| Objective:To investigate the molecular mechanism of a novel membrane bound IL-15 isoform (mb-IL-15) inducing epithelial-mesenchymal transformation in human renal cancer cells (RCCs).Methods:1) Flow cytometry cell surface staining and immunoprecipitation were used to detect the expression of the novel mb-IL-15 on primary human renal carcinoma cell lines(ACHN,786-0).2) With stimulation of the soluble IL-15Ra chain (s-IL-15Ra) to the mb-IL-15, Immunofluorescence was used to observe the morphological changes of 786-0 cells, Western blot and quantitative PCR to detect epithelial and mesenchymal phenotype changes, migration assay and invasion assay to analyze cell migration and invasion changes.3) To analyze expression levels of Src, Erkl/2 and Akt kinases after stimulation to the mb-IL-15, western blot was used at different time and different doses of s-IL-15Rα;4) Immunofluorescence and co-immunoprecipitation were performed to detect the expression and location changes of β-catenin and the glycogen synthase kinase 3β (GSK3β).5) The employment of si-FAK was used in the migration and invasion assays to analyze the role of FAK kinase in cell migration and invasion.6) mRNA and protein expressions of IL-15 were investigated in primary renal cancer tissues and adjacent normal tissues by Real-time PCR and immunohistochemistry (IHC).Results:1) Human renal cell carcinomas (RCCs) expressed a membrane bound IL-15 isoform (mb-IL-15) displaying an unusual molecular weight of 27kDa.2) The mb-IL-15 stimulation with the soluble IL-15Ra chain (s-IL-15Ra) triggered in RCC cells the epithelial-mesenchymal transdifferentiation process (EMT) as shown by the down-regulation of the epithelial markers E-cadherin and ZO-1 and the up-regulation of the mesenchymal markers vimentin and N-cadherin, displaying a fibroblast-like morphology from a cobblestone-like morphology.3) The signalway of EMT was characterized by the phosphorylation of Src, Akt, Erkl/2. The use of the Src specific inhibitor PP2 showed that activation of Akt and Erkl/2 pathways depended on Src phosphorylation, while use of specific inhibitors of Erkl/2 (PD98059) and of Akt (MK2206) showed that only the latter pathway triggered EMT inducing the down-regulation of E-cadherin and the up-regulation of vimentin.4) Further dissection of the Akt pathway showed that its activation was associated to the sIL-15Rα dependent phosphorylation of the phosphatidylinositol 3-kinase (PI3K) and of the glycogen synthase kinase 30 (GSK3β) leading to the nuclear translocation of β-catenin.5) Stimulation of the mbIL-15 with its soluble ligand induced the phosphorylation of the Focal Adhesion Kinase (FAK) increasing the migratory and invasive properties of RCC cells. And Src activation was upstream of FAK and cannot be inhibited by FAK specific si-RNA.6) IL-15 mRNA and protein expression levels in renal cell carcinoma compared with normal kidney tissue were significantly increased, determined by qRT-PCR and IHC respectively (n=15).Conclusion:1) Human RCCs express a novel mb-IL-15 that induces EMT, in response to the s-IL-15Rα chain.2) Src-dependent Akt/GSK3β/β-catenin pathway is required for mb-IL-15/s-IL-15Rα-dependent induction of EMT in RCC cells.3) Stimulation of mb-IL-15 with s-IL-15Rα chain promotes cellular migration and invasion in RCC cells through the Src/FAK pathway.4) Mb-IL-15 may contribute to the development and metastasis of RCCs. |
