Clinical Significance Of Ezrin Expression In Human Lung Carcinoma And The Effects Of Ezrin On The Biological Behaviors Of Lung Cancer Cell Lines

Lung carcinoma is the leading cause of cancer death both in men and in women. Metastasis is the primary cause of cancer related mortality,approximately 90%of all cancer deaths arise from metastasis formation.Now,it's well known that tumor metastasis is a multi-factorial,multi-step and complex process under the control of genes. Hence,identification of the key regulatory molecules that are involved in tumor metastasis will be crucial not only for understanding the molecular mechanisms underlying tumor dissemination,but also for finding a putative biomarker of tumor prognosis and for formulating more effective therapeutic targets.Ezrin is a membrane cytoskeletal cross-linker.There is increasing evidence that various tumor has ezrin mutation and abnormal expression.However,the role played by ezrin in lung carcinoma has not been clearly delineated.This thesis represents the first all-round study on the role and the mechanism of ezrin in lung carcinoma.Objective:The aims of this study were to investigate the ezrin expression pattern in human lung carcinoma and the correlation with clinicopathologic characteristics.Furthermore, its molecular mechanism in lung tumor genesis and development was explored.Methods:1.Ezrin expression was examined by immunohistochemical staining in tumor tissues from 75 lung cancer cases and the correlation with clinical characteristics was analyzed.2.Semi quantity RT-PCR and western-blotting were used to detect the expression of ezrin mRNA and protein in lung carcinoma cell lines.The distribution of ezrin expression was also detected under Confocal Laser Scanning Microscope(CLSM).So, the lung cancer cell lines with the highest ezrin expression and the lowest ezrin expression were screened out.3.In vitro,the ability of proliferation,motility,adhesion and invasion between the highest ezrin expression and the lowest ezrin expression cell line was compared with flow cytometer,a scratch wound model and transwell invasive experiment.4.The model of human lung adenocarcinoma A549 and Calu-3 cell line were established and identified in nude mice.The ezrin expression and tumor growth were compared between the two models.5.The possible mechanism of ezrin in tumor metastasis was investigated with human lung cancer specimens and in vitro and in vivo treated with paclitaxel and LY294002.Results:1.The positive incidence of ezrin expression(77.3%) was significantly lower in lung carcinoma tissues than that in normal tissues(100%)(P＜0.05).No significantly association with age,gender,smoking status,histological type,differentiation and stages was found(P＞0.05).But ezrin expression was associated with lymph node involvement, distant metastasis(P＜0.05).Otherwise,the sub-cellular redistribution of ezrin from cell membrane to cell plasma was detected in lung cancer and was significantly correlated with lymph node metastasis(P＜0.05).2.Both RT-PCR and western-blotting analysis showed that all the three lung cancer cell lines with similar genetic background PG(parental cell),PG-LH7(low metastatic potential sub-line) and BE₁(high metastatic potential sub-line) have ezrin expression.The high-metastasis potential sub-line BE₁ showed the lowest ezrin expression locating only in cytoplasm,while the low-metastasis potential sub-line PG-LH₇ showed the highest ezrin expression locating in both membrane and cytoplasm; Ezrin were also detected in the five lung cancer cell lines with different genetic background.Of them,A549 lung adenocarcinoma cell line exhibited the lowest ezrin expression,and Calu-3 exhibited the highest ezrin expression.3.The ability of proliferation,motility,adhesion and invasion is higher in A549 expressing low ezrin than that in Calu-3 expressing high ezrin.4.In the model of both A549 and Calu-3 in nude mice,the rate of successful inoculation was 100%.The tumor possessed the characteristic of human lung carcinoma in pathology,chromosome map and high expression of CK.Otherwise,the tumor maintained the same ezrin expression as respective cell line.In comparison with the Calu-3 xenograft,A549 xenograft has earlier tumor formation and rapid growth,but neither has been discovered distant metastasis.5.The IC₅₀ of LY294002 in A549 cell line was 29.2μM.After treated with IC₂₀ LY294002 3.4μM,the expression of ezrin was the same as that of the control group (P＜0.01),while the pAKT expression decreased significantly.In vivo,each treated group get excellent results,furthermore combined therapy was significantly better than single therapy(P＜0.05).No severe toxicity and side-effect was observed.As in vitro,the positive expression percentage of ezrin was not changed compared with that of the control group(P＜0.01),while the pAKT expression decreased significantly.Conclusion:1.Ezrin expression is down-regulated in lung cancer,and its locational change from cell membrane to cell plasma may be associated with the tumor genesis and development of lung carcinoma.Ezrin may be a good target for anti-metastasis therapy.2.The ezrin expression reduced along with the increase of invasive and metastasis ability in the three cell lines with similar genetic background;Among the cell lines with different genetic background,the one expressing low ezrin showed increased ability of proliferation,motility,adhesion and invasion.These suggest that ezrin may perform a suppressive role in tumor progression.3.Ezrin may be an upstream regulator of AKT and may involve in suppressing tumor growth through the other pathway except PI3K/ATK or through the way of PI3K/ATK indirectly.