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Modeling HER2 effects on cell behavior from mass spectrometry phosphotyrosine data.

Kumar N, Wolf-Yadlin A, White FM, Lauffenburger DA - PLoS Comput. Biol. (2006)

Bottom Line: An understanding of how phenotype is determined requires the distributed characterization of signaling processes (e.g., phosphorylation states and kinase activities) in parallel with measures of resulting cell function.Finally, model analysis identified nine especially informative phosphorylation sites on six proteins that recapitulated the predictive capability of the full model.Thus, a PLSR modeling approach reveals critical signaling processes regulating HER2-mediated cell behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
Cellular behavior in response to stimulatory cues is governed by information encoded within a complex intracellular signaling network. An understanding of how phenotype is determined requires the distributed characterization of signaling processes (e.g., phosphorylation states and kinase activities) in parallel with measures of resulting cell function. We previously applied quantitative mass spectrometry methods to characterize the dynamics of tyrosine phosphorylation in human mammary epithelial cells with varying human epidermal growth factor receptor 2 (HER2) expression levels after treatment with epidermal growth factor (EGF) or heregulin (HRG). We sought to identify potential mechanisms by which changes in tyrosine phosphorylation govern changes in cell migration or proliferation, two behaviors that we measured in the same cell system. Here, we describe the use of a computational linear mapping technique, partial least squares regression (PLSR), to detail and characterize signaling mechanisms responsible for HER2-mediated effects on migration and proliferation. PLSR model analysis via principal component inner products identified phosphotyrosine signals most strongly associated with control of migration and proliferation, as HER2 expression or ligand treatment were individually varied. Inspection of these signals revealed both previously identified and novel pathways that correlate with cell behavior. Furthermore, we isolated elements of the signaling network that differentially give rise to migration and proliferation. Finally, model analysis identified nine especially informative phosphorylation sites on six proteins that recapitulated the predictive capability of the full model. A model based on these nine sites and trained solely on data from a low HER2-expressing cell line a priori predicted migration and proliferation in a HER2-overexpressing cell line. We identify the nine signals as a "network gauge," meaning that when interrogated together and integrated according to the quantitative rules of the model, these signals capture information content in the network sufficiently to predict cell migration and proliferation under diverse ligand treatments and receptor expression levels. Examination of the network gauge in the context of previous literature indicates that endocytosis and activation of phosphoinositide 3-kinase (PI3K)-mediated pathways together represent particularly strong loci for the integration of the multiple pathways mediating HER2's control of mammary epithelial cell proliferation and migration. Thus, a PLSR modeling approach reveals critical signaling processes regulating HER2-mediated cell behavior.

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A Mass Spectrometry Approach Measured 248 Phosphorylation ProfilesThe title at the top of each entry indicates the phosphosite measured. Median normalized phosphorylation (see Methods) is plotted at 0, 5, 10, and 30 min. For each phospho-site, four conditions were measured: parental + HRG (80 ng/ml), 24H + HRG (80 ng/ml), parental + EGF (100 ng /ml), and 24H + EGF (100 ng/ml). Error bars indicate ± standard deviation. The data were obtained from [10].
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pcbi-0030004-g001: A Mass Spectrometry Approach Measured 248 Phosphorylation ProfilesThe title at the top of each entry indicates the phosphosite measured. Median normalized phosphorylation (see Methods) is plotted at 0, 5, 10, and 30 min. For each phospho-site, four conditions were measured: parental + HRG (80 ng/ml), 24H + HRG (80 ng/ml), parental + EGF (100 ng /ml), and 24H + EGF (100 ng/ml). Error bars indicate ± standard deviation. The data were obtained from [10].

Mentions: As previously described, we developed and employed a mass spectrometry approach to measure the effect of HER2 overexpression in 184A1 human mammary epithelial cells (HMEC) [10]. Two closely related cell lines with known receptor expression levels were used; a parental cell line (P) with 200,000 EGFR, 20,000 HER2, and 20,000 HER3, and a HER2-overexpressing cell line (24H) with 200,000 EGFR, 600,000 HER2, and 30,000 HER3 per cell [10]. Both cell lines have very low levels of HER4. Thus, the 24H cell line was used to assess effects of HER2 overexpression, with the parental cell line serving as a baseline for these measurements. HMECs were treated with saturating levels of EGF or HRG, and under each treatment condition the tyrosine phosphorylation of 62 phosphosites was quantified at 0, 5, 10, and 30 min. Figure 1 displays the 248 time courses collected. Our measurements revealed the dynamic activation of molecules commonly associated with ErbB signaling (e.g., extracellular regulated kinase 1 [ERK1] and SH2-containing protein [Shc]) and others less commonly associated with the ErbB network (e.g., human transferrin receptor [TfR], ephrin A2 receptor [EphA2], and the previously unidentified KIAA 1217). Comparison with previously published maps of ErbB and migration-associated signaling networks reveals broad network coverage with the 62 measured signals [7,11].


Modeling HER2 effects on cell behavior from mass spectrometry phosphotyrosine data.

Kumar N, Wolf-Yadlin A, White FM, Lauffenburger DA - PLoS Comput. Biol. (2006)

A Mass Spectrometry Approach Measured 248 Phosphorylation ProfilesThe title at the top of each entry indicates the phosphosite measured. Median normalized phosphorylation (see Methods) is plotted at 0, 5, 10, and 30 min. For each phospho-site, four conditions were measured: parental + HRG (80 ng/ml), 24H + HRG (80 ng/ml), parental + EGF (100 ng /ml), and 24H + EGF (100 ng/ml). Error bars indicate ± standard deviation. The data were obtained from [10].
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1761044&req=5

pcbi-0030004-g001: A Mass Spectrometry Approach Measured 248 Phosphorylation ProfilesThe title at the top of each entry indicates the phosphosite measured. Median normalized phosphorylation (see Methods) is plotted at 0, 5, 10, and 30 min. For each phospho-site, four conditions were measured: parental + HRG (80 ng/ml), 24H + HRG (80 ng/ml), parental + EGF (100 ng /ml), and 24H + EGF (100 ng/ml). Error bars indicate ± standard deviation. The data were obtained from [10].
Mentions: As previously described, we developed and employed a mass spectrometry approach to measure the effect of HER2 overexpression in 184A1 human mammary epithelial cells (HMEC) [10]. Two closely related cell lines with known receptor expression levels were used; a parental cell line (P) with 200,000 EGFR, 20,000 HER2, and 20,000 HER3, and a HER2-overexpressing cell line (24H) with 200,000 EGFR, 600,000 HER2, and 30,000 HER3 per cell [10]. Both cell lines have very low levels of HER4. Thus, the 24H cell line was used to assess effects of HER2 overexpression, with the parental cell line serving as a baseline for these measurements. HMECs were treated with saturating levels of EGF or HRG, and under each treatment condition the tyrosine phosphorylation of 62 phosphosites was quantified at 0, 5, 10, and 30 min. Figure 1 displays the 248 time courses collected. Our measurements revealed the dynamic activation of molecules commonly associated with ErbB signaling (e.g., extracellular regulated kinase 1 [ERK1] and SH2-containing protein [Shc]) and others less commonly associated with the ErbB network (e.g., human transferrin receptor [TfR], ephrin A2 receptor [EphA2], and the previously unidentified KIAA 1217). Comparison with previously published maps of ErbB and migration-associated signaling networks reveals broad network coverage with the 62 measured signals [7,11].

Bottom Line: An understanding of how phenotype is determined requires the distributed characterization of signaling processes (e.g., phosphorylation states and kinase activities) in parallel with measures of resulting cell function.Finally, model analysis identified nine especially informative phosphorylation sites on six proteins that recapitulated the predictive capability of the full model.Thus, a PLSR modeling approach reveals critical signaling processes regulating HER2-mediated cell behavior.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.

ABSTRACT
Cellular behavior in response to stimulatory cues is governed by information encoded within a complex intracellular signaling network. An understanding of how phenotype is determined requires the distributed characterization of signaling processes (e.g., phosphorylation states and kinase activities) in parallel with measures of resulting cell function. We previously applied quantitative mass spectrometry methods to characterize the dynamics of tyrosine phosphorylation in human mammary epithelial cells with varying human epidermal growth factor receptor 2 (HER2) expression levels after treatment with epidermal growth factor (EGF) or heregulin (HRG). We sought to identify potential mechanisms by which changes in tyrosine phosphorylation govern changes in cell migration or proliferation, two behaviors that we measured in the same cell system. Here, we describe the use of a computational linear mapping technique, partial least squares regression (PLSR), to detail and characterize signaling mechanisms responsible for HER2-mediated effects on migration and proliferation. PLSR model analysis via principal component inner products identified phosphotyrosine signals most strongly associated with control of migration and proliferation, as HER2 expression or ligand treatment were individually varied. Inspection of these signals revealed both previously identified and novel pathways that correlate with cell behavior. Furthermore, we isolated elements of the signaling network that differentially give rise to migration and proliferation. Finally, model analysis identified nine especially informative phosphorylation sites on six proteins that recapitulated the predictive capability of the full model. A model based on these nine sites and trained solely on data from a low HER2-expressing cell line a priori predicted migration and proliferation in a HER2-overexpressing cell line. We identify the nine signals as a "network gauge," meaning that when interrogated together and integrated according to the quantitative rules of the model, these signals capture information content in the network sufficiently to predict cell migration and proliferation under diverse ligand treatments and receptor expression levels. Examination of the network gauge in the context of previous literature indicates that endocytosis and activation of phosphoinositide 3-kinase (PI3K)-mediated pathways together represent particularly strong loci for the integration of the multiple pathways mediating HER2's control of mammary epithelial cell proliferation and migration. Thus, a PLSR modeling approach reveals critical signaling processes regulating HER2-mediated cell behavior.

Show MeSH
Related in: MedlinePlus