The Role Of PD-L1 In Primary Resistance To EGFR-TKIs And Its Mechanism In Non-small Cell Lung Cancer

Objectives:Approximately 30%of patients with epidermal growth factor receptor(EGFR)-activating mutations have no response to EGFR-tyrosine kinase inhibitors(TKIs)(primary resistance).However,little is known about the molecular mechanism involved in primary resistance to EGFR-TKIs in EGFR-mutant.Programmed death ligand-1(PD-L1)plays important regulatory roles in intracellular functions and leads to acquired resistance to EGFR-TKIs in NSCLC.Here,we investigated the mechanistic role of PD-L1 in primary resistance to EGFR-TKIs in EGFR-mutant NSCLC.Methods:The mRNA expression level of PD-L1 and the sensitivity to gefitinib in H1975,HCC827 and PC-9 cells were determined by RT-qPCR and MTT assay,respectively.Molecular manipulations(silencing or over-expressing)were performed to assess the effect of PD-L1 on sensitivity to gefitinib,and a mouse xenograft model was used for in vivo confirmation.Western blot and RT-qPCR analysis were used to analyse expression of epithelial-mesenchymal transition(EMT)markers and phosphorylation of Smad3.The effect of PD-L1 on migratory and invasive abilities was evaluated by using Transwell assay.Results:(1)The IC50 values of H1975,HCC827 and PC-9 cell lines were 10.312 μM,0.083 μM and 0.009 μM,respectively.The mRNA expression levels of PD-L1 in these three cells were:H1975>HCC827>PC-9(P<0.001).(2)In HCC827 cells,the IC50 of the control group and the PD-L1 over-expression group were 0.096 μM and 10.032 μM,respectively;the IC50 of the control group(Sh-PD-L1-NC)and the PD-L1 interference group(Sh-PD-L1-1,Sh-PD-L1-2)were 0.125 μM,0.009 μM,and 0.016 μM,respectively.(3)In the un-administered group,there was no significant difference in tumor growth between the PLVX-NC and PLVX-PD-L1 group(P>0.05);in the gefitinib-administered group,the inhibitory effect on tumor growth of PLV-PD-L1 group was stronger than that of PLVX-NC group(P<0.001).At the end of the experiment,in the un-administered group,there was no significant difference in tumor volume and weight between the PLVX-NC and PLVX-PD-L1 group(P>0.05).In the gefitinib-administered group,the tumor volume and weight of the PLVX-PD-L1 group was more than that in the PLVX-NC group(P<0.001)(4)In the HCC827 cell line,compared with the control group,the expression of E-cadherin was down-regulated and the expression of N-cadherin,Vimentin and Snail were up-regulated in the PD-L1 over-expression group;compared with the control group,the expression of E-cadherin was up-regulated and the expression of N-cadherin,Vimentin and Snail were down-regulated in the PD-L1 interference group.The number of migrating and invading cells in the PD-L1 over-expression group was more than that in the control group(P<0.001).The number of migrating and invading cells in the PD-L1 interference group was less than that in the control group(P<0.001).(5)In the HCC827 cell line,the expression of p-Smad3 in the PD-L1 over-expression group was higher than that in the control group;the expression of p-Smad3 in the PD-L1 interference group was higher than that in the control group.Under the stimulation of TGF-β1,the expression of PAI-1 luciferase reporter plasmid in PD-L1 interference group was lower than that in the control group.(6)Under the stimulation of TGF-β1,the PD-L1 interference group showed the weaker up-regulation of p-Smad3 and Vimentin and down-regulation of E-cadherin compared with the control group;compared with the control group,under the stimulation of SIS3,the PD-L1 over-expression group showed the weaker down-regulation of p-Smad3 and Vimentin and up-regulation of E-cadherin.(7)Under the stimulation of TGF-β1,the PD-L1 interference group showed the weaker increase of cell migration and invasion compared with the control group;under the stimulation of SIS3,the PD-L1 over-expression group showed the weaker decrease of cell migration and invasion compared with the control group.Conclusion:The expression of PD-L1 is related to the sensitivity to gefitinib in EGFR-mutant NSCLC,and PD-L1 may induce EMT through the TGF-β/Smad signaling pathway to induce resistance to gefitinib in EGFR-mutant NSCLC.