| Background/ObjectiveKindlins are a newly found protein family including Kindlin-1, Kindlin-2 and Kindlin-3. Loss of function mutation of KIND1 gene leads to a defect in actin-ECM linkage, which causes Kindler syndrome, an autosomal recessive disorder characterized by neonatal blistering, sun sensitivity, atrophy, abnormal pigmentation, and fragility of the skin. Some special cases showed hemorrhagic colitis, erosions and ulcers with pseudomembranous on mucosa of the descending and sigmoid colon and the rectum. The Kindlin family proteins show high homology of their amino acid sequence and share the same protein structure that all of them have two FERM (Four point one-ezrin-radxin-moesin) domains interrupted with a PH (pleckstrin homology) domain. FERM domain is a conservative structure existing in proteins that connect the cytoplasmic domains of transmembrane proteins to the actin cytoskeleton and modulate cell signaling and cell membrane structures while PH domain is a feature of a number of cytoskeletal-associated and/or cell-signaling molecules. The special structure of Kindlin-1 and its localization at focal adhesion imply Kindlin-1 may interact with Integrins and modulate Integrin mediated cell adhesion and migraton. Interestingly, Kindlin-1 mRNA was found overexpressed in 70% of human cancers and 60% of lung cancer patients. What’s more, Kindlin-1 can be upregulated by TGF-131 and suppress expression of E-cadherin, upregulating Snail expression simultaneously, which contributes to EMT (epithelial-mesenchymal transition). In our study, we will focus on Kindlin-1 expression during colon cancer progression and its role in Integrin mediated colon cancer cell adhesion, migration and invasion and elucidate the signaling pathway in which Kindlin-1 involves and contributes to colon cancer progression.Methods1. Immunohistochemistry was performed to examine Kindlin-1 expression in benign polyps, adenomas, primary colon cancers on different Dukes stage and paired normal colon mucosas/mesentery lymph node metastases, distant organ metastases, which represent different stages of colon cancer progression. 2. In serial sections, expression of Kindlin-1 and proliferative marker Ki-67 was examined by immunohistochemistry to analyze their correlation. 3. GST pull-down assay and co-immunoprecipitation were used to examine the interaction of Kindlin-1 with Integrinβ1. Colocalization of Kindlin-1 and Integrinβ1 in SW480 cells and colon cancer tissues was detected by double immunofluorescence staining. 4. Genetic manipulation was performed to up-regulate or knock down expression of Kindlin-lin colon cancer cells and followed by cell adhesion, cell migration, cell invasion and MTT assays to test Integrin mediated cell adhesion and migration, cell invasion and proliferation respectively. 5. After Genetic manipulation on colon cancer cells, Western blot was performed to examine the regulatory effects of Kindlin-1 on key proteins in Wnt/β-catenin pathway. Luciferase reporter gene assay was used to test affection of Kindlin-1 on Wnt/β-catenin target transcriptional factor Tcf by calculating ratio of Topflash/Fopflash.Results1. Kindlin-1 expression increases with colon cancer progression: in normal colon mucosa, Kindlin-1 immunoactivity was either absent or barely detectable in progenitor cells at the bottom of normal mucosa crypts. Kindlin-1 expression was nearly negative in benign polyp but began to increase in adenomas with atypical hyperplasia. Interestingly, an even stronger expression of Kindlin-1 was found in primary colorectal carcinoma and the strongest in metastasis cancers (p＜0.0001). In colon cancers on different Dukes stage, Kindlin-1 expression in colon cancer on Dukes A was lower than that in colon cancer on Dukes B, Dukes C and Dukes D (p＜0.05). In addition, Kindlin-1 was weakly expressed in mucinous adenocarcinomas compared with that in adenocarcinomas (p＜0.0001). 2. Kindlin-1 expression was positively correlated with that of proliferative marker Ki-67 (p＜0.001) and MTT assay showed that SW480 cells transfected with Flag-Kindlin-1 proliferate much more quickly than those transfected with empty vector Flag (p＜0.05). 3. GST pull down assay showed GST-Integrinβ1 tail pulled down exogenous Flag-Kindlin-1 transfected into COS-7 cells and co-immunoprecipitation showed endogenous Kindlin-1 interacted with endogenous Integrin 131 in colon cell lines. Consistent with this, Kindlin-1 and Integrinβ1 were found colocalized at focal adhesion and lamellipodia in SW480 cells and at the invasive front of human colon cancer tissues. 4. Transfection of Flag-Kindlin-1 into SW480 cells enhanced Integrin mediated cell adhesion (p＜0.05) and migration (p＜0.001) on Collagen typeⅠwhile knocking down of Kindlin-1 by siRNA in SW480 cells impaired cell adhesion (p＜0.05) and migration (p＜0.01). In addition, overexpression of Kindlin-1 in SW480 cell by gene transfection promoted cell invasion on Matrigel (p＜0.01). 5. Knocking down of Kindlin-1 (p＜0.01) in HCT116 cells down regulated phosphorylated GSK-3β(p＜0.05), which enhanced activity of GSK-3βand thereby down regulatedβ-catenin (p＜0.01) and its down stream proteins including cyclin D1 (p＜0.01) and Snail (p＜0.001) simultaneously. In SW480 cells, similar results were obtained by knocking down Kindlin-1 expression with another two siRNAs. Along with the downregulation of Snail, E-cadherin is re-expressed in SW480 cells. On the other hand, transfection of Kindlin-1 into SW480 cells up-regulated Wnt/β-catenin downstream proteins c-myc (p＜0.05), cyclin D1 (p＜0.01) and Snail (p＜0.05). Luciferase assay showed that Topflash/Fopflash ratio was much higher in HCT116 cells transfected with Flag-Kindlin-1 than in those tansfected with empty Flag vector (p＜0.05), which suggested that Kindlin-1 activate Wnt/β-catenin-Tcf/Lef pathway.Conclusion1. Kindlin-1 expression increases with human colon cancer progression.2. Kindlin-1 interacts with Integrinβ1 and enhances Integrin-mediated colon cancer cell adhesion, migration and invasion.3. Kindlin-1 activates Wnt/β-catenin pathway by inhibiting GSK-3βactivity and up-regulates its down steam protein c-myc and cyclin D1 expressions, therefore promoting cell prolifetation.4. Kindlin-1 up-regulates Snail and down-regulates E-cadherin expression through either activating Wnt/β-catenin pathway or inhibiting GSK-3βthat can phosphorylate Snail directly and lead to its degradation by ubquitin-mediated pathway, thus contributing to colon cancer EMT. |
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