| Migfilin is a recently identified, widely expressed LIM domain protein, with unique dual subcellular localization in the nucleus and along actin-based filaments in the cytosol. It is a component of cell-ECM adhesions, where it links the cell-ECM adhesions to the actin cytoskeleton and functions in actin cytoskeleton remodeling and cell shape modulation. However, the underlying mechanisms are not fully elucidated. In this study, we have determined the functions of migfilin in esophageal cancer cells, and the mechanisms involved.Western blot and immunohistochemical analysis were performed in human esophageal squamous cell carcinoma (ESCC) tissue specimens and their normal counterparts respectively. The results demonstrate that increased expression and nuclear-to-cytoplasmic translocation of migfilin in ESCC compared to matched adjacent normal epithelium. However, real time RT-PCR analysis in37ESCC samples with clear lymph node status showed a significantly negative correlation of migfilin expression levels with clinical metastasis status, implicates that increased expression of migfilin in ESCC may participate in suppressing its malignant phenotype and nodal metastasis.To gain insight into the effect of migfilin on the motility potential of esophageal cancer cells, we overexpressed migfilin in KYSE450cells. The results showed that overexpression of migfilin promoted the adhesion of esophageal cancer cells to fibronectin and formation of filopod, and significantly attenuated cell migration and invasion. Reciprocally, silenced migfilin from KYSE150cells potently promoted migration as well as invasion. These results indicate a suppressive role of migfilin in esophageal cancer cell migration and invasion.We next explored which signaling pathway is involved in migfilin-mediated suppression of cell invasion and migration and found β-catenin, an important component in E-cadherin-mediated cell adhesion and Wnt-dependent signal transduction, was significantly decreased in the migfilin stable transfectants and transient transfectants in HEK293T cells substantially suppressed β-catenin-induced transcripition activity in a dose-dependent manner. On contrary, knockdown of migfilin by siRNA in KYSE150cells prompted the cytoplasmic accumulation of P-catenin as well as increased its transcription activity. These data demonstrate that migfilin induced downregulation of P-catenin and negatively regulated Wnt/p-catenin signaling.Moreover, transfection of a mutant β-catenin at Ser37which is a critical phosphorylation site of GSK3P, GSK3β inhibitor LiC1or proteasome inhibitor MG132reversed the migfilin-mediated P-catenin degradation and transcription inhibition, implicating that GSK3β activity and proteasomal degradation system are involved in migfilin-mediated destabilization of P-catenin.We also demonstrated that migfilin promoted P-catenin ubiquitin degradation both by reinforcing the association between P-catenin and GSK3β and increasing GSK3P activity. Migfilin interacted with P-catenin and enhanced the association of GSK3βwith β-catenin which facilitated the phosphorylation of P-catenin by GSK3β. Overexpression of migfilin attenuated the activity of Akt and phosphorylation of GSK3β while phosphatidylinositol3-kinase (PI3K) inhibitor LY294002abolished activation of Akt induced by knockdown of migfilin, indicating that the effects were mediated by the PI3K/Akt signaling pathway. Additionally, exogenously expressed P-catenin partially restored migfilin-induced suppression of cell invasion.Collectively, these results suggest that while migfilin is widely overexpressed in ESCC, its expression level is inversely correlated with clinical metastasis status, and migfilin inhibits ESCC cell invasion at least in part through promoting degradation of β-catenin via the PI3K/Akt/GSK3β-dependent pathway.
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