Gab2 Activates Breast Cancer Stem Cells And Plays Key Role In HER2-induced Drug Resistance

Objective:HER2 is a member of the epidermal growth factor receptor family.HER2 is overexpressed in 20%-30% breast cancer patients,especially plays a key role in invasive breast cancer.HER2 has been implicated in inducing mammary tumorigenesis as well as mediating aggressive tumor growth and metastasis.HER2 overexpression is related with poor prognosis and chemoresistance in breast cancer patients.Grb2-associated binding protein 2(Gab2)is a member of Grb2-associated binder family proteins(Gabs),which is a docking protein with highly conserved during evolution.Gab2 is amplified in numerous cancers,including breast cancer.Scaffold proteins play an important role in signaling cascade.Tyrosyl phosphorylated Gab2 can induce the activation of several signaling pathways,such as PI3K/AKT and MAPK/ERK.In this study,our results showed that Gab2 was upregulated in HER2-overexpressed breast cancer cells.Knocking down of Gab2 reduced the activity of PI3K/AKT and MAPK/ERK pathways and suppressed the cell proliferation.Knocking down of Gab2 reversed the tamoxifen resistance of HER2-overexpressed breast cancer cells.Furthermore,Gab2 ablation significantly decreased the stemness of breast cancer cells.Our data first indicated that Gab2 served as an important regulator of breast cancer stem cells in HER2-overexpressed breast cancer.Thus,it suggests that Gab2 may be a potential therapeutic target for HER2-overexpressed breast cancer.Methods:(1)Using Real-time PCR and Western blot assay to validate the expression of m RNA and protein in T47D-control and T47D-HER2 cell lines.(2)Western blot,Colone formation and MTT technology were used to examine the cell proliferation when knocking down of Gab2 in HER2-overexpressed breast cancer cells.(3)Using Sphere formation assay,Real-time PCR and Western blot to detect the effect of Gab2 on the stemness of breast cancer cells.(4)Constructing animal model to validate the effect of Gab2 on tumor growth in vivo.(5)Using MTT and Colone formation assay to detectthe viability of T47D-control,T47D-HER2 and T47D-HER2-sh Gab2 cells after tamoxifen treatment.Results:(1)Using Real-time PCR and Western blot assay,we identified the m RNA and protein levels of Gab2 were overexpressed in HER2 overexpression breast cancer cells.(2)Knocking down of Gab2 can suppress the activity of PI3K/AKT and MAPK/ERK pathways.Knocking down of Gab2 can also inhibit cell proliferation.(3)Gab2 ablation suppressed the stemness of HER2 overexpression breast cancer cells.(4)In vivo,knocking down of Gab2 could inhibit the growth of tumor.(5)Knocking down of Gab2 can reverse the tamoxifen resistance of HER2 overexpression breast cancer cells.Conclusions: This study unveiled a potential function of Gab2 in breast cancer stem cells(BCSCs)in HER2-overexpressed breast cancer cells.Knocking down of Gab2 suppressed the stemness of breast cancer cells.Downregulation of Gab2 also impressed the activity of MAPK/ERK and PI3K/AKT pathways,decreased cell proliferation and clonogenicity ability.Gab2 ablation reversed tamoxifen resistance of HER2-overexpressed breast cancer cells.These findings indicated that Gab2 might be a potential target in the clinical therapy of HER2-overexpressed breast carcinoma.