Quantitative tyrosine phosphoproteomics of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor-treated lung adenocarcinoma cells reveals potential novel biomarkers of therapeutic response

Xu Zhang, Tapan Maity, Manoj K. Kashyap, Mukesh Bansal, Abhilash Venugopalan, Sahib Singh, Shivangi Awasthi, Arivusudar Marimuthu, Harrys Kishore Charles Jacob, Natalya Belkina, Stephanie Pitts, Constance M. Cultraro, Shaojian Gao, Guldal Kirkali, Romi Biswas, Raghothama Chaerkady, Andrea Califano, Akhilesh Pandey, Udayan Guha

Research output: Contribution to journalArticle

Abstract

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.

Original languageEnglish (US)
Pages (from-to)891-910
Number of pages20
JournalMolecular and Cellular Proteomics
Volume16
Issue number5
DOIs
StatePublished - May 1 2017

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Biomarkers
Epidermal Growth Factor Receptor
Protein-Tyrosine Kinases
Tyrosine
Phosphorylation
Therapeutics
Mutation
Adenocarcinoma of lung
Cells
Isotope Labeling
Cell Line
Conserved Sequence
Missense Mutation
Cell culture
Isotopes
Labeling
Mass spectrometry
Mass Spectrometry

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Cite this

Quantitative tyrosine phosphoproteomics of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor-treated lung adenocarcinoma cells reveals potential novel biomarkers of therapeutic response. / Zhang, Xu; Maity, Tapan; Kashyap, Manoj K.; Bansal, Mukesh; Venugopalan, Abhilash; Singh, Sahib; Awasthi, Shivangi; Marimuthu, Arivusudar; Jacob, Harrys Kishore Charles; Belkina, Natalya; Pitts, Stephanie; Cultraro, Constance M.; Gao, Shaojian; Kirkali, Guldal; Biswas, Romi; Chaerkady, Raghothama; Califano, Andrea; Pandey, Akhilesh; Guha, Udayan.

In: Molecular and Cellular Proteomics, Vol. 16, No. 5, 01.05.2017, p. 891-910.

Research output: Contribution to journalArticle

Zhang, X, Maity, T, Kashyap, MK, Bansal, M, Venugopalan, A, Singh, S, Awasthi, S, Marimuthu, A, Jacob, HKC, Belkina, N, Pitts, S, Cultraro, CM, Gao, S, Kirkali, G, Biswas, R, Chaerkady, R, Califano, A, Pandey, A & Guha, U 2017, 'Quantitative tyrosine phosphoproteomics of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor-treated lung adenocarcinoma cells reveals potential novel biomarkers of therapeutic response', Molecular and Cellular Proteomics, vol. 16, no. 5, pp. 891-910. https://doi.org/10.1074/mcp.M117.067439
Zhang, Xu ; Maity, Tapan ; Kashyap, Manoj K. ; Bansal, Mukesh ; Venugopalan, Abhilash ; Singh, Sahib ; Awasthi, Shivangi ; Marimuthu, Arivusudar ; Jacob, Harrys Kishore Charles ; Belkina, Natalya ; Pitts, Stephanie ; Cultraro, Constance M. ; Gao, Shaojian ; Kirkali, Guldal ; Biswas, Romi ; Chaerkady, Raghothama ; Califano, Andrea ; Pandey, Akhilesh ; Guha, Udayan. / Quantitative tyrosine phosphoproteomics of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor-treated lung adenocarcinoma cells reveals potential novel biomarkers of therapeutic response. In: Molecular and Cellular Proteomics. 2017 ; Vol. 16, No. 5. pp. 891-910.
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T1 - Quantitative tyrosine phosphoproteomics of Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor-treated lung adenocarcinoma cells reveals potential novel biomarkers of therapeutic response

AU - Zhang, Xu

AU - Maity, Tapan

AU - Kashyap, Manoj K.

AU - Bansal, Mukesh

AU - Venugopalan, Abhilash

AU - Singh, Sahib

AU - Awasthi, Shivangi

AU - Marimuthu, Arivusudar

AU - Jacob, Harrys Kishore Charles

AU - Belkina, Natalya

AU - Pitts, Stephanie

AU - Cultraro, Constance M.

AU - Gao, Shaojian

AU - Kirkali, Guldal

AU - Biswas, Romi

AU - Chaerkady, Raghothama

AU - Califano, Andrea

AU - Pandey, Akhilesh

AU - Guha, Udayan

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