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Screening of Plasma Membrane Protein Biomarkers of Human Lung Adenocarcinoma Using2D-LC-MS/MS Combined with iTRAQ Technologies

Author: ZhangPengFei
Tutor: XiaoZhiQiang
School: Central South University
Course: Pathology and Pathophysiology
Keywords: human lung adenocarcinoma iTRAQ plasmamembrane quantitative proteomics biomarker
CLC: R734.2
Type: PhD thesis
Year: 2012
Downloads: 107
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Abstract

Lung cancer is one of the most frequently occurring malignancies with increasing incidence and is the leading cause of mortality in cancer-related deaths in China and worldwide. According to its histological types, lung cancer can be divided into four subtypes, including lung squamous carcinoma, lung adenocarcinoma, large cell lung cancer, and small cell lung cancer. Lung adenocarcinoma (AdC) accounts for approximately20-30%of overall lung cancer incidence. In recent years, the incidence rate of human primary lung adenocarcinoma has clearly been on the increase. Patients with lung adenocarcinoma have shown both low survival rate and very poor prognosis, with the overall5-year lung adenocarcinoma free actuarial survival rate at only16%. The main reason behind is the secrecy of lung adenocarcinoma pathogenesis and there is no apparent symptom at early stages. Most patients are already in the advanced stage at the time of their first hospital visit and by then there is no chance for any effective treatment. Because of this, even after operations, most patients die from lung adenocarcinoma metastasis and distant metastasis. So, early diagnosis and recurrence and metastasis prediction of Lung adenocarcinoma are critically important for the effective prevention and treatment. From the theoretical viewpoint, biomarker is the most sensitive index for the diagnosis of malignant tumorz and for the monitoring of disease activities in patients. Therefore, looking for new biomarkers of lung adenocarcinoma is an effective pathway of improving the cure and survival rates for cancer patients, and these biomarkers, if ever found, could facilitate the diagnosis and treatment of lung adenocarcinoma.In order to screen early diagnosis markers of lung adenocarcinoma, we selected both lung adenocarcinoma tissues and paraneoplastic normal lung tissues (PNLTs). We first adopted differential centrifugation, sucrose density centrifugation and aqueous two-phase partition technology to purify plasma membrane (PM). Next, using isobaric tag for relative and absolute quantitation (iTRAQ) combined with the2D-LC-MS/MS analysis method, we isolated and identified differentially expressed PM proteins in primary lung adenocarcinomas and PNLTs. After that, partially differential PM proteins including FLOT1(Flotillin-1), CAV1(Caveolin-1) and ITGB1(Integrinβ1) were validated by Western blot. Finally, to further study the associations of expression levels of FLOT1, CAV1and ITGB1with clinical pathological factors and evaluate their clinicopathological significance, immunohistochemical techniques were employed to analyze the levels of these differential proteins in archived paraffin imbedded tissues. Statistical analyses were utilized to ascertain the relationships among expression levels, clinical pathological factors, patient’s relapse, and outcomes.In this study, we have not only established an effective method for purifying plasma membrane by using differential centrifugation, sucrose density centrifugation, and aqueous two-phase partition technology, but also developed a novel strategy to isolate and identify PM proteins by iTRAQ-tagging combined with2D-LC-MS/MS analysis. A total of41differential proteins, including36differential PM proteins, in the above two tissues have been identified,18of which were found to be up-regulated and23down-regulation in lung adenocarcinoma tissues compare to PNLTs. Our Western blot results indicated that FLOT1and ITGB1were significantly up-regulated and CAV1was markedly down-regulated in lung adenocar-cinoma tissues compared to PNLTs. Immunohistochemical analysis results further confirmed that FLOT1and ITGB1were up-regulated in lung adenocarcinoma tissues versus PNLTs, and CAV1was down-regulated in lung adenocarcinoma tissues versus PNLTs. Furthermore, FLOT1, ITGB1and CAV1were up-regulated in lymph nodes metastasis lung adenocarcinoma tissues compare with primary lung adenocarcinoma tissues. Statistical analysis results suggested that the increase of these three PM proteins expression levels in tumors was significantly associated with lymph metastasis and advanced clinical stage. Kaplan-Meier cure and Cox regression analysis indicated that FLOT1, ITGB1and CAV1expression levels correlated well with relapse and survival rates, suggesting that these three PM proteins’over-expression was reliable indicators of the increased relapse and decreased survival rates. FLOT1, ITGB1and CAV1highly expressed in lung adenocarcinoma are risk factors of lymph node metastasis and recurrence and independent prognostic factors of lung adenocarcinoma.To the best knowledge of the present author, it is the first time that biomarkers were identified in human lung adenocarcinoma tissue by the two-phase partition approach coupled with iTRAQ-labeling and2D-LC-MS/MS techniques. It is also the first time to report that FLOT1expression level correlates with relapse and survival rates of lung adenocarcinoma patients. Our findings will facilitate in screening biomarkers and understanding pathogenesis of lung adenocarcinoma.

Screening of Plasma Membrane Protein Biomarkers of Human Lung Adenocarcinoma Using2D-LC-MS/MS Combined with iTRAQ Technologies

Abstract

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