EGFR-TKIs Resistances Increase PD-L1 Expression And Promote De Novo Synthesis Of Fatty Acid In EGFR Mutant Non-Small Cell Lung Cancer

Background and ObjectionEGFR-TKIs were used as first-line therapies for the treatment of lung cancer with EGFR mutation.However,novel mutant forms of EGFR gene and complex compensation networks among bypass signal pathways still greatly limit the clinical efficacy of EGFR-TKIs.Thus,it is of great significance to explore new strategy for the treatment of EGFR-TKIs resistant lung cancer to improve the survival and prognosis of patients acquired EGFR-TKIs resistance.Recently,more and more researches showed that targeting tumor immune escape and metabolic reprogramming could effectively inhibit tumor growth.Therefore,investigating the PD-L1 expression and metabilic changes in EGFR-TKIs resistant lung cancer could provide novel strategies for the treatment of lung cancer patients who acquired EGFR-TKIs resistance.MethodsThe change of PD-L1 in lung cancer tissues before and after EGFR-TKI resistance was determined based on datasheets from TCGA database and lung tumor speimens before and after acquired EGFR-TKIs resistance.HGF,c-Met amplification and EGFRT790M mutant EGFR-TKIs resistance cells and subcutaneous tumor models were established to clarify the expression of PD-L1 on tumor cell surface.Moreover,killing capabilities of immune cells to the above-mentioned cells and subcutaneous tumors were also evaluated in vivo and in vitro.RNA sequence and RT-qPCR were used to determine the metabolic difference between EGFR-TKIs resistant lung cancer cells,PC-9GR5 and its parent cells,PC-9.Meanwhile,datasheet from TCGA database was used to analyze the influence of metabolic related genes on the survival of patients.Small molecule inhibitor and Si-RNA were used to inhibit activity of ACC to disturbe the de novo synthesis process of fatty acid in PC-9GR5 and PC-9 cells,and the changes of cell growth and proliferation,intracellular reactive oxygen species,cell cycle,apoptosis and EGFR-TKI sensitivity were determined.In addition,440 cytokines chips were used to detect the supernatant of PC-9GR5 cells to unfold the resistant mechanism to EGFR-TKIs.And the effect of resistant mechanism in PC-9GR5 cell on the de novo synthesis process of fatty acid were also evaluated.ResultsWe found that the expression of PD-L1 on EGFR mutant tumors was increased after acquired EGFR-TKIs resistance,and the expression of HGF and c-Met was positively correlated with PD-L1 expression.The in vitro experiment results showed that HGF and c-Met amplification could increase the expression of PD-L1 in lung cancer cells through the activation of PI3K and MAPK pathway,and moderate the killing capability of T cells to tumor cells.Meanwhile,the EGFR-T790M mutation upregulates the expression of PD-L1 through PI3K,MAPK and NF-κB pathway.Knockout or blocking of PD-L1 protein could restore the killing effect of immune cells to EGFR-TKIs resistant cells and subcutaneous tumors.The results based on RNA sequence and RT-qPCR indicated abnormal lipid metabolism on PC-9GR5 cells,and a serises of de novo fatty acid synthesis related genes,ACC,FASN,ACLY,were increased.Moreover,the results of K-M analysis showed that EGFR mutant patients with high expression of ACC seems to have shorter overall survial time than that of patients with low expression of ACC.The in vivo and in vitro results showed that inhibition of ACC could disturb the de novo synthesis of fatty acids and effectively decrease the growth and proliferation of EGFR-TKI resistant lung cancer cells,PC9GR5,and its subcutaneous tumors.Meanwhile,increasion of the reactive oxygen level,proportion of cells in G2/M phrase as well as the percentage of apoptosis cells were also detected after inhibiting the activity of ACC in PC-9GR5 cells.In addition,ACC inhibitor combined with EGFR-TKI could effectively decrease the proliferation of PC9GR5 cells.Based on the results of cytokine array,high secretion levels of FGF2 and FGF21 were detected in PC-9GR cells.Moreover,inhibition of FGF/FGFR signal pathway could effectively decreased the proliferation as well as downregulated SREBP-1,ACC and ACLY expression in PC-9GR5 cells.ConclusionEGFR-TKIs resistance enhances the expression of PD-L1 and promotes immune escape of lung cancer cells with EGFR mutation;Knockout or blocking of PD-L1 protein could restore the killing capability of immune cells to EGFR-TKI resistant cells and subcutaneous tumors.HGF and c-Met amplification increases the expression of PD-L1 in lung cancer cells through activation of PI3K and MAPK pathway,while EGFR-T790M mutation upregulates PD-L1 expressiont through PI3K,MAPK and NFκB pathway.Abnormal activation of lipid metabolism were detected on PC-9GR5 cells;Inhibition of ACC could disturb the de novo synthesis of fatty acids and effectively decrease the growth and proliferation of EGFR-TKIs resistant lung cancer cells and subcutaneous tumors,ACC inhibitor combined with EGFR-TKI effectively decrease the proliferation of EGFR-TKI resistant lung cancer cells.Inhibition of FGF/FGFR signal pathway could decrease the proliferation of EGFR-TKIs resistant lung cells,and downregulated genes expression related to de novo synthesis process of fatty acid.