title: Quantitative secretome analysis reveals that COL6A1 is a metastasis-associated protein using stacking gel-aided purification combined with iTRAQ labeling.
abstract: In cancer metastasis, secreted proteins play an important role in promoting cancer cell migration and invasion and thus also in the increase of cancer metastasis in the extracellular microenvironment. In this study, we developed a strategy that combined a simple gel-aided protein purification with iTRAQ labeling to quantify and discover the metastasis-associated proteins in the lung cancer cell secretome. Secreted proteins associated with lung cancer metastasis were produced using CL1-0 and CL1-5 cells with different metastatic abilities. Quantitative secretomics analysis identified a total of 353 proteins, 7 of which were considered to be metastasis-associated proteins. These included TIMP1, COL6A1, uPA, and AAT, all of which were higher in CL1-5, and AL1A1, PRDX1, and NID1, which were higher in CL1-0. Six of these metastasis-associated proteins were validated with Western blot analysis. In addition, pathway analysis was performed in building the interaction network between the identified metastasis-associated proteins. Further functional analysis of COL6A1 on the metastatic abilities of CL1 cells was also carried out. An RNA interference-based knock-down of COL6A1 suppressed the metastatic ability of CL1-5 cells; in contrast, a plasmid-transfected overexpression of COL6A1 increased the metastatic ability of CL1-0 cells. This study describes a simple and high throughput sample purification method that can be used for the quantitative secretomics analysis of metastasis-associated proteins.
authors.affiliation: Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
authors.forename: Kuo-Hsun
authors.initials: KH
authors.lastname: Chiu
authors.forename: Ying-Hwa
authors.initials: YH
authors.lastname: Chang
authors.forename: Yu-Shun
authors.initials: YS
authors.lastname: Wu
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Lee
authors.forename: Pao-Chi
authors.initials: PC
authors.lastname: Liao
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement; D002853:Chromatography, Liquid / Q000379:methods; D024142:Collagen Type VI / Q000302:isolation & purification / Q000378:metabolism*; D019295:Computational Biology; D017077:Culture Media, Conditioned / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry* / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins / Q000032:analysis* / Q000302:isolation & purification; D025941:Protein Interaction Mapping; D040901:Proteomics / Q000379:methods; D034741:RNA, Small Interfering / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D013194:Staining and Labeling / Q000379:methods*
chemical_list: C553839:Col6a1 protein, human; D024142:Collagen Type VI; D017077:Culture Media, Conditioned; D009363:Neoplasm Proteins; D034741:RNA, Small Interfering