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

GSK-3β upregulated the expression of NKG2D ligands. PCR (A) and Western blot (C) were performed in 4T1 cells to determine the expression of H60 and Rae1 or Qa-1 after treatment with inhibitor TWS119 (2.5 μmol/L). (B) Quantitative analysis of the PCR in 2A. PCR (D) and Western blot (F) were performed in 4T1 to determine the expression of H60 and Rae1 after treatment with GSK-3β activator, LY294002 (30 μmol/L). (E) Quantitative analysis of the PCR in 2D. *, ** indicate values of P < 0.05, P < 0.01, respectively.

GSK-3β improved NK cell cytotoxicity and suppressed migration of 4T1 cells by upregulating the expression of NKG2D. After the treatment with TWS119 and LY294002, the expression of NKG2D in primary NK cells (A) and in mice NK cell line KY-1 (B) was assessed by RT-PCR and Western blot. (C) The cytotoxicity of NK cells was measured by Calcein AM-release assay. (D) The migration ability of 4T1 cells was detected by transwell migration assay. The migrated cells were quantified for 4 random fields. *, ** indicate values of P < 0.05, P < 0.01, respectively.

GSK-3β enhanced NK cell function by attenuating the intracellular ROS production. (A) ROS levels of 4T1 cells, treated with TWS119 and LY294002, were determined by flow cytometry. (B) The expression of H60 and Rae1 was analyzed by RT-PCR and real time-PCR after H₂O₂ (250 μmol/L) and NAC (5 mmol/L) treatment. (C, D, E) The expression of NKG2D, the cytotoxicity of NK cells, and IFN-γ concentration was evaluated by RT-PCR, real time-PCR, Calcein release assay, and ELISA. *, ** indicate values of P < 0.05, P < 0.01, respectively.

Attenuating mitochondrial ROS was mainly responsible for the improved function of NK cells after GSK-3β treatment. (A) Mitosox^TM fluorescence probe was used to observe mitochondrial ROS activity after treatment with TWS119, LY294002, H₂O₂, and NAC. (B) The expression of NOX1-4 was amplified by RT-PCR and NOX4 levels were further analyzed by Western blot. (C) The activity of MRCC I and III in 4T1 cells was determined by a multiscan spectrum microplate spectrophotometer. *, ** indicate values of P < 0.05 and P < 0.01, respectively.

eIF2B functions as a downstream molecule of PI3K/AKT/GSK-3β/ROS pathway to upregulate NKG2D ligands. (A) eIF2B, CCND1, and NFAT1 levels were investigated by RT-PCR. (B) mRNA levels of GSK-3β and eIF2B, protein levels of pSer9-GSK-3β, pSer535-eIF2B, H60, and Rae1 were quantified. (C) The expression of p-eIF2B induced by H₂O₂, NAC, and SOD (1,000 U/mL) as well as TWS119, LY294002, and Wortmannin (2 μmol/L, another inhibitor of PI3K/AKT) was detected by Western blot. (D) The protein levels of p-GSK-3β, p-eIF2B, H60 and Rae1 expressed in tumor tissue homogenate after intraperitoneal injection of TWS119 and LY294002 (5 mg/kg) was assayed by Western blot. (E) Consecutive sections of above tissues were stained by DAB. The mean densities in the positive areas were quantitatively by the IPP software. *, indicates values of P < 0.05.