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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

Unsupervised clustering of lung cancer cell lines based on SH2 binding.(A) Rosette data clustered by SH2 domain and cell line. Each row represents a single SH2 domain and each column represents a single cell line. Biological characteristics (EGFR mutation, K-RAS mutation, erlotinib sensitivity) are shown above in black and white. For erlotinib sensitivity, positive/sensitive: IC50 <10 nM; intermediate/moderately sensitive: 10–1000 nM; negative/insensitive: >1000 nM. (B) Far-Western data clustered by SH2 domain-specific bin and cell line. Each row represents a single MW bin (20 bins/lane) for a particular SH2 domain and each column represents a single cell line.
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pone-0013470-g002: Unsupervised clustering of lung cancer cell lines based on SH2 binding.(A) Rosette data clustered by SH2 domain and cell line. Each row represents a single SH2 domain and each column represents a single cell line. Biological characteristics (EGFR mutation, K-RAS mutation, erlotinib sensitivity) are shown above in black and white. For erlotinib sensitivity, positive/sensitive: IC50 <10 nM; intermediate/moderately sensitive: 10–1000 nM; negative/insensitive: >1000 nM. (B) Far-Western data clustered by SH2 domain-specific bin and cell line. Each row represents a single MW bin (20 bins/lane) for a particular SH2 domain and each column represents a single cell line.

Mentions: To investigate the relatedness of different cell lung cancer cell lines, quantitative SH2 binding values were subjected to unsupervised hierarchical clustering analysis (see Methods). Results are shown in heat map format in Fig. 2A and the raw image data is shown in Suppl. Fig. S1. Data with low signal/background were discarded; data for the remaining 70 probes were median-centered for clustering; red indicates higher than median binding, green lower. The processed data can be found in Suppl. Table S2. Data can also be accessed using a web-based viewer (http://proteome.moffitt.org/sh2/). In this analysis, cell lines harboring mutant EGFR cluster together in three distinct sub-clusters, while two large clusters (of four and eight cell lines) consist entirely of lines with wild-type (wt) EGFR. These results suggest that SH2 profiling can identify subsets of lung cancer cells, and that such clusters appear related to EGFR mutation status.


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)

Unsupervised clustering of lung cancer cell lines based on SH2 binding.(A) Rosette data clustered by SH2 domain and cell line. Each row represents a single SH2 domain and each column represents a single cell line. Biological characteristics (EGFR mutation, K-RAS mutation, erlotinib sensitivity) are shown above in black and white. For erlotinib sensitivity, positive/sensitive: IC50 <10 nM; intermediate/moderately sensitive: 10–1000 nM; negative/insensitive: >1000 nM. (B) Far-Western data clustered by SH2 domain-specific bin and cell line. Each row represents a single MW bin (20 bins/lane) for a particular SH2 domain and each column represents a single cell line.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2957407&req=5

pone-0013470-g002: Unsupervised clustering of lung cancer cell lines based on SH2 binding.(A) Rosette data clustered by SH2 domain and cell line. Each row represents a single SH2 domain and each column represents a single cell line. Biological characteristics (EGFR mutation, K-RAS mutation, erlotinib sensitivity) are shown above in black and white. For erlotinib sensitivity, positive/sensitive: IC50 <10 nM; intermediate/moderately sensitive: 10–1000 nM; negative/insensitive: >1000 nM. (B) Far-Western data clustered by SH2 domain-specific bin and cell line. Each row represents a single MW bin (20 bins/lane) for a particular SH2 domain and each column represents a single cell line.
Mentions: To investigate the relatedness of different cell lung cancer cell lines, quantitative SH2 binding values were subjected to unsupervised hierarchical clustering analysis (see Methods). Results are shown in heat map format in Fig. 2A and the raw image data is shown in Suppl. Fig. S1. Data with low signal/background were discarded; data for the remaining 70 probes were median-centered for clustering; red indicates higher than median binding, green lower. The processed data can be found in Suppl. Table S2. Data can also be accessed using a web-based viewer (http://proteome.moffitt.org/sh2/). In this analysis, cell lines harboring mutant EGFR cluster together in three distinct sub-clusters, while two large clusters (of four and eight cell lines) consist entirely of lines with wild-type (wt) EGFR. These results suggest that SH2 profiling can identify subsets of lung cancer cells, and that such clusters appear related to EGFR mutation status.

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