Abstract
To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.
Highlights
From the ‡Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha 410008, China; §Department of General Surgery and Operative Surgery, School of Medicine, University of South China, Hengyang 421001, China; ¶Department of Cardiothoracic Surgery, the Second Xiangya Hospital, Central South University, Changsha 410011, China; ʈDepartments of Radiation Oncology, Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, Georgia 30322
Identification of Differentially Expressed Proteins during Human Bronchial Epithelial Carcinogenesis Using Isobaric tags for relative and absolute quantitation (iTRAQ) Labeling and 2D LC-MS/MS—A total of 387 nonredundant proteins were repeatedly identified by triplicate iTRAQ labeling and 2D LC-MS/MS analyses, 87.1% of which were identified with Ն2 peptide matches
The detailed information including information of peptide sequences, protein quantification date, average iTRAQ ratio, and distinct and common peptides with a group of proteins for these identified proteins is shown in supplementary Table S2, and the CID spectra of 50 proteins based on the single peptide identification are shown in supplementary Fig. S2
Summary
Lung Squamous Cell Cancer and Early Detection played in the bronchial epithelia of the patients with LSCC and/or smokers [5], and that could be used to identify key proteins associated with the ongoing carcinogenic process. Our previous studies using proteomics based on 2-DE and MS identified the differential tissue and serum proteins in LSCC leading to discovery of potential biomarkers for diagnosis or prognosis of LSCC (10 –13). A number of proteomic studies on lung cancer have been reported (6 –17), little is known about the changes of protein expressional profiles in the human bronchial epithelial carcinogenic process [18], and there are no clinically established biomarkers available for early detection of LSCC. Comparative proteomics analysis of successive stages of human bronchial epithelial carcinogenesis is the most direct and persuasive way to find biomarkers for early diagnosis of LSCC. Since 1996, LCM has emerged as a good choice for purifying target cells from tissues [19]
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