Identification of Candidate Biomarkers for Early Detection of Human Lung Squamous Cell Cancer by Quantitative Proteomics

Lung cancer is the most frequently occurring malignancy with increasing incidence and is the leading cause of mortality in cancer-related deaths in China and worldwide (1, 2). Although great improvement has been made in diagnosis and treatment of lung cancer, the overall patients' survival is still very low and does not exceed 15% (3). The poor prognosis of this cancer is mainly explained by the fact that the diagnosis is generally made only at advanced stages because of the lack of reliable, early diagnostic biomarkers and the limited understanding of its carcinogenic mechanisms. Therefore, identification of biomarkers for early detection of lung cancer is mandatory, in turn leading to more effective treatment and reduction of mortality. Lung squamous cell carcinoma (LSCC)1 originated from the bronchial epithelial cells is the most common histological type of lung cancer. It is known that carcinogenesis of LSCC is a multistage process and the result of multistep accumulation of genetic and epigenetic alterations (4). With exposure to environmental carcinogens, bronchial epithelial carcinogenesis often progresses in the following manner: hyperplasia, squamous metaplasia (SM), atypical hyperplasia (AH), cancer in situ (CIS) and invasive cancer (5). LSCC is the end-point of a whole range of morphological abnormalities that are displayed 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. Analysis of differentially expressed proteins in LSCC using proteomics revealed that expression and modified levels of proteins have some predictive power for clinical outcome and personalized risk assessment (6–9). 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). Although 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. A major obstacle, however, to the analysis of tissue specimens is tissue heterogeneity, which is particularly relevant to bronchial preneoplastic lesions as these tissues only include a little of target cells. Several approaches have been employed to obtain homogeneous cell populations from a heterogeneous tissue, such as short-term cell culture and laser capture microdissection (LCM). Since 1996, LCM has emerged as a good choice for purifying target cells from tissues (19). Isobaric tags for relative and absolute quantitation (iTRAQ) in combination with two dimensional liquid chromatography tandem MS (2D LC-MS/MS) analysis is emerging as one of the more powerful quantitative proteomics methodologies in the search for tumor biomarkers (20–23). In the iTRAQ technology, tagging is on primary amines, thus potentially allowing the tagging of most tryptic peptides. The multiplexing ability afforded by the iTRAQ reagents, which are available in four to eight different tags, is ideally suited for our study because it provides us with a means to simultaneously compare proteomes in successive stages of human bronchial epithelial carcinogenesis. To search biomarkers for early detection of LSCC and explore the possible mechanisms of bronchial epithelial carcinogenesis, in this study iTRAQ tagging followed by 2D LC-MS/MS was performed to identify differential proteins among LCM-purified bronchial epithelial carcinogenic tissues, and some differentially expressed proteins identified by proteomics were selectively validated. Furthermore, values of the three differential proteins (GSTP1, HSPB1, and CKB) with progressively expressional alterations in the bronchial epithelial carcinogenic process for early detection of LSCC were assessed by immunohistochemistry and receiver operating characteristic (ROC) curve analysis, and the roles of GSTP1 in human bronchial epithelial carcinogenic process were analyzed. We first time show that GSTP1, HSPB1, and CKB are potential biomarkers for early detection of LSCC, and demonstrate that GSTP1 is involved in human bronchial epithelial carcinogenesis.