Girdin Mediates The Invasion Of Lung Cancer Cells Via The HGF/MET Signaling Pathway

Lung cancer is one of the most malignant tumors in the world,ranked first in cancer-related death causes.Non-small cell lung cancer(NSCLC)accounts for80%-90% of all lung cancer cases.In recent years,there is an upward trend in global morbidity and mortality of lung cancer,especially in developing countries including China.Girdin is a non-receptor guanosine exchanging factor(GEF)that regulates the GTP enzyme activity of the heterotrimeric GTP binding protein,contributing cell invasion,angiogenesis,and the development of the tumor.Previous studies have confirmed that the expression of Girdin is high in a wide variety of tumor cells,but the reports on the roles of Girdin in lung cancer pathogenesis are relatively fewer.Aim: This study aims to study the role of Girdin in the HGF-induced invasion of the lung cancer cells and explore the underlying molecular mechanism.Methods:(1)We used immunohistochemistry to explore the correlation between Girdin and c-MET,a receptor for HGF;(2)We demonstrated the significance of MET in lung cancer pathogenesisusing bioinformatic analysis;(3)Western blot was performed to detect the downstream signaling transductions of the receptor;(4)Wound healing assay and Transwell assaywere used to estimate the role HGF/MET signaling pathway in cells migration and invasion,respectively;(5)Girdin sh RNA plasmid was transfected into cells to downregulate the expression of Girdin and Western blot was used to examine the effect of Girdin downregulation on HGF/MET signaling pathway;(6)Wound healing assay and Transwell assay were performed to detect the cells migration and invasion ability after Girdin sh RNA transfection;(7)We performed immunofluorescence to detect the subcellularlocalization of Girdin and MET in cells;(8)The immunoprecipitation assay was performed to investigate the interaction between MET and Girdin;(9)The expression of the endogenous Girdin protein was knocked down by Lentivirus-mediated sh RNA method,and a rescue experiment was performed to explore the effects of over-expression of Girdin WT(wild-type)and Girdin FA(mutant type)on HGF/MET signaling pathway and cell invasion of lung cancer cells.Results:(1)Both MET and Girdin were highly expressed in malignant lung cancer sections;(2)Patients with both high MET and Girdin had significant lower surviving rate than those with low MET or Girdin expression;(3)HGF triggered auto-phosphorylation of MET and initiated the downstream signaling pathway including Akt phosphorylation and Erk phosphorylation;(4)HGF induced lung cancer cells migration and invasion ability;(5)After the decrease of Girdin expression,thephosphorylation level of the downstream Akt decreased.;(6)Girdin downregulation sharply inhibited cells migration and invasion ability;(7)HGF induce co-localization between Girdin and MET on the plasma membrane,and the interaction between Girdin and MET is enhanced;(8)The GEF function of Girdin is essential for the role of Girdin in lung cancer cell invasion as the F1685 A mutant of Girdin failed to mediate the HGF/MET signaling and HGF-induced invasion.Conclusions: Our study proved that Girdin mediates HGF-induced lung cancer cells migration by affecting HGF/MET downstream signaling pathway.Therefore,our results represent an attractive target site for designing therapeutic agent to disrupt MET/Girdin interface for lung cancer treatments.