Apply Lectin Array and Quantitative Tissue Glycoproteome Analysis to Develop a Glycoprotein Biomarker Panel for Early Diagnosis and Prognosis of Non-Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC), comprising ~80% of all lung cancers is one of the most prominent causes of cancer-related mortality worldwide. The asymptomatic characteristics and limited diagnosis approaches lead to delayed detection and high mortality of lung cancer in the clinic. The stage of lung cancer is highly correlated to prognosis and the degree of cancer spread to lymph nodes (LN) is the determining factor in the accurate staging that would be the basis for treatment. Thus, it is urgent to search good biomarkers for early detection of cancer and micrometastasis in NSCLC. Protein glycosylation is a ubiquitous post-translational modification found in all domains of life. It has been estimated that approximately half of all human proteins are glycoproteins that contribute to various normal physiologies pathologies, including human cancers. The nine FDA-approved protein biomarkers for the diagnosis and management of cancer are serum glycoproteins, supporting the enormous potential of glycoprotein as biomarkers for cancers. In this proposal, we will identify/quantified stage and metastasis-related glycoprotein biomarkers (early stage without LN involvement vs. late stage with LN involvement or distant metastasis) for NSCLC by lectin microarray, lectin chromatography followed by iTRAQ coupling with HCD/ETD quantitative tissue glycoproteome analyses. We will integrate the differential tissue proteome/glycoproteome, pleural effusion (PE) proteome (benign vs. malignant), lung cancer cell secretome (low vs. high invasiveness), and pathway analysis to search the potential tissue- and also body fluid-accessible glycoprotein biomarkers for early detection and prognosis of NSCLC. The potential glycoprotein markers will be verified and validated by using large scale of clinical samples. To address the molecular mechanisms of these stage/metastasis-related glycoprotein markers, we will combine molecular biology, cell biology, and xenograft mice models to characterize the regulations of these glycoproteins and their corresponding glycosyltransferase involved in lung cancer progression. This proposal is the first study to establish the stage/metastasis-related tissue glycoproteome for NSCLC. The long term goal of this proposal is to develop a clinical useful glycoprotein marker panel for early diagnosis/prognosis and address the mechanism of cancer-related glycoprotein/glycosyltransferase for therapeutic development in NSCLC. Our specific aims are as follows: 1. Generate the distinct stage and metastasis-related tissue glycoproteome dataset for NSCLC. a. Screen and enrich the stage/metastasis-related tissue glycoproteins from paired NSCLC tissues (tumor and adjacent normal) with or without EGFR mutation (L858R, exon 19 deletion and T790M) by lectin microarray, lectin chromatography and immunoprecipitation. b. Establish the distinct stage and metastasis-related glycoprotein profiles for NSCLC tissues by iTRAQ coupling with HCD/ETD quantitative proteomics technology. c. Verify the stage/metastasis-related glycoproteins (~10 targets) by western blot and lectin staining. d. Pathway analysis of stage and metastasis-related proteome/glycoproteome. 2. Develop a glycoprotein panel for early detection and prognosis of NSCLC. a. Combine differential tissue proteome/glycoproteome, PE proteomes, and lung cancer cell secretome to search stage and metastasis-related glycoprotein marker candidates those are originating from cancer cells and could be detected in body fluids. b. Validate and analyze the clinical significance of stage or metastasis-related glycoprotein marker candidates by immunohistochemistry staining, western blot, ELISA and lectin ELISA using large scale of clinical specimens (n≧250). 3. Study the roles of cancer-related glycoprotein and/or glycosylation related enzymes in cancer cell line and xenograft mice model. a. Characterize the biological function of promising glycoprotein and its corresponding glycosyltransferase/de-glycosylation enzymes in lung cancer cell and xenograft mouse model. b. Define the relationship between promising glycoprotein and well-known cancer progression signaling or discover the novel interaction network regarding promising glycoprotein markers by SILAC-based quantitative proteomics strategy.

Project ID:PC10408-1724
External Project ID:MOST104-2320-B182-027