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Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling.

Machida K, Eschrich S, Li J, Bai Y, Koomen J, Mayer BJ, Haura EB - PLoS ONE (2010)

Bottom Line: Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib.SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases.The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

ABSTRACT

Background: Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/principal findings: We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/significance: This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.

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Related in: MedlinePlus

SH2 profiling of cells treated with erlotinib or dasatinib.Change in SH2 binding in presence of EGFR TKI (erlotinib) or SRC TKI (dasatinib) for four lung cancer cell lines (H292, H358, H441 and HCC827). Cells were treated for 1 h with inhibitors prior to analysis. (A) The log2 fold change in SH2 binding upon erlotinib and dasatinib treatment (relative to untreated) as determined by rosette assay is shown for all probes, ranked in order of fold change in response to erlotinib. Each cell line is represented in a separate panel. For each SH2 domain probe, values for changes upon erlotinib treatment are shown as red bars, for dasatinib as blue bars. Colored arrows indicate position of SH2 domains for RAS activators (blue), PI3Ks (green), Crk family (red), and Nck family (purple). (B) Bar graph of mean log2 fold change for indicated SH2 domain groups and cell lines treated with erlotinib (E) or dasatinib (D). (C) Dendrograms obtained from hierarchical clustering of log2 fold changes (TKI-treated vs. untreated).Results for rosette assay are shown on top and far-Western blotting below. See Suppl. Fig. S5A for heatmaps.
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pone-0013470-g007: SH2 profiling of cells treated with erlotinib or dasatinib.Change in SH2 binding in presence of EGFR TKI (erlotinib) or SRC TKI (dasatinib) for four lung cancer cell lines (H292, H358, H441 and HCC827). Cells were treated for 1 h with inhibitors prior to analysis. (A) The log2 fold change in SH2 binding upon erlotinib and dasatinib treatment (relative to untreated) as determined by rosette assay is shown for all probes, ranked in order of fold change in response to erlotinib. Each cell line is represented in a separate panel. For each SH2 domain probe, values for changes upon erlotinib treatment are shown as red bars, for dasatinib as blue bars. Colored arrows indicate position of SH2 domains for RAS activators (blue), PI3Ks (green), Crk family (red), and Nck family (purple). (B) Bar graph of mean log2 fold change for indicated SH2 domain groups and cell lines treated with erlotinib (E) or dasatinib (D). (C) Dendrograms obtained from hierarchical clustering of log2 fold changes (TKI-treated vs. untreated).Results for rosette assay are shown on top and far-Western blotting below. See Suppl. Fig. S5A for heatmaps.

Mentions: Rosette binding data (log2 fold changes in treated versus untreated groups) were visualized in waterfall plots ranking changes in SH2 binding (Fig. 7A, Suppl. Fig. S4A). In HCC827, erlotinib caused complete collapse of pTyr signaling, as large decreases in SH2 binding are observed for almost all probes. The most significant reductions in binding occurred for the Grb2, Grap2, Vav2, and Vav1 SH2 domains. Broadly similar changes were observed upon dasatinib treatment, suggesting an overlap of mechanism, yet fold changes were less for most probes, consistent with its less potent effects on EGFR phosphorylation [9]. Similar to HCC827, binding of almost all SH2 domain probes was markedly reduced in TKI-treated H358 cells, and there was even greater similarity between the responses to erlotinib and dasatinib. In H441, which is resistant to TKI, erlotinib and dasatinib evoke similar overall patterns of change as H358. However the decrease in SH2 binding was blunted in H441 compared to H358 and HCC827, and the binding of a larger number of SH2 domains increased in the presence of both inhibitors compared to untreated cells. Finally, H292 showed the least dramatic changes in SH2 domain binding, and the overall pattern of response to TKI treatment in this cell line was very different from the other three. Furthermore, the erlotinib and dasatinib profiles were more dissimilar compared to the other cell lines tested. The same samples were also analyzed by far-Western blotting using a limited set of SH2 probes (Suppl. Fig. S4B). These data showed that responses to erlotinib and dasatinib were quite similar in the H358 and H441 cells, again arguing that the two agents have similar targets in these cells.


Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling.

Machida K, Eschrich S, Li J, Bai Y, Koomen J, Mayer BJ, Haura EB - PLoS ONE (2010)

SH2 profiling of cells treated with erlotinib or dasatinib.Change in SH2 binding in presence of EGFR TKI (erlotinib) or SRC TKI (dasatinib) for four lung cancer cell lines (H292, H358, H441 and HCC827). Cells were treated for 1 h with inhibitors prior to analysis. (A) The log2 fold change in SH2 binding upon erlotinib and dasatinib treatment (relative to untreated) as determined by rosette assay is shown for all probes, ranked in order of fold change in response to erlotinib. Each cell line is represented in a separate panel. For each SH2 domain probe, values for changes upon erlotinib treatment are shown as red bars, for dasatinib as blue bars. Colored arrows indicate position of SH2 domains for RAS activators (blue), PI3Ks (green), Crk family (red), and Nck family (purple). (B) Bar graph of mean log2 fold change for indicated SH2 domain groups and cell lines treated with erlotinib (E) or dasatinib (D). (C) Dendrograms obtained from hierarchical clustering of log2 fold changes (TKI-treated vs. untreated).Results for rosette assay are shown on top and far-Western blotting below. See Suppl. Fig. S5A for heatmaps.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2957407&req=5

pone-0013470-g007: SH2 profiling of cells treated with erlotinib or dasatinib.Change in SH2 binding in presence of EGFR TKI (erlotinib) or SRC TKI (dasatinib) for four lung cancer cell lines (H292, H358, H441 and HCC827). Cells were treated for 1 h with inhibitors prior to analysis. (A) The log2 fold change in SH2 binding upon erlotinib and dasatinib treatment (relative to untreated) as determined by rosette assay is shown for all probes, ranked in order of fold change in response to erlotinib. Each cell line is represented in a separate panel. For each SH2 domain probe, values for changes upon erlotinib treatment are shown as red bars, for dasatinib as blue bars. Colored arrows indicate position of SH2 domains for RAS activators (blue), PI3Ks (green), Crk family (red), and Nck family (purple). (B) Bar graph of mean log2 fold change for indicated SH2 domain groups and cell lines treated with erlotinib (E) or dasatinib (D). (C) Dendrograms obtained from hierarchical clustering of log2 fold changes (TKI-treated vs. untreated).Results for rosette assay are shown on top and far-Western blotting below. See Suppl. Fig. S5A for heatmaps.
Mentions: Rosette binding data (log2 fold changes in treated versus untreated groups) were visualized in waterfall plots ranking changes in SH2 binding (Fig. 7A, Suppl. Fig. S4A). In HCC827, erlotinib caused complete collapse of pTyr signaling, as large decreases in SH2 binding are observed for almost all probes. The most significant reductions in binding occurred for the Grb2, Grap2, Vav2, and Vav1 SH2 domains. Broadly similar changes were observed upon dasatinib treatment, suggesting an overlap of mechanism, yet fold changes were less for most probes, consistent with its less potent effects on EGFR phosphorylation [9]. Similar to HCC827, binding of almost all SH2 domain probes was markedly reduced in TKI-treated H358 cells, and there was even greater similarity between the responses to erlotinib and dasatinib. In H441, which is resistant to TKI, erlotinib and dasatinib evoke similar overall patterns of change as H358. However the decrease in SH2 binding was blunted in H441 compared to H358 and HCC827, and the binding of a larger number of SH2 domains increased in the presence of both inhibitors compared to untreated cells. Finally, H292 showed the least dramatic changes in SH2 domain binding, and the overall pattern of response to TKI treatment in this cell line was very different from the other three. Furthermore, the erlotinib and dasatinib profiles were more dissimilar compared to the other cell lines tested. The same samples were also analyzed by far-Western blotting using a limited set of SH2 probes (Suppl. Fig. S4B). These data showed that responses to erlotinib and dasatinib were quite similar in the H358 and H441 cells, again arguing that the two agents have similar targets in these cells.

Bottom Line: Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib.SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases.The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

View Article: PubMed Central - PubMed

Affiliation: Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut, United States of America.

ABSTRACT

Background: Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/principal findings: We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/significance: This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.

Show MeSH
Related in: MedlinePlus