Roles Of Kindlin-2 And Prdx4 In Tumor Cell Invasion And Tumorigenesis

Kindlin protein family is a kind of vinculin.The molecular weight of mammalian Kindlins protein is about 75 kDa.Till now,three sub members,kindlin-1,Kindlin-2 and kindlin-3,have been identified.Kindlins are kind of very important factors on regulating the activation of integrin and adhesion of cells to extracellular matrix(ECM).The mainly effect of integrin family is mediate cell-extracellular matrix adhesion.Current studies have demonstrated that Kindlins play an important role on cell adhesion by interacting with integrin and involved in neovascularization,dermatological diseases,and the function of immune system,as well as tumor invasion and drug resistance of cancer cells.Serving as signaling molecule,Kindlins participate in regulation of cell adhesion,migration,proliferation and differentiation.The C-terminal of Kindlin-2 containing FERM domain structure,(F1,F2 and F3 three substructure domain),the F2 substructure domain interrupted by a PH domain.F3 substructure domain combined with cytoplasmic tails of β1 and β3 integrin.Kindlin-2 together with Talin plays a role in activation of integrin.The N-terminal of Kindlin-2 regulated actin dynamics and intracellular signaling pathways directly or indirectly by connecting with Integrin Linked Kinase(ILK)and Migfilin.Kindlin-2 is an essential component of focal adhesions which are the important structures to mediated epithelial cells combined with extracellular matrix.Kindlin-2 recruits Migfilin and Filamin to focal adhesions,and involved in the related signal transduction.As reported in other studies,the mice dead in 7.5 days in embryonic period by knockout Kindlin-2,which cause serious separation of the ectoderm and endoderm.Kindlin-2 localized in focal adhesions as cytoskeleton protein,connecting the integrin to the actin cytoskeleton and influence cell migration,and even the progression of cancer.Some studies shown that Kindlin-2 was upregulated in malignant melanoma,and enhanced the cell adhesion and migration.In mesothelioma and human endothelial cells,Kindlin-2 can advance the cell movement and invasion.Moreover,knockout or down-regulation of Kindlin-2 inhibits the abilities of gastric cancer cells,epithelial,fibroblasts and esophageal squamous carcinoma cells migration.Some papers claimed that the change of Kindlin-2 expression is related to the occurrence and development of some kinds of tumor.Kindlin-2 protein was highly expressed in breast cancer,malignant mesothelioma,gastric cancer,leiomyomas and esophageal squamous cancer.Kindlin-2 was expressed in leiomyosarcoma at a low level,while was highly expressed in leiomyoma.Down-regulation of Kindlin-2 by using siRNA inhibits the migration and invasion of cancer cells by regulating integrins.Gong et al.reported that the correlation with Kindlin-2 and cisplatin in the resistance of prostate cancer.Kindlin-2 highly expressed cell line tolerate to cisplatin,while Kindlin-2 knockout cell lines present a high sensitivity to cisplatin treatment.Clinical studies have shown that Kindlin-2 can be used to assess prognosis and grading of malignant degree of breast cancer,malignant mesothelioma,gastric cancer,dark skin cancer and pancreatic cancer.Kindlin-2 may provide a promising target for the therapy of cancer.However,the mechanism of Kindlin-2 regulates the occurrence,invasiveness and metastasis of tumor is still unclear.Based on the above understanding,we design the experiments to screen out the new binding proteins of kindling-2.We used the specific Kindlin-2 antibody to pulldown the split product of prostatic cancer PC-3 cells.The sample was analyzed by 2-D DIGE,the result indicated Prdx4,which belongs to Prdx family was the potential interaction protein of Kindlin-2.PRDXs are a multi-functional protein family widely expressed in biological body.The molecular weight of PRDXs is about 20-30 kDa.Peroxidase protein Prdx4 played an important role in oxidation-reduction reaction.The mechanism of PRDXs is the substrate of catalase oxidase a hydrogen peroxide specific-Cys-SPH residue of family member.According to the amount of cysteine residues which involve the redox reaction,the family was divided into three kinds:typical 2-Cys-Prdx(Prdx1-4)、atypical 2-Cys-Prdx(Prdx5)and 1-Cys-Prdx(Prdx6).PRDXs involved in different biogenesis,including Oxidant to detoxify,cell proliferation,cell differentiation and gene expression.Furthermore,more and more evidences proved the expression of Prdx4 was related to occurrence and development of tumor,even someone suggested regarding Prdx4 as the biomarker of prostatic cancer.Base on some studies,Prdx4 was upregulated in lung cancer.Prdxl and Prdx4 were highly expressed in bladder cancer and lung cancer tissue,Prdx3 and Prdx4 were highly expressed in prostate cancer organizations.Meanwhile,When Prdx4 was down-regulated or knockout,the growth of gastric cancer was restricted and result in spermatophore apoptosis.In previous studies,we use co-immunoprecipitation and the binding assay to investigate the interaction between kindlin-2 and prdx4.Moreover,confocal microscopy shows stress-induced co-localization of kindlin-2 and prdx4.In conclusion,we speculate that the interaction between kindling-2 and Prdx4 may play a regulating role in the occurrence and development of tumor.Through the researches of the interaction between them,we have better understanding of the occurrence and development of tumor,and may provide new way to the therapy of tumor.As the importance of the interaction between Kindlin-2 and Prdx4,we designed and fulfilled the following experiments:To investigate the effect of Kindlin-2 protein on the migration of human prostate cancer,we tested the expression level of Kindlin-2 and Prdx4 in several cell lines.Western blotting shown,the highest expression of Kindlin-2 and lowest expression of Prdx4 was in PC-3 cells,so we chose PC-3 cells to built Kindlin-2 shRNA PC-3 stable expression cells lines or HA-Prdx4 PC-3 stable expression cell lines.The expression of Kindlin-2 protein was down-regulated in Kindlin-2 shRNA PC-3 cell lines,whereas the expression of Prdx4 protein was up-regulated in HA-Prdx4 PC-3 cells lines,which was confirmed by western blotting.Prdx4 protein expression was not chang when Kindlin-2 protein was down-regulated by shRNA.This suggests Kindlin-2 with Prdx4 play a physiological function through the relation between proteins,but not on gene transcriptional level.The function of Kindlin-2 and Prdx4 was investigated in transwell migration and invasion assays.When Kindlin-2 was knocked down by shRNA,the migration of PC-3 cells were remarkably inhibited compared with control group(P<0.01).The migration was significantly increased in Prdx4 over-expressed PC-3 cells compared with control group(P<0.05).The invasion was remarkably inhibited in Kindlin-2 shRNA PC-3 cells compared with control group(P<0.01).The invasion was significantly increased in Prdx4 over-expressed PC-3 cells compared with control group(P<0.01).The significant reduction of migration and invasion in Kindlin-2 shRNA PC-3 cells suggested that Kindlin-2 may play an important role in the transformation of PC-3 cells in vivo.To test this hypothesis,Control shRNA PC-3 and Kindlin-2 shRNA PC-3 were separately injected into two groups of the left armpit of nude mice.Four weeks later,the group injected with Control shRNA PC-3 cells showed symptoms of developing tumors,such as loss of body weight and lack of activity.Furthermore,the sizes of tumor in Kindlin-2 shRNA PC-3 cells were significantly smaller than control mice.Therefore,these observations indicated that knockdown of Kindlin-2 inhibited the growth of tumor in a mouse model.Finally,Immunohistochemistry was performed to detect the expression of Ki67 and Caspase 3 levels in two groups of tumor tissues.The expression of Ki67 in Kindlin-2 shRNA PC-3 group was dramatically repressed,and the expression of Caspase 3 was significantly increased compared with control group.The metastasis of lymph node in Kindlin-2 shRNA PC-3 group was repressed.Furthermore,Plasmid pcDNA3.0-HA-Prdx4 was transfected into Hela cells,then the Hela cells were infected with LV-Prdx4 RNAi vector to establish a stable Prdx4 shRNA Hela cells lines.The change in the expression of Prdx4 protein was validated by western blotting.The expression of Prdx4 protein was up-regulated in Hela cells after transfection with pcDNA3.0-HA-Prdx4 plasmid(P<0.05),whereas it was down-regulated in Prdx4 shRNA Hela cells(P<0.05).The wound healing assay,transwell migration and invasion assays were performed to detect the migration and invasion of Hela cells,respectively.The ability of migration and invasion was significantly increased in Prdx4 over-expressed Hela cells(P<0.01).When Prdx4 was knocked down by shRNA,the migration and invasion of Hela cells wereRemarkably repressed compared with control group(P<0.01).We can draw the following conclusions through the experiments:1.Knockdown of Kindlin-2 expression inhibit the migration and invasion of PC-3 cells.2.Up-regulation of Prdx4 facilitate the migration and invasion of PC-3 and Hela cells.3.Knockdown of Kindlin-2 expression reduces the formation of tumor,and represses the metastasis of lymph node.4.Down-regulation of Prdx4 inhibit the migration and invasion of Hela cells.