The Effect Of Egfrv â…¢ On Human Glioma Cell Migration And Its Molecular Mechanism

Glioblastoma multiform (GBM) is a highly malignant, lethal primary brain tumor. The therapeutic efficacy of surgery is not ideal because it infiltrates the surrounding normal tissue, thus GBMs have also associated with poor prognosis and short life expectance. One significant characteristic of GBM(with a frequency of about 40%-50%) is Epidermal growth factor receptor (EGFR) gene amplification, mutation and re-arrangement, among which Epidermal growth factor receptor variant III (EGFRvIII) is the most common that has been associated with cell migration of GBM cases. EGFRvâ…¢is the derangement in the extracellular domain of EGFR, which is also referred to as AEGFR or de2-7 EGFR (a deletion encompassing exons 2-7 of EGFR 801 bp base, then relink exon 1 and 8 to form a glycine, Gly), resulting in an in-frame deletion of 267 amino acids in the extracellular domain. Thus, EGFRvIII is ligand-independently autophosphorylated, conferring the abnormal cellular behaviors of GBMs including cell proliferation, invasion and migration. We have previously shown EGFRvâ…¢plays a role in promoting glioma cell migration. Still, the causal mechanism has not been elucidated. It is the main emphasis of our research.EGFRvâ…¢-promoted glioma cell migration is closely linked to high level of tyrosine phosphorylation in Focal adhesion kinase (FAK) at Y397. FAK is a non-receptor protein-tyrosine kinase and functions as an important regulator in cellular processes such as cell survival, proliferation, migration and invasion. In addition, FAK is also a regulator of glioma cell invasion. The inhibition of FAK could trigger cell death and block cell migration. And we have also previously shown that FAK is associated with cell growth and invasion in U87AEGFR cells. Extracellular signal-regulated kinase (ERK1/2) is a crucial downstream signaling molecule of FAK. As an important signaling molecule of the RAS/Mitogen-activated protein kinase (MAPK) family, ERK may enter the cell nucleus when it is activated to upregulate transcription factors and affect cell migration. Furthermore, malignant human gliomas are pathophysiologically characterized by their insidious infiltration of the brain resulting from activating ERK 1/2 expression and secretion. It is known that EGFRvIII constitutively activates the ERK pathway in human glioma cells. Many antineoplastic drugs used clinically target the inhibition of the ERK pathway. The study was mainly to explore whether the activation of FAK and its downstream molecules would mediate the cell migration enhanced by EGFRvIII.In all of this study, we first stably designated transfectants which we transfected the plasmid of GFP, the mutation plasmid of AEGFR and wtEGFR into human glioma U87 cell line, respectively, as U87GFP, U87AEGFR and U87EGFR cells. We found that 1) phosphorylation levels of both FAK (FAK pY397) and EGFR (EGFR pY1068) were increased in U87AEGFR cells but not in U87 and U87GFP cells and 2) FAK RNAi can dramatically inhibit cell migration in U87AEGFR cells. The data were also consistent with those in U87EGFR cells. To investigate the mechanism of EGFRvIII-promoted cell migration, we tested the hypothesis that EGFRvâ…¢could stimulate the phosphorylation of FAK by binding to FAK, and that the resulting activation of FAK and its downstream molecules involved in the EGFRvâ…¢-enhancing cell migration. We demonstrated that EGFRvâ…¢formed a complex with FAK using Immunoprecipitation and Immuncfluorescence microscopy. In order to explore how the complex formation of EGFRvâ…¢and FAK affected downstream molecules, we detected ERK, AKT, JNK and their phosphorylation level after treating with FAK RNAi, we found that the knockdown of FAK expression reduced the phosphorylation level of ERK 1/2. Furthermore, we have performed in vitro cell migration to make sure the role of ERK1/2 in the EGFRvIII stimulated glioma cell migration. We treated U87AEGFR cells using U0126 (MEK1/2 inhibitor) for 1h and the assays confirmed that cell migration in U87AEGFR cells were inhibited by 68%, which demonstrated that the role of ERK1/2 was involved in the EGFRvIII-FAK complex promoted cell migration signaling pathway.In conclusion, the results in our study indicate that EGFRvâ…¢forms a complex with FAK, resulting in enhanced phosphorylation level of FAK Y397 and EGFR Y1068,then the EGFRvIII-FAK complex transduces the signal via ERK1/2 to promote glioma cell migration. These factors may act together to promote glioma cell migration in U87AEGFR cells, thus explaining in part the EGFRvâ…¢-promoted migration of glioma cells. Therefore, our data also provide insights into potential therapeutic targets for glioma via decreasing EGFRvâ…¢levels as well as its modification.