Identification Of Novel Biomarkers For Early Detection Of Human Lung Squamous Cell Cancer By Laser Capture Microdissection Combined With 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. Although great improvement has been made in diagnosis and treatment of lung cancer, the overall patients' survival is still very low and less than15%. The poor prognosis of this cancer is mainly explained by the fact that the diagnosis is generally made only at advanced stages due to 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) originated from the bronchial epithelial cells is the most common histological type of lung cancer. It is known that carcinogenesis of LSCC is a multi-stage process and the result of multistep accumulation of genetic and epigenetic alterations. 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. To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with2D LC-MS/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,102differentially expressed proteins were identified. Among these differentially expressed proteins, some proteins are progressively upregulation, some proteins are progressively downregulation in human bronchial epithelial carcinogenic process, and the other proteins are upregulation or downregulation in a certain stage of the process of carcinogenesis. To get more insight on the biological significance of the differentially expressed proteins in bronchial epithelial carcinogenic process, hierarchical clustering was performed on102differentially expressed proteins. All differentially expressed proteins were hierarchically grouped into8clusters and3groups. GO analysis showed that Group1(clusters1and2) was dominated by proteins associated with development, apoptosis, translation and metabolic process, group2(clusters6) consisted of proteins related to biological processes in LSCC, such as multi-organism process, regulation and negative regulation of apoptosis, response to biotic stimulus and actin filament bundle formation, group3(clusters3,4,5,7and8) were enriched with catabolic process, cellular process, apoptosis, protein transport, localization, cell proliferation and humoral immune response. KEGG pathway analysis revealed that the proteins in3groups were involved in cancer-associated signaling pathways such as MAPK signaling pathway, apoptosis, cell cycle and p53signaling pathway, and ErbB signaling pathway. The proteins within the same cluster are co-regulated proteins, and may have similar biological functions during bronchial epithelial carcinogenesis. The differentially expressed proteins may play certain role in bronchial epithelial carcinogenesis by these signaling pathways.Among102differentially expressed proteins, HSPB1was progressively upregulation, GSTP1and CKB were progressively downregulation in human bronchial epithelial carcinogenic process. Therefore, we choose HSPB1, GSTP1, and CKB an in-depth study. The expression of these three proteins in various stage tissues of bronchial epithelial carcinogenesis were detected by Western blotting, the results were concordant with that of the proteomics. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including normal bronchial epithelium, squamous metaplasia, atypical hyperplasia, cancer in situ and invasive cancer, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combined detection of HSPB1, GSTP1, and CKB could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of96%and a specificity of92%in discriminating NBE from preneoplatic lesions, a sensitivity of99%and a specificity of98%in discriminating NBE from invasive LSCC, and a sensitivity of92%and a specificity of91%in discriminating preneoplatic lesions from invasive LSCC, respectively.To investigate the role of GSTP1in carcinogenesis of lung squamous cell carcinoma, we took immortalized human bronchial epithelial cell line16HBE as the study object, established a stable silencing GSTP1expression16HBE cell line (16HBE/PLO.1-GSTP1-shRNA) and the empty vector transfected control cell line using siRNA techlique, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation by MTT assay, anchorage dependent and independent colony formation assays, flow cytometry analysis and hoechst33258staining of apoptotic cells. The results showed that cell growth rate was significantly higher, the number of plate colonies and soft agar colonies was significantly increased, the rate of apoptosis was significantly reduced in6HBE cells with knockdown of GSTP1than in the control cells induced by B[a]P. Taken together, these results demonstrated that knockdown of GSTP1increased the susceptibility of human bronchial epithelial cell transformation induced by B[a]P, supporting that GSTP1downregulation involved in human bronchial epithelial carcinogenesis. GSTP1downregulation may be the susceptibility biomarker of prediction lung squamous cell carcinoma for smokers.In this study, Comparative Proteomics Analysis was performed in various stage tissues of bronchial epithelial carcinogenesis using LCM combined with quantitative proteomics technology. We identified102differentially expressed proteins and studied their biological functions and signaling pathway which were related to human bronchial epithelial carcinogenesis. The results demonstrated that the combined detection of HSPB1, GSTP1, and CKB could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC; GSTP1, HSPB1and CKB are novel potential biomarkers for early detection of LSCC. We first found that knockdown of GSTP1in6HBE cells can increase the sensitivity of bronchial epithelial cells to malignant transformation induced by chemical carcinogens B[a]P, GSTP1downregulation plays an important role in lung squamous carcinoma carcinogenesis.