The Effects Of EGFR-TKIs And Sanguinarine On MHC-? In NSCLC And The Possible Mechanism

ObjectiveLung cancer is the most common cancer in China.86%of lung cancer patients in China are non-small cell Lung Cancer(NSCLC).The theory of driving gene mutation and the successful development of targeted therapy drugs have put forward a new concept of precise medical treatment for NSCLC.Epidermal growth factor receptor(EGFR)mutation accounts for 28%of NSCLC driving gene mutation subtypes in China(statistics of 2015).Tyrosine Kinase Inhibitors(TKIs)targeting EGFR are currently the preferred drug for NSCLC patients with EGFR mutations.However,due to the progress of TKIs resistance,all three generations of approved EGFR-TKIs have developed drug resistance.The targeted therapies have not fundamentally improved the long-term survival rate of NSCLC patients with EGFR mutations.In recent years,the emergence of immune checkpoint inhibitory drugs has brought dawn for the treatment of advanced tumors,in which PD-1/PD-L1 has made significant breakthroughs in the treatment of some kinds of tumor,and the long-term survival rate has been greatly improved.However,the insensitivity to anti-PD-1/PD-L1 agents in NSCLC patients with mutated EGFR has restricted the application of anti-PD-1/PD-L1 therapy in NSCLC patients with mutated EGFR.Recently,It has been reported that MHC-I molecule in EGFR mutant tumors is down-regulated and correlated with EGFR mutation status,which may partly explain why patients with EGFR mutation are insensitive to PD-1/PD-L1 antibody.In this study,we found that the mutant activation of EGFR affects the membrane expression of MHC-I and antigen presentation system in clinical specimens and in vitro experiments.Since several clinical trials of TKIs combined with anti-PD-1/PD-L1 drugs were discontinued due to serious adverse reactions,our findings may provide a theoretical basis for the combination of anti-PD-1/PD-L1 drugs and AKT/mTOR pathway inhibitors which are downstream of EGFR to improve the efficacy of immunotherapy,and then discussed the feasibility of the combination of drugs.Sanguinarine is a natural compound derived from Papaveraceae purpura and celandine.Both purpura and celandine have curative effect on clearing toxin and soothing itchy skin,suggesting that their ingredients may affect immune function.In recent years,it has been reported that Sanguinarine has anti-tumor effects in various tumor cell lines and animal cancer models[6],but its anti-tumor mechanism is not very clear.In this study,through the computer molecular simulation,immunology and molecular biology experiments,it is proved that Sanguinarine can inhibit the activation of proto-oncogenes EGFR and AKT,which in turn affects the expression of MHC-I molecules,suggesting that it may enhance the efficacy of immunotherapy for lung cancer.Methods1.Immunohistochemical staining of CD8 and HLA-ABC was performed in specimens of 34 patients with EGFR mutation and 20 patients with wild type EGFR.Immunohistochemical staining method are described later in the text.After staining,Hscores were obtained from two pathologists scoring and computer applications(ImageJ,Image-Pro Plus)were used to compute the gray density.2.The stable expression cell line was constructed by transferring EGFR mutant plasmid into normal human pulmonary epithelial cell line Beas-2B.The relative expression of MHC-I,PD-L1 on membrane activation were detected by Western Blot,flow cytometry and immune-fluorescence.The effects of EGFR-TKIs on the expression and distribution of MHC-I in NSCLC cell lines were detected by WB,flow cytometry and qRT-PCR.3.By applying EGFR downstream signaling pathway inhibitors in cell lines,the effects of downstream signaling pathway inhibitors on the expression and membrane distribution of MHC-I were detected by WB,flow cytometry and qRT-PCR.4.The effects of Sanguinarine on the expression of MHC-I molecules were verified by computer aided molecular docking,immunological and molecular biological experimental methods.Results1.Immunohistochemical staining results of tissue specimens:CD8+staining showed that there was no statistical difference in the number of CD8+T cells infiltrated between wild-type EGFR group and mutant group;HLA-ABC staining showed that there was no statistical difference in the gray scale of HLA-ABC staining between wild-type EGFR group and mutant group.The membrane expression of HLA-ABC in mutant EGFR group was significantly lower than that in wild-type group(P<0.05).2.When the normal lung epithelial cell lines transfected with mutant EGFR,MHC-I on the cell membrane was decreased and PD-L was increased(P<0.05).In NSCLC cell lines,MHC-I on the surface of cell membrane was increased in the EGFR-TKIs treatment group compared with control group(P<0.05).And in NSCLC cell lines,EGFR-TKIs increase the expression of proteasome core enzymes PSMB8/9 and transporter-associated with antigen processing(TAP2)which are required for antigen production and presentation.3.When NSCLC cell lines were treated with AKT/mTOR inhibitors,MHC-I on cell membrane surface increased significantly compared with control group(P<0.05).The effect of increasing MHC-I in cell membrane was relieved when PSMB8/9 inhibitor was applied simultaneously,and the difference was statistically significant(P<0.05).AKT/mTOR inhibitors increase the expression of proteasome core enzymes PSMB8/9 and transporter-associated with antigen processing(TAP2).4.The Molecular Dockings showed that Sanguinarine has a higher affinity for mutated EGFR,and its affinity for AKT is also higher.By binding to two proto-oncogene proteins,the activation of these two kinases is inhibited,results in up-regulating MHC-I expression and membrane transportation.Conclusion1.In tissue specimens,the infiltration levels of CD8+T cells in wild-type EGFR group and mutant group are similar,and there is no significant difference in HLA-ABC expression in wild-type EGFR group and mutant group.Compared with the wild-type group,the expression of HLA-ABC membrane in mutant EGFR group decreases significantly.2.Activation of EGFR in normal pulmonary epithelial cell lines can reduce MHC-I and increase PD-L1 on the cell membrane.In NSCLC cell lines,the application of EGFR-TKIs increases MHC-I on cell membran.3.In NSCLC cell lines,the use of AKT/mTOR signaling pathway inhibitors in downstream EGFR signaling pathway can increase MHC-I on the cell membrane,and the effect of AKT/mTOR inhibitors is discharged when PSMB8/9 inhibitors are applied simultaneously.4.By inhibiting the activation of EGFR and AKT,Sanguinarine increases the expression and membrane distribution of MHC-I molecules.