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Differential Protein Expression Profiling by iTRAQ−2DLC−MS/MS of Lung Cancer Cells Undergoing Epithelial-Mesenchymal Transition Reveals a Migratory/Invasive Phenotype

Venkateshwar G. Keshamouni,*^† George Michailidis, Catherine S. Grasso,^§ Shalini Anthwal,^† John R. Strahler,^§ Angela Walker,^§ Douglas A. Arenberg,^† Raju C. Reddy,^† Sudhakar Akulapalli,^# Victor J. Thannickal,^† Theodore J. Standiford,^† Philip C. Andrews,^§ and Gilbert S. Omenn^‡

Divisions of Pulmonary and Critical Care Medicine and Molecular Medical Genetics, Department of Internal Medicine, Michigan Proteomics Consortium, National Resource for Proteomics and Pathways, and Department of Biological Chemistry, Department of Statistics, and Department of Human Genetics, University of Michigan, Ann Arbor, Michigan, 48109, and Cell Signaling and Angiogenesis Laboratory, Boys Town National Research Hospital, University of Nebraska Medical Center, Omaha, Nebraska 68131

J. Proteome Res., 2006, 5 (5), pp 1143–1154

DOI: 10.1021/pr050455t

Publication Date (Web): March 24, 2006

To whom correspondence should be addressed. Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical Center, 6301 MSRB III, 1150 W. Medical Center Drive, Ann Arbor, MI 48109. Phone: (734) 936-7576. Fax: (734) 764-4556. E-mail: vkeshamo@med.umich.edu.

†

Divisions of Pulmonary and Critical Care Medicine, University of Michigan.

Department of Statistics, University of Michigan.

Michigan Proteomics Consortium, National Resource for Proteomics and Pathways, and Department of Biological Chemistry, University of Michigan.

Cell Signaling and Angiogenesis Laboratory, Boys Town National Research Hospital, University of Nebraska Medical Center.

‡

Molecular Medical Genetics, University of Michigan.

Department of Human Genetics, University of Michigan.

Abstract

Transforming growth factor-β (TGF-β) induces epithelial-mesenchymal transition (EMT) of epithelial cells in both normal embryonic development and certain pathological contexts. Here, we show that TGF-β induced-EMT in human lung cancer cells (A549; adenocarcinoma cells) mediates tumor cell migration and invasion phenotypes. To gain insights into molecular events during EMT, we employed a global stable isotope labeled profiling strategy using iTRAQ reagents, followed by 2DLC−MS/MS, which identified a total of 51 differentially expressed proteins during EMT; 29 proteins were up-regulated and 22 proteins were down-regulated. Down-regulated proteins were predominantly enzymes involved in regulating nutrient or drug metabolism. The majority of the TGF-β-induced proteins (such as tropomyosins, filamin A, B, & C, integrin-β1, heat shock protein27, transglutaminase2, cofilin, 14-3-3 zeta, ezrin-radixin-moesin) are involved in the regulation of cell migration, adhesion and invasion, suggesting the acquisition of a invasive phenotype.

Keywords: iTRAQ • quantitative proteomics • TGF-β • lung cancer • epithelial-mesenchymal transitions • cell-migration • filamin • transglutaminase • HSPB1 • β1-integrin

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