The Clinical Significance Of RKIP Expression In Non-small Cell Lung Cancer And Its Mechanism Contributing To Migration And Invasion

Due to the environmental pollution, smoking，ionizing radiation andother factors, the incidence of lung cancer has increased greatly during thepast decades. Lung cancer is the leading cause of death related to neoplasticdiseases worldwide. Especially in China, the incidence of lung cancer ranksthe top of all malignant tumors. Although significant progress has beenachieved in comprehensive treatment of lung cancer, its5-year survival rateremains merely10%-15%. Non-small cell lung cancer （NSCLC）accounts for approximately85%lung cancer stratified by histological type.The latest researches show that performance status, the stage of disease, age,sex, weight loss are independent risk factors, but no biological marker isreliable enough for individualized therapy regimen or for predicting prognosis.There is an urgent need to uncover the molecular mechanisms of invasion andmetastasis of NSCLC and to find biological markers for effective prevention,diagnosis and treatment of NSCLC.Raf-1kinase inhibitor protein （RKIP） belongs to a group of highlyconserved proteins named by phosphatidylethanolamine-binding protein（PEBP）. Recent studies show that RKIP can not only inhibitRAF-MEK-ERK signaling pathway, but also interfere with the activity ofNF-κB and G protein-coupled receptor kinase. Expression of RKIP isnegatively associated with the occurrence, development, invasion, migrationand metastasis of many tumors including prostate cancer, breast cancer, livercancer, melanoma, colorectal cancer, stomach cancer and cervical cancer etc.,however, it is rarely reported in lung cancer.Invasion and migration of NSCLC, which would weaken the treatmenteffect and predict poor outcome, is the leading cause of death. The invasionand metastasis of NSCLC are complicated which is involved in the interactions between cancer cell and organism. In this study, we collectedthe tissue samples from NSCLS patients, examined for mRNA and protein ofRKIP via real-time PCR and IHC respectively, and analyzed the difference inRKIP expressions in NSCLC and in normal tissue around the cancer. Wedemonstrated that the mRNA level of RKIP decreased significantly inNSCLC, and lower level of RKIP mRNA correlated with poorerdifferentiation and advanced pathologic TNM stage. Moreover, byoverexpression of RKIP in vitro, we noted that RKIP upregulation inhibitsthe invasion and migration of the A549cells which is a lung carcinoma cellline probably through the suppression of epithelial mesenchymal transition（EMT）. Chapter1The Clinical significance of RKIP expression in non-smallcell lung cancer.Objective: Raf-1kinase inhibitor protein （RKIP） expression wasassociated with the onset, development, invasion, migration and metastasis ofnumerous tumor types. Association between RKIP expression, theclinical-pathological features and prognosis were rarely investiged inresectable non-small cell lung cancer （NSCLC）.Method: Real-time PCR （RT-PCR） was performed to detect theexpression of RKIP mRNA in126pairs of lung tumor tissues （TT） andsurrounding normal tissues （sNT）. Correlation between RKIP mRNAexpression and clinicopathological features was evaluated by statisticalanalysis. Tissue microarray （TMA）, Immunohistochemistry （IHC） wasperformed to detect the expression RKIP protein in pairs tissues of70cases ofa complete five-year follow-up data. Association of RKIP expression with theprognosis of70NSCLC was analyzed.Results: In the126patients examined, RKIP mRNA expression wassignificantly lower in lung TT than the sNT （P<0.05）. Our results furtherdemonstrated that lower level of RKIP mRNA expression was associated withpoorer N-stage （P=0.019） and advanced pathological TNM stage（P=0.015）. however, no significant association was observed between theexpression status of RKIP mRNA and clinicopathologic factors, such asgender, age, histological type, and the size of the tumor （P>0.05）. In the70patients with complete five-year follow-up data. Lower expression of RKIPprotein was associated with poorer outcome of NSCLC as illustrated by K-Msurvival curves （P<0.05）. However, RKIP was not identified as anindependent prognostic factor according to the Cox regression analysis（P>0.05）.Conclusions: The level of RKIP mRNA was found to be significantlydownregulated in NSCLC, and the lower mRNA levels correlated with poorer differentiation, advanced pathologic TNM stage in patients with NSCLC. Thelower expression of RKIP, which was associated with poorer outcome,however cannot be a positively prognostic factor in NSCLC Chapter2Overexpression of RKIP inhibits cell invasion and migrationin A549cells through suppressing EMT.Objective: It has been reported that Raf-1kinase inhibitor protein （RKIP）is associated with the invasion and metastasis of prostate cancer and bowelcancer, however, only few studies focusing on the effects of RKIP in lungcancer. Therefore, we intend to determine whether RKIP would alter theinvasion and metastasis of non-small cell lung cancer （NSCLC） andrelated mechanisms.Methods: A549cells was transfected with RKIP-GV141plasmid foroverexpression of RKIP. Colony formation assay and MTT assay wereperformed to measure the effects of RKIP on the proliferation and cellviability assay of A549cells. Transwell, Migration Assay and wound healingassay were performed to analyse the effects of RKIP on the invasion andmetastasis of A549cells. E-cadherin and vimentin were measured by Westernblot to conform RKIP affects invasion and metastasis of NSCLC viainhibiting Epithelial-mesenchymal transition （EMT）.Results: RKIP was highly expressed in the A549cells transfected withRKIP-GV141plasmid as determined by RT-PCR and Western blot, while itwas barely expressed in non-transfected cells. Colony formation assayrevealed that RKIP inhibited proliferation of A549cells. MTT assay revealeddecreased cell viability of A549cells with overexpression of RKIP. Woundhealing assay showed degressive ability of metastasis. Transwell andMigration Assay showed decreased ability of invasion and migration. Highexpression of E-cadherin and low expression of vimentin indicated that RKIPaffects invasion and metastasis of NSCLC via inhibitingEpithelial-mesenchymal transition.Conclusion: Overexpression of RKIP in A549cells would decrease thecellular proliferation, viability, invasion ability and metastasis ability probablyvia inhibiting EMT through upregulating E-cadherin expression and downregulating vimentin expression.