Role And Mechanism Of Gab2 In The Invasion And Metastasis Of Breast Cancer

Objective: In this study,we detected the proteins expression of Gab2,matrix metalloproteinases and epithelial-mesenchymal transition markers in breast cancer tissues and breast cancer cell lines MCF-7 and MDA-MB-231 by knocking down or overexpression the expression of Gab2 protein,to investigate the effects and mechanism of Gab2 in invasion and metastasis of breast cancer.Therefore to provide experimental evidence for breast cancer invasion and metastasis.Methods: Immunohistochemistry was applied to evaluate the expression of Gab2 in80 cases of histologically diagnosed breast cancer and adjacent non-tumor tissues and to analysis the difference expression of Gab2 in invasive breast cancer and adjacent non-tumor tissues and to analyse the correlation with clinicopathological parameters of breast cancer;To detect the proteins expression of MMP-2,MMP-9,E-cadherin and Vimentin in breast cancer and analysis the correlation between them.Western blot analysis was used to detected the expression of Gab2 in MDA-MB-231 cells and MCF-7 cells;a Stealth si RNAs against human Gab2 were transiently transfected into MDA-MB-231 cells and an overexpression of Gab2 plasmid transfected into MCF-7 cells,then Western blot analysis was used to detecte the proteins expression of MMP-2,MMP-9,E-cadherin and Vimentin in transfected cells,Transwell invasion assay was used to detecte the invasive ability of breast cancer cells with Gab2 knocking down or Gab2 overexpression;Western blot analysis was used to analysis the phosphorylation of ARK5,Akt and GSK-3β stimulating by EGF and the protein expression of Snail in breast cancer cells with Gab2 knocking down or Gab2 overexpression.Results: The expression of Gab2 protein in breast cancer was significantly higher than in adjacent non-tumor tissues(P=0.002);the expression of Gab2 closely correlated with ER,histological grade and lymph node metastasis(P=0.013,P=0.016,P=0.027);The expression of Gab2 protein was positive correlated with MMP-2,MMP-9 and Vimentin protein(P=0.001,P=0.001,P=0.026),negative correlated with E-cadherin protein(P=0.030).Gab2 expression was markedly higher in MDA-MB-231 cells compared with that in MCF-7 cells.The expression of Gab2 protein was decreased in MDA-MB-231 cells after transfection with interference RNA plasmid and the expression of Gab2 protein was increased in MCF-7 cells after transfection with Gab2 plasmid.Transwell invasion assay showed that invasion of MDA-MB-231 cells were dramatically reduced with Gab2 knocking down(P=0.012),Conversely,invasion of MCF-7 cells were enhanced with Gab2overexpression(P=0.007).Western blot revealed that E-cadherin expression significantly increased(P=0.000)and proteins expression of MMP-2,MMP-9 and Vimentin were decreased after Gab2 reduction(P=0.001,P=0.000,P=0.004),Conversely,Gab2 overexpression dramatically reduced E-cadherin protein expression(P=0.000),while enhanced the proteins expression of MMP-2,MMP-9and Vimentin(P=0.001,P=0.000,P=0.002);The expression of Snail and the phosphorylation of Akt,ARK5 and GSK-3β were inhibited in MDA-MB-231 cells with the stimulation of EGF after Gab2 reduction,Conversely,Gab2 overexpression dramatically enhanced Snail and phosphorylation of Akt,ARK5 and GSK-3β with the stimulation of EGF.Conclusion:(1)The expression of Gab2 protein in breast cancer was higher with lymph node metastasis that revealed Gab2 protein closely correlated with the invasion and metastasis of breast cancer.(2)Gab2 might be involved in the regulation of Akt-ARK5 signaling pathway,supporting a role of Gab2 in promoting breast cancer invasion and metastasis via MMP-2 and MMP-9 protein production.(3)Gab2 might influence the activation of GSK-3β and the expression of Snail to promot breast cancer invasion and metastasis by induce EMT.