SAHA Upregulats The Expression Of MHC-I Molecules And Enhance Anti-Tumor Immune Response In NSCLC Cells By Activating STAT1 And Smad2/3 Signals

Objectives: Low expression of MHC-I molecules is an important mechanism of tumor immune escape.Our previous study found that histone deacetylase inhibitor SAHA(Suberoylanilidehydroxamicacid)can significantly up-regulate the expression of MHC-I molecules on the surface of non-small cell lung cancer(NSCLC)cells.It is speculated that SAHA can enhance the immune response to anti-NSCLC tumor.In this study,we will explore the mechanism of SAHA up-regulating the expression of MHC-I molecules and observe the effect of SAHA on anti-tumor immune response in vivo and in vitro,in order to provide new therapeutics ideas for NSCLC.Methods:(1)Human lung cancer A549,NCI-H520 cells and mouse lung cancer Lewis cells were treated with SAHA.The expression of MHC-I,CD80 and CD86 was detected by flow cytometry.Western blot was used to detect the expression of STAT1,Smad2/3,MHC-I,P-STAT1 and P-Smad2/3,as well as the nuclear / cytoplasmic ratio of P-STAT1 and P-Smad2/3.(2)siRNA down-regulated the expression of STAT1 and Smad2/3,and the expression of MHC-I in tumor cells stimulated by SAHA was detected by Westernblot and flow cytometry.(3)C57BL/6 mice were subcutaneously inoculated with Lewis cells to establish lung cancer model,and intratumoral injection of SAHA or PBS,was performed with or without intraperitoneal injection of pathway inhibitors(Nifuroxzaide and ITD-1).The tumor volume was measured and calculated.(4)Intratumoral injection of SAHA significantly increased the proportion of CD8+T and IFN-γ + CD8+T cells in TIL,and blocked STAT1 and Smad2/3 pathway,the increase of CD8+T and IFN-γ + CD8+T cells decreased significantly.(5)C57BL/6 mice were immunized with Lewis cells.Splenic CD8+T cells were sorted by magnetic beads and co-cultured with SAHA-pretreated or untreated Lewis cells.The proliferation of CD8+T cells and apoptosis of Lewis cells were explored by flow cytometry.Results:(1)After SAHA treatment,the expressions of MHC-I,CD80 and CD86 on the surface of A549,NCI-H520 and Lewis cells were significantly up-regulated.Moreover,the expression levels of STAT1,Smad2/3,P-STAT1,P-Smad2/3 and the nuclear /cytoplasmic ratio of P-STAT1 and P-Smad2/3 were significantly increased.(2)Blocking STAT1 and Smad2/3 signals by siRNA can significantly inhibit the expression of MHC-I molecules induced by SAHA.(3)Intratumoral injection of SAHA markedly inhibited tumor growth,and blocking STAT1 and Smad2/3 pathways obviously weakened the anti-tumor effect mediated by SAHA.(4)Intratumoral injection of SAHA significantly increased the proportion of CD8+T and IFN-r+CD8+T cells in TIL,while blocking STAT1 and Smad2/3 pathway,(5)The proliferation ability of Lewis cells pretreated with SAHA to stimulate CD8+T cells(derived from mouse spleen immunized with Lewis cells)was significantly enhanced,and the apoptosis rate of Lewis cells increased significantly after co-culture.Blocking the signal pathway of STAT1 and Smad2/3 can reverse the above phenomenon.Conclusion:(1)SAHA up-regulates the expression of MHC-I molecules on the surface of non-small cell lung cancer cells by activating STAT1 and Smad2/3 signals,and enhances the anti-tumor immune response mediated by CD8+T cells.(2)SAHA is expected to be used as an adjuvant therapy for non-small cell lung cancer.