B7-H3 confers stemness characteristics to gastric cancer cells by promoting glutathione metabolism through AKT/pAKT/Nrf2 pathway

Background:Cancer stem-like cells(CSCs)are a small subset of cells in tumors that exhibit self-renewal and differentiation properties.CSCs play a vital role in tumor formation,progression,relapse,and therapeutic resistance.B7-H3,an immunoregulatory protein,has many protumor functions.However,little is known about the mechanism underlying the role of B7-H3 in regulating gastric cancer(GC)stemness.Our study aimed to explore the impacts of B7-H3 on GC stemness and its underlying mechanism.Methods:GC stemness influenced by B7-H3 was detected both in vitro and in vivo.The expression of stemness-related markers was examined by reverse transcription quantitative polymerase chain reaction,Western blotting,and flow cytometry.Sphere formation assay was used to detect the sphere-forming ability.The underlying regulatory mechanism of B7-H3 on the stemness of GC was investigated by mass spectrometry and subsequent validation experiments.The signaling pathway(Protein kinase B[Akt]/Nuclear factor erythroid 2-related factor 2[Nrf2]pathway)of B7-H3 on the regulation of glutathione(GSH)metabolism was examined by Western blotting assay.Multi-color immunohistochemistry(mIHC)was used to detect the expression of B7-H3,cluster of differentiation 44(CD44),and Nrf2 on human GC tissues.Student’s t-test was used to compare the difference between two groups.Pearson correlation analysis was used to analyze the relationship between two molecules.The Kaplan-Meier method was used for survival analysis.Results:B7-H3 knockdown suppressed the stemness of GC cells both in vitro and in vivo.Mass spectrometric analysis showed the downregulation of GSH metabolism in short hairpin B7-H3 GC cells,which was further confirmed by the experimental results.Meanwhile,stemness characteristics in B7-H3 overexpressing cells were suppressed after the inhibition of GSH metabolism.Furthermore,Western blotting suggested that B7-H3-induced activation of GSH metabolism occurred through the AKT/Nrf2 pathway,and inhibition of AKT signaling pathway could suppress not only GSH metabolism but also GC stemness.mIHC showed that B7-H3 was highly expressed in GC tissues and was positively correlated with the expression of CD44 and Nrf2.Importantly,GC patients with high expression of B7-H3,CD44,and Nrf2 had worse prognosis(P=0.02).Conclusions:B7-H3 has a regulatory effect on GC stemness and the regulatory effect is achieved through the AKT/Nrf2/GSH pathway.Inhibiting B7-H3 expression may be a new therapeutic strategy against GC.