types. Taken in sum, the high correlation between EGFR family member dysregulation and cancer progression highlights the need for mechanistic understanding of the underlying cellular signaling networks, both for improved basic knowledge of cancer and to find new and more effective drug targets. Although HER2 appears to have no intrinsic ligand‐binding capability, it can interact reversibly with ligand‐activated EGFR or HER3 to form active heterodimers that perturb and often enhance the downstream signals that govern cell proliferation and migration. To understand cellular signaling in the context of cell regulation, it is helpful to quantify phosphorylation dynamics of regulatory sites and their consequent association with downstream cell functions. To identify important EGF‐ and heregulin (HRG)‐induced protein phosphorylation events that control cell migration and proliferation in the context of HER2 overexpression, we utilized a mass spectrometry approach to measure temporal dynamics of protein tyrosine phosphorylation sites following EGFR or HER3 stimulation in the presence and absence of HER2 overexpression ([Figure 1][1]). As a result of these analyses, 332 phosphorylated peptides from 175 proteins were identified, including a total of 20 phosphorylation sites on EGFR, HER2, and HER3, including nine tyrosine and two serine sites on EGFR, eight tyrosine phosphorylation sites on HER2, and one tyrosine phosphorylation site on HER3 ([Figure 1B–D][1]). Downstream of the receptors, quantitative data were obtained for 36 phosphorylation sites on 15 different proteins in the EGFR canonical signaling pathway. Coverage of the cell adhesion/migration pathway included quantitative information on 41 phosphorylation sites distributed along 16 proteins, including nine tyrosine phosphorylation sites on δ‐catenin. To assess the effect of increased HER2 expression levels on cell motility and the canonical EGF‐activated pathways, the phosphorylation level for sites observed in EGF‐stimulated 24H (HER2‐overexpressing) cells was divided by the phosphorylation level for the same site and stimulation time in EGF‐stimulated parental cells, producing a fold change in phosphorylation level for a given site and time. This process was also performed for HRG‐stimulated 24H cells to identify differential pathway utilization between EGFR–HER2 and HER2–HER3 heterodimer signaling. Several mechanistic insights were obtained from these comparisons. For instance, from this study, it is clear that EGF stimulation of HER2‐overexpressing cells promoted migration by the phosphorylation of proteins from multiple pathways (e.g., PI3K, MAPK, catenins, and FAK), whereas HRG stimulation of HER2‐overexpressing cells activated only a very specific subset of proteins in the canonical migration pathway, in particular, FAK, Src, paxillin, and p130Cas. In order to identify clusters of tyrosine‐phosphorylated peptides exhibiting similar temporal dynamics, as well as to globally visualize the high‐dimensional information we have obtained, we used the self‐organizing map (SOM) algorithm to interrogate the phosphoproteomics data set. Within the final SOM, four statistically significant clusters were identified. By comparing the average phosphorylation profiles within each cluster, tyrosine phosphorylation sites could be linked to activated EGFR family member homo‐ and heterodimers at the cell surface. To correlate signaling data with cellular response, we quantified both cell migration and cell proliferation in the human mammary epithelial cell parental and 24H cell lines. Partial least‐squares regression analysis of the proteomic and phenotypic data sets produced a vector of coefficients indicating the importance of each signaling metric with respect to cellular behavior. In addition, PLSR provided a reduced‐dimension map, with axes defined as linear combinations of our original signaling metrics ([Figure 6A][2]), on which...