Quantitative Proteome Analysis Of Metastasis-associated Molecules In Human Lung Adenocarcinoma

Lung cancer is one of malignant tumors with very high morbidity and mortality.According to histological types,lung cancer is divided into four subtypes including lung squamous carcinoma,lung adenocarcinoma,small cell lung cancer and large cell lung cancer.In recent years,the incidence rate of human primary lung adenocarcinoma(AdC) has clearly been on the increase.And the patients of lung AdC have very low survival rate and very poor prognosis.Cancer cell metastasis is the major cause of high mortality and poor prognosis in lung AdC.In clinic,early-finding and controlling cancer cell metastasis is effective strategy to decrease the mortality.However,cancer cell metastasis is a very complicated process which is involved with many factors, many steps and many genes.A variety of positive and negative genes may be involved in this highly sophisticated process,which play important role in promoting or inhibiting cancer cell metastasis.Furthermore,these genes should be translated into proteins to exert the function of controlling cancer cell metastsis.Therefore,screening metastasis-related proteins directly is to provide the foundation for finding predictive metastatic biomarkers and clinical treat targets.To screen metastasis-associated biomarkers of lung AdC,laser capture microdissection(LCM) was used to purify the cancer cells from human primary lung AdC with(LNM AdC) and without metastasis(non-LNM AdC) according to clinical diagnosis of lymph node metastasis and distant metastasis.Then two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was performed to isolate the total proteins of the pooled microdissected cancer cells from non-LNM AdC and LNM AdC.The differential proteins between non-LNM AdC and LNM AdC were analyzed by Decyder software and further identified by mass spectrometry(MS).The partial differential proteins including S100A9,B23,annexin A1,annexin A2 and annexin A3 were validated by Western blot.To further study the associations between expression levels of the partial differential proteins with clinical pathological fators and evaluate their clinicopathological significance, immunohistochemical technique was performed to analyze the expression levels of these proteins in archived paraffin imbedded tissues.And statistical analysis evaluated the relationships of their expression levels and clinical pathological factors and patients' relapse and outcome.In the present study,2D-DIGE patterns of microdissected non-LNM AdC and LNM AdC were established,and 20 differential proteins in the above two tissues were identified,13 out of which were up-regulated and 7 were down-regulated in LNM AdC compared to non-LNM AdC.Western blot results indicated that S100A9,annexin A1,annexin A2 and annexin A3 were significantly up-regulated in LNM AdC compared to non-LNM AdC;B23 was significantly down-regulated in LNM AdC compared with non-LNM AdC.Immunohistochemical analysis further indicated S100A9,annexin A1, annexin A2 and annexin A3 were up-regulated in LNM AdC compared with non-LNM AdC;B23 was down-regulated in LNM AdC compared with non-LNM AdC.Furthermore,annexin A1,annenxin A2 and annexin A3 were up-regulated in positive lymph nodes compared with primary lung adenocarcinoma.Statistical analysis indicated the increase of the three annexins expression levels in these tumors was significantly associated with lymph node metastasis and advanced clinical stage.Kaplan-Meier curve and Cox regression analysis indicated annexin A1,annexin A2 and annexin A3 expression levels were correlated with relapse and survival.This is to say,the three annexins over-expression were associated with increased relapse rate and decreased survival rate.It was the first time that metastasis-associated proteins were identified in human primary lung AdC by LCM coupled with 2D-DIGE and MS techniques. Our findings will facilitate understanding of human lung AdC metastasis and provide some direct proof for mining markers for predicting metastasis and patients' outcome so as to improve the diagnosis and treatment of lung AdC.