| Lung cancer(including Non-Small-Cell lung cancer(NSCLC)and Small-Cell lung cancer(SCLC))remains the leading cause of cancer-related death in both male and female population in the world.Although great efforts have been made towards improving early diagnosis and effective treatment,including findings of new bio-markers,establishment of modified operation and development of specific drags,the prognosis is still poor.The WT1 gene is an important regulator of cell growth and development in the embryo kidney,adult urogenital system and central nervous system.WT1 was recently proposed to act as a chameleon gene in human malignancies.Initially,WT1 was described as a tumor suppressor gene in Wilms' tumor.Based on accumulating evidence,however,WT1 has demonstrated to function as an oncogene in other types of cancers including leukemia and solid tumor.What kind of role WT1 plays in NSCLC,why WT1 acts a dichotomous manner or,so called,tissue specificity and whether it serves as a valuable biomarker of NSCLC diagnosis and prognosis or not,these questions are of great importance for the patients and cancer therapy,which is still far from satisfaction.Objectives:To investigate the role and the underlying molecular machanism of WT1 on NSCLC,Methods:Two sets of NSCLC samples and corresponding adjacent areas(159 cases from Sept.2007 to Aug.2009 and 77 cases from Sept.2007 to Apr.2011)were used for clinical study.A549,H1299,H1568 and H1650 lung cancer cell lines were employed in our in vitro and in vivo experiments.4-week-old Balb/cnu/nu mice were used to establish xenograft model.Stable clones with over-expression and knock-down of WT1 were generated with plasmid and shRNA via lentivirus technology.Sanger Sequencing and Real-Time PCR were performed for KRAS mutation and mRNA detection;the Genowide array was used to screen out potential targets affected by WT1;MTT and Ki-67 staining were generated for poliferation test;TUNEL and Human apoptotic Proteomic Profiling were carried out for apoptosis detection;wound healing assay,transwell assay and PCR array focusing on metastasis pathway were applied for migration and invasion test;Luciferase Reporter assay was engaged to confirm the regulatory function of WT1.Results:The result of the Genowide array indicated that WT1 was widely involved in the regulation of different genes and pathways,including MAP-Kinase,NF-?B and apoptosis pathways.Overexpression of WT1 and cMyc were both detected in NSCLC and associated with worse OS in mutant KRAS samples.NSCLC patients carrying KRAS mutations had worse prognosis.Upregulated WT1 and cMyc was found to be related to shorter OS in patients with KRAS mutant tumors.In vitro experiments demonstrated that WT1 promotes cell proliferation and has an antiapoptotic function in RAS mutant NSCLC,but not in RAS wild-type.In addition,a functional link was found where the WT1 protein targets the cMyc promoter,only in mutant RAS cells.In the second part of the metastasis study,the level of the WT1 mRNA was negatively correlated with that of E-cadherin(CDH1)and both of them were associated with pathological stage,metastasis and survival rate of 159 NSCLC patients.A series of genes were regulated by WT1,including CDH1,MMP13,NR4A3 et.al.WT1 suppressed CDH1 transcription via direct binding to its promoter,which may be a mechanism why WT1 could enhance the invasive ability of H1 568 and H1 650 NSCLC cell lines.Conclusion:In summary,we found(1)positive correlation between WT1 and cMyc and negative correlation of WT1 and CDH1 in NSCLC samples;(2)measuring WT1 and cMyc expression may serve as useful biomarkers to identify subgroup with KRAS mutation for individualized therapy;(3)WT1 and CDH1 maybe valuable markers for NSCLC metastasis;(4)WT1 promotes cell proliferation and has antiapoptotic function by affecting multiple apoptosis-related genes and by directly targeting the cMyc promoter in RAS mutant NSCLC;(5)WT1 promotes invasion and metastasis of NSCLC cells probably via suppression of CDH1... |
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