The PI3K/Akt/GSK-3β/ROS/eIF2B pathway promotes breast cancer growth and metastasis via suppression of NK cell cytotoxicity and tumor cell susceptibility

Objective: To examine the effect of pSer9-GSK-3β on breast cancer and to determine whether the underlying metabolic andimmunological 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 NKcells. The expression of GSK-3β, natural killer group 2 member D (NKG2D) ligands, eIF2B was quantified by PCR and Western 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 complexI/III kits. Results: Our investigation revealed that TWS119 downregulated NKG2D ligands (H60a and Rae1), suppressed the cytotoxicity ofNK cells, and promoted the migration of 4T1 murine breast cancer cells. Nevertheless, LY290042, which attenuates p-GSK-3βformation by inhibiting the PI3K/Akt pathway, reversed these effects. We also found that higher expression of pSer9-GSK-3βinduced higher levels of ROS, and observed that abnormality of mitochondrial respiratory chain complex I/III function inducedthe dysfunction of GSK-3β-induced electron transport chain, naturally disturbing the ROS level. In addition, the expression ofNOX3 and NOX4 was significantly up-regulated, which affected the generation of ROS and associated with the metastasis of breastcancer. Furthermore, we found that the expression of pSer535-eIF2B promoted the expression of NKG2D ligands (Mult-1 andRae1) 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.