CXCL12/CXCR4-induced Invadopodia Nvadopodia Formation And Invasion Mediate Kinase In Glioma Cells Ediated By Arg

Invasive growth is one of the most prominent biological features of malignant gliomas, which basically leads to the spread of glioma cells in the Central Nervous System and causes the death. Recent studies show that during invasion and migration, actin is polymerized in the invasive tumor cells and form a membrane protrusion, called invadopodia. This structure can recruit and secrete related enzymes such as matrix metalloproteinases(MMPs) to degrade the surrounding extracellular matrix, promoting invasion and migration of tumor cells. It has been known that invadopodia plays a key role in the process of invasion and metastasis of tumor cells.During the formation of invadopodia, cortactin phosphorylation is very important for invadopodia maturation and the acquirement of its function. As one of the first discovered human oncogene, Src is known to have the ability to phosphorylates cortactin to regulate invadopodia maturation and to be fully functional. Recently, studies in breast carcinoma show that Arg, a non-receptor tyrosine kinase, can also work as a downstream target of activated Src, that is, phosphorylated by Src. More importantly, activated Arg is also able to phosphorylates cortactin. Compared with Src, Arg has been showed to act more strongly in the phosphorylation of cortactin. The first reason is that Arg has a stronger affinity for cortactin than Src. Secondly, with the effect of EGF(Epidermal Growth Factor), the Y272 autophosphorylation domain of Arg can be autophosphorylated, which is not affected by its upstream regulatory factors of Src. Therefore, Arg might be more critical than Src in the cortactin phosphorylation and its function is reasonable to be further studied in the process of invadopodia maturation and acquire its function.CXCL12(also known as stromal cell-derived factor-1, SDF-1) binds with chemokine receptor CXCR4(chemokine CXC motif receptor 4) in glioma cells, which induced glioma cells migration and invasion. Studies show that, CXCL12 has the ability to induce tumor cells to secrete matrix metalloproteinases. Based on above studies and our preliminary studies, we hypothesize that CXCL12 / CXCR4 may regulate invadopodia maturation through Arg-cortactin pathway, thereby inducing glioma cell invasion and migration.This study aims to investigate that Arg mediates CXCL12 / CXCR4-induced glioma cell invasion and its potential regulation mechanism, including the following four sections:1. To investigate the role of CXCL12/ CXCR4 in glioma cell invasion and migration. Transwell invasion assay and Invadopodia formation assay were used to analyze the effect of CXCL12/ CXCR4 on glioma cell invasion. The level of cortactin phosphorylation regulated by CXCL12 was determined with Western blotting. Compared with the control group, the invasiveness of glioma cells significantly increased after CXCL12 added, and invadopodia formation and the degraded area of matrix for each of the tumor cells also increased. By Western blotting, the amount of cortactin expression in tumor cells had no significant change after CXCL12 added, but the level of p Y-421 cortactin was up-regulated. AMD3100, an inhibitor specific to CXCR4, could decrease the invasiveness of glioma cells dramatically. It also reduced area of the matrix degraded by invadopodia for each of tumor cells and decreased the level of cortactin phosphorylation as well. In this study, the role of CXCL12 / CXCR4 was demonstrated in regulating the formation of invadopodia by upregulating the level of phosphorylated cortactin.2. The role of Arg in glioma cell invasion. The recombinant lentivirus was constructed for silencing Arg, the glioma cell line with Arg stably knocked down was established after transfected with recombinant lentivirus. Human U87 glioma cells were employed in this study and were assigned into three groups: Arg silenced by sh RNA group(sh RNA-ARG), sh RNA-negative control treated group(sh RNA-NC) and non-silencing sh RNA treated groups(sh RNA-N). q RT-PCR and western blotting were used to detect Arg expression at m RNA and protein level, respectively. Transwell invasion assay and invadopodia formation assay were used to determine the invasiveness of glioma cells. Co-Immunoprecipitation and immunofluorescence were used to observe the positional relationships between Arg and cortactin in glioma cells. The expression of cortactin and phosphorylated cortactin at tyrosine 421(p Y-421cortactin) were detected with western blotting. Results showed that, compared with the control group, the invasiveness of glioma cells in sh RNA-Arg group was reduced significantly, and the area of matrix degraded by invadopodia of each cell was also decreased. Double immunofluorescent staining revealed that Arg co-localized with cortactin at cell protruded membrane. At the meanwhile, the combination between cortactin and Arg in glioma cells was further demonstrated by Co-IP. The level of cortactin phosphorylation was significantly reduced in sh RNA-Arg group, detected by Western blotting. It was suggested that Arg functioned in regulating the invadopodia formation and matrix degradation. Silenced Arg expression could block cortactin phosphorylation and lead to inhibition of glioma cell invasion.3. Arg mediated CXCL12 / CXCR4-induced glioma cell invasion. In order to confirm that Arg can work as a downstream factor of CXCL12 / CXCR4, glioma cells with Arg stably knocked down were applied. Three groups were assigned: Arg KD(glioma cells with Arg stably knocked down) + CXCL12、Arg KD + PBS(Phosphate Buffer Solution) and Arg NC(glioma cells with original expression of Arg) + CXCL12. Transwell invasion assay and invadopodia formation assay were used to evaluate the invasiveness of each group. Western blotting was applied to detect the expression of cortactin and phosphorylated cortactin in each experimental group. Results showed that compared with glioma cells with original Arg expression, increased invasiveness induced by CXCL12 was abolished in Arg KD glioma cells. Moreover, CXCL12 could not induce cortactin phosphorylation in Arg KD glioma cells(P<0.05).In conclusion:1. CXCL12/CXCR4 was capable of inducing glioma cell invasion, and probably by regulating cortactin phosphorylation.2. Silenced Arg would inhibit glioma cell invasion for the sake of blocked cortactin phosphorylation.3. Arg mediates CXCL12/CXCR4-induced glioma cell invasion, CXCL12 / CXCR4 regulates invadopodia maturation through Arg-cortactin pathway, thereby inducing glioma cell invasion.