Objective:To examine the effect of pSer9-GSK-3β on breast cancer and to determine whether the underlying metabolic and immunological mechanism is associated with ROS/eIF2B and natural killer (NK) cells.Methods:We employed TWS119 to inactivate GSK-3β by phosphorylating Ser9 and explored its effect on breast cancer and NK cells.The expression of GSK-3β,natural killer group 2 member D (NKG2D) ligands,eIF2B was quantified by PCR and West blot.We measured intracellular reactive oxygen species (ROS) and mitochondrial ROS using DCFH-DA and MitoSOXTM probe,respectively,and conducted quantitative analysis of cellular respiration on 4T1 cells with mitochondrial respiratory chain complex Ⅰ/Ⅲ kits.Results:Our investigation revealed that TWS119 downregulated NKG2D ligands (H60a and Rae 1),suppressed the cytotoxicity of NK cells,and promoted the migration of 4T1 murine breast cancer cells.Nevertheless,LY290042,which attenuates p-GSK-3β formation by inhibiting the PI3K/Akt pathway,reversed these effects.We also found that higher expression of pSer9-GSK-3β induced higher levels of ROS,and observed that abnormality of mitochondrial respiratory chain complex Ⅰ/Ⅲ function induced the dysfunction of GSK-3β-induced electron transport chain,naturally disturbing the ROS level.In addition,the expression of NOX3 and NOX4 was significantly up-regulated,which affected the generation of ROS and associated with the metastasis of breast cancer.Furthermore,we found that the expression of pSer535-eIF2B promoted the expression of NKG2D ligands (Mult-1 and Rael) following by expression of pSer9-GSK-3β and generation of ROS.Conclusions:The PI3K/Akt/GSK-3β/ROS/eIF2B pathway could regulate NK cell activity and sensitivity of tumor cells to NK cells,which resulted in breast cancer growth and lung metastasis.Thus,GSK-3β is a promising target of anti-tumor therapy.