| Malignant melanoma(MM)is a type of cancer originated from uncontrolled proliferation of melanocytes.It notes for its high potential to invasion and metastasis,as well as its resistance to conventional radiotherapy and chemotherapy.V-Raf murine sarcoma viral oncogene homolog B1(BRAF)is a member of the RAF protein kinase family and an intermediate in the RAS-RAF-MEK-ERK signaling pathway.It has been found mutated in more than 60%of melanomas with the most common type of valine-to glutamic acid substitution at residue 600(V600E).The activation of BRAF V600E in melanoma can promote tumor cell proliferation,migration and invasion.However,mutations in BRAF alone are not sufficient to cause melanoma;it is also frequently found in benign nevi.Moreover,despite of the successes of BRAF inhibitors in therapy of melanoma patients with BRAF mutation,there are only half of these patients demonstrated effective in the process of treatment and almost all patients will be eventually resistant to inhibitors.Overall,these facts indicate that single gene alteration is insufficient for the explanation of melanoma invasion and metastasis.LKB1,known as a tumor suppressor,plays multiple roles in promoting tumor progression.In lung,LKB1 loss concomitant with BRAF mutation lead adenoma to lung carcinoma,however its cooperation with BRAF still remains unclear in melanoma.Therefore,the aim of this study was to investigate the role of LKB1 loss in BRAF mutation melanoma invasion.ObjectiveTo explore the relationship between LKB1 and MMP-2 expression in human melanoma and its relationship with clinicopathological features;To observe the effects of LKB1 loss cooperating with BRAF mutation on melanoma cell invasion and migration and involved molecular mechanism.Methods 90 cases of human melanoma FFPE tissues were divided into BRAF V600E mutation and BRAF wild type based on the results of ARMS-PCR analysis,and then all tissues were subjected to IHC staining for LKB1 and MMP-2simultaneously.The relationship between LKB1 expression and clinicopathological features of melanoma was statistical analyzed by SPSS software.LKB1 expression was inactivated by RNA interference in BRAF mutation A375 cells and BRAF wild type MeWo melanoma cells respectively.A series of experiments including wound scratch test、Transwell assay are used to observe the changes of melanoma cell invasiveness and migration.Signal pathway molecules involved in invasion will be detected by western blot.Results1.IHC staining with BRAF V600E mutation-specific antibody displayed 100%sensitivity and 96.8%specificity.Further statistical analysis showed that results from the two methods were consistent(kappa=0.948 p<0.001),positive predictive value(PPV)and negative predictive value(NPV)were 93.1%、100%.2.In BRAF wild type human melanoma tissues,there exists no relationship between LKB1 and MMP-2,and LKB1 expression did not correlate with any clinicopathological parameters.While in those BRAF V600E mutated cases,LKB1 low level expression was not only associated with high level MMP-2 expression(Spearman’s rank correlation test,r=-0.304,p<0.05)but also with distant metastasis and lymph node metastatic status(contingency table Chi-squared test,p<0.05).3.LKB1 was successfully knockdown in A375 and Me Wo cells.4.SiLKB1 transfected Me Wo cells showed no change on migration and invasive behavior,compared with SiCtrl transfected MeWo cells.5.SiLKB1 transfected A375 cells showed significant enhancement on migration and invasive behavior,compared with SiCtrl transfected A375 cells.6.The expression of MMP-2、phosphorylated Akt and phosphorylated mTOR were upregulated in SiLKB1 transfected A375 cells,but no change in SiLKB1 transfected MeWo cells.Conclusions1.IHC is a simple,fast and economic screening method for BRAF V600E mutation analysis in malignant melanoma.2.In BRAF mutated melanoma,the expression of LKB1 negatively correlated with the expression of MMP-2,and associated with tumor distant and lymph node metastasis.3.LKB1 loss cooperating with BRAF mutation promotes invasion and migration of melanoma cells.4.LKB1 loss cooperates with BRAF V600E to promote melanoma cell invasion and migration by up-regulation MMP-2 via PI3K/Akt/mTOR pathway. |
