| Background and ObjectionLung cancer is the most common cancer and the leading cause of cancer death worldwide.The epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKIs)could effectively extend the overall survival for certain percentage of patients with lung cancer.However,EGFR secondary mutation in threonine 790(T790M),MET amplification,human epidermal growth factor receptor 2(HER2)amplification,hepatocyte growth factor(HGF),activation of insulin-like growth factor 1 receptor(IGF-1R),Epithelial-Mesenchymal Transition(EMT),loss of phosphatase PTEN and other mechanisms induce drug resistance of EGFR-TKIs in lung cancer.Therefore,it is of great significance to find novel and effective treatments for patients with lung cancer,especially those who acquired resistance to EGFR-TKIs.As a check-point immune therapy,anti-PD-1/PD-L1 pathway therapies have made a great breakthrough in the treatment of a variety of malignant tumors.The NCCN guidelines for lung cancer in 2017 recommend the use of anti PD-1/PD-L1 therapy for tumors with high expression of PD-L1.What's more,the researches relating to the relationship between EGFR mutation and PD-L1 expression has become one of the frontier research topics in the treatment of lung cancer.It's reported that EGFR-TKIs could decrease the expression of PD-L1 in lung cancer with EGFR mutation,the changes of PD-L1 expression in lung cancer after acquired resistance to EGFR-TKIs,however,are rare reported.Whether EGFR-TKIs resistant patients would benefit from anti-PD-L1 therapy remains to be clarified.We analyzed the relative datasheet in TCGA database to unfold the tumor and tumor environment differences between EGFR-TKIs sensitive and resistant tumors.Moreover,we found that EGFR-TKIs resistances could increase the PD-L1 expression in lung cancer in vitro and in vivo,and investigated the regulatory mechanisms among them.We further evaluated the effect of anti PD-L1 therapy for EGFR-TKIs resistant lung cancer in vivo and in vitro.Our study would illustrate the mechanisms of PD-L1 expression induced by EGFR-TKIs resistance,provide the experimental basis for anti PD-1/PD-L1 axis therapy in the treatment of EGFR-TKIs resistant lung cancer,and provide a novel treatment method for those patients who acquired resistance to EGFR-TKIs.MethodsMET amplified-EGFR-TKIs resistant lung cancer cell lines,PC-9R cells,HGF and 293FT cells transfected with EGFR-19Del and/or EGFR-T790M mutation plasmids were used to establish MET amplification,HGF and EGFR-T790M mutation mediated EGFR-TKIs resistant cell models,respectively.Then Western Blot,flow cytometry,RT-qPCR and other technology are used to compare the PD-L1 expression of cells mentioned above.And all the cell models are co-cultured with human T lymphocytes and tested by lactate dehydrogenase(LDH)cytotoxicity assay to evaluate their immune escape ability.Lung cancer cells with PD-L1 gene deletion and its negative control cells were used to build xenograft models and treated with human T lymphocytes to evaluate the immune escape ability of tumors in vivo.The TCGA datasheet were analyzed to compare the difference of tumor mutation loads,expression of negative immune regulator molecules such as PD-L1,tumor-infiltrating lymphocytes(TILs),immune cytolytic activity(CYT)in tumor environment between EGFR-TKIs sensitive and resistant lung cancers.ResultsThe experimental results showed that MET amplification,HGF and EGFR-T790M increase PD-L1 expression in the surface of tumor cells,while the relative regulatory mechanisms are different.PI3K/Akt and MAPK signal pathway are involved in the expression of PD-L1 induced by MET amplification and HGF,while PI3K/Akt,MAPK and NF-kappa B signal pathway are involved in the expression of PD-L1 induced by EGFR-T790M.The experiment results in vivo and vitro showed that MET amplification,HGF and EGFR-T790M promote immune escape of lung cancer by moderating the cytotoxicity of T lymphocytes against tumor cells through upregulation the expression of PD-L1,while downregulating the expression of PD-L1 by deletion the PD-L1 gene in tumor cells or anti-PD-L1 monoclonal antibodies could reverse cytotoxic effect of T lymphocyte.The datasheet in TCGA database indicated that heavier tumor mutation loads and higher expression of immune suppressive molecules,such as PD-1,PD-L1 expression in EGFR-TKIs sensitive tumors,comparing to resistant tumors.Conclusion(1)Higher tumor mutation loads,stronger immune suppressive effect in EGFR-TKIs resistant lung cancer tumors,comparing to sensitive tumors(2)MET amplification promote immune escape of non-small cell lung cancer cells by upregulation of PD-L1 expression through PI3K/Akt and MAPK signal pathways(3)HGF promote immune escape of non-small cell lung cancer cells by inducing the expression of PD-L1 through PI3K/Akt and MAPK signal pathways(4)EGFR-T790M mutation promote immune escape of lung cancer by inducing the expression of PD-L1 through PI3K/Akt,MAPK,and NF-kappa B signal pathways(5)Anti PD-1/PD-L1 axis could restore the cytotoxicity of T lymphocytes toward EGFR-TKIs resistant lung cancer... |
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