The Regulatory Roles Of CD147 In Tumor Cell Migration Through Cdc42 Signaling

Malignant tumor metastasis is the leading cause of death in cancer patients,accounting for more than 90% of tumor related deaths.The cell migration is a prerequisite for tumor metastasis and plays a vital role in the multistep invasion-metastasis process.Tumor migration ability is closely related to the change of cancer gene.CD147(Cluster of differentiation 147),belongs to the immunoglobulin superfamily,is a highly glycosylated transmembrane protein.CD147 is widely expressed in many tissues and cells,especially in a variety of malignant tumor tissues and cell lines,and is involved in the adhesion and metastasis of tumor cells.The present study suggests that the formation of filopodia is prerequisite for cell movement,filopodia are finger-like membrane structure,can provide adhesive sites for cells,allowing cells to attach with extracellular matrix(ECM),and generate a net force to drive the cell forward movement.However,the downstream signal conduction of CD147,as a transmembrane protein,especially its involvement in the filopodia formation correlation signal,are still not revealed.The activation of Cell division cycle 42(Cdc42)signal is the initiating intracellular step of filopodia formation.Cdc42 is a member of the Rho GTPase family.There is a cycle between active state(GTP-bound)and inactive state(GDP-bound)of Rho GTPase.Cdc42 induced filopodia formation via two different mechanisms.Convergent elongation model is one of the two mechanisms,which inducing filopodia formation dependent on the actin-related protein-2/3(Arp2/3)complex,and the alternate mechanism is called tip nucleation model,thus to promote migration and metastasis process involved in tumor cells.CD147 as a tumor with high expression of transmembrane protein,whether interacted with intracellular Cdc42 or regulated Cdc42 activation to initiate filopodia formation process,also has not been reported.To this end,our thesis systematically studied the interaction between cell membrane protein CD147 and filopodia formation signal initiating protein Cdc42,evaluate the regulation of CD147 on Cdc42 activation and formation of filopodia,and revealed phosphorylation sites of CD147 involved the activation of Cdc42 signaling,providing the structure basis for CD147 regulated tumor cells migration.On this basis,we made specific phosphorylation of CD147 antibody to detect the association of CD147 phosphorylation and the malignant degree of the tumors in the cell and tissue levels,in order to provide a new signal transduction mechanism for CD147 in tumor cell migration promotion.First,we revealed the relationship between CD147 and cell filopodia formation.The overexpression of CD147,promote the prostate cancer LNcap cells to form a large number of elongated filopodia,with significant higher number and length of filopodia than the control group.Further immunofluorescence staining proved that overexpression of CD147 promotes filopodia markers of Fascin1(localized all along the filopodia shafts)and mDia2(localized at filopodia tips)increased.These results suggested that the overexpression of CD147 induce filopodia formation.Meanwhile,through the transwell experiment,we tested the effect of CD147 on cell migration.Overexpressed CD147 cells migration to high concentration serum increased significantly,demonstrating that CD147 promotes cell migration and filopodia formation.Then,we examined the role of CD147 in the regulation of filopodia formation pathway associated proteins.We found that overexpression of CD147 promoted Cdc42 level,the upstream signal of the filopodia formation,increased the expression level of ArpC2 and N-WASP,downstream protein of Cdc42,and significantly increased the expression level of filopodia markers,Fascin1 and mDia2,suggesting overexpression of CD147 activates filopodia formation signal.Activated Cdc42 possessed the ability binding to CRIB domain of its downstream of PAK,so using GST-PAK-CRIB fusion protein we demonstrated the overexpression of CD147 significantly increased the levels of Cdc42-GTP,Cdc42 activation form,in the GST-pulldown experiments,suggesting that CD147 opens the molecular switch for Cdc42 activation.Further,through shRNA interference,we explored whether Cdc42 is involved in CD147 induced filopodia formation.We found that Cdc42 gene interference reversed the promotion effects of Cdc42 signaling pathway related proteins by CD147 overexpression,including ArpC2,N-WASP,Fascin1 and mDia2.Microscopic observations suggested that Cdc42 gene interference blocked the promotion effects of CD147 on the number and length of filopodia,which was close to the control group.Transwell experiments also proved that Cdc42 down-regulation reversed the roles of CD147 in promoting cell migration.These results suggested that Cdc42 is involved in CD147 induced filopodia formation,and CD147 can activate the Cdc42 signal.CD147,as a transmembrane glycoprotein of the immunoglobulin superfamily,its molecular structure is most of located in the extracellular and intracellular structure contains only 39 amino acids as cytoplasmic tail.While Cdc42 is mainly localized in the plasma membrane and Golgi apparatus,which also provides the premising condition for the interaction between Cdc42 and CD147.We further examined the interaction between these two proteins,and discussed the identification possibility of CD147 intracellular domain to Cdc42.In immunofluorescence staining in tumor cells,CD147 can colocalize with Cdc42.In CO-IP experiments,the immunoprecipitation of endogenous CD147 protein can co-precipitate endogenous Cdc42 and immunoprecipitation of overexpressed CD147 protein,can also co-precipitate endogenous Cdc42,suggesting CD147 and Cdc42 exist in the same complex.Through the GST-pulldown experiment,we detected the direct interaction between CD147 intracellular domain and Cdc42.The above results indicated that CD147 intracellular domain and Cdc42 has a direct biophysical interaction,provides a structural basis of CD147 for Cdc42 activation.CD147 cytoplasmic domain contains two serine phosphorylation sites,S246 and S252.Since protein phosphorylation is an important way to activate the protein,we further examined the related phosphorylation site for the involvement of CD147 in promoting cell migration and filopodia formation functions.We compared the functions between wild-type CD147 and phosphorylation site mutation of CD147 S246 A,S252A or S246/252 A.246A and wild type CD147 functioned similar,but S252 A or S246/252 A mutation strongly inhibited the formation of filopodia,suggesting that the S252 mutation change induction effects of wild-type CD147 on filopodia,suggesting that phosphorylation of S252 of CD147 play a key role in promoting filopodia formation.Through the GST--pulldown experiment,we detected S252 A and S246/252 A of CD147 mutant lost the direct interaction with Cdc42,suggesting that S252 is the key phosphorylation site to open the recognition of Cdc42 signal by CD147.On this basis we suggest that S252 phosphorylation site play a decisive role in CD147 induced filopodia formation and cell migration promotion process,we prepared CD147-S252 phosphorylated antibodies and tested the difference in CD147 protein S252 phosphorylation level among normal cells,low grade tumor cells and metastatic high malignant tumor cells.It was hard to detect CD147 S252 phosphorylation in normal and low malignant tumor cells,but significant increase in S252 phosphorylation CD147 proteins can be detected in high metastatic malignant tumor cells.With the enhancement of malignancy degree,the level of activated Cdc42 increased significantly.It is suggesting that S252 phosphorylation of CD147 is associated with tumor metastasis and Cdc42 activation.Through clinical samples,we also found the same trends.These results indicated that phosphorylation of CD147 determines the activation level of Cdc42,and then determines the degree of tumor malignancy.In summary,our study found that CD147 not only promotes the expression of Cdc42,but also direct interacts and activates Cdc42 signaling through its intracellular domain,further to induce the formation of filopodia,thereby to promote cancer cell migration.Serine 252 phosphorylation site in CD147 intracellular domain directly involves in activating and opening CD147/Cdc42 signal.Our results provide a new signal transduction pathway for CD147 targeted tumor metastasis,and provide new ideas and strategies for the treatment of cancer.