Tumor Microenvironment-related Factors On NSCLC Resistance To EGFR-tyrosine Kinase Inhibitor Gefitinib And The Underlying Mechanism

A significant advance has been made in target therapy for specific onco-driver genes in lung cancer.EGFR as the drug target of tyrosine kinase inhibitor(TKI)can greatly ease the clinical symptoms and extend the progression free survival(PFS)in EGFR mutation non-small cell lung cancer(NSCLC)patients.However,clinical application of EGFR-TKI inevitably develop drug resistance and thus disease relapse,which are the biggest hurdles to the clinical use of TKI drugs.EGFR-TKI resistance as an important phenotype in cancer cells refers to various mechanisms including secondary mutations in onco-driver genes,upregulation of parallel signaling pathways,histological transformation and epithelial mesenchymal transition(EMT),etc.Among those,tumor microenvironment related factors and cancer stem cells(CSCs)are believed to play key role in drug resistance and receive intensive attention in NSCLC study.Tumor microenvironment has significant impact on cell phenotypes.The hypoxia environment in the solid tumor tissue is found to be associated with survival,tumor cell proliferation,apoptosis,migration and invasion,etc.,which promote the tumor malignancy.In addition,inflammation related cytokines in tumor microenvironment are highly relevent to EGFR-TKI resistance in NSCLC.Our current study focus on the influences of hypoxia and inflammation condition constituted microenvironment in gefitinib resistance development,and microenvironment related resistance mechanism in EGFR-TKI intrinsic and adapative resistant NSCLC cells.In this study,ALDEFLUOR assay was used to detect the aldehyde dehydrogenase(ALDH)expression in NSCLC cells with different onco-driver genes mutations,and tumorshpere forming assay was used to validate the stemness of ALDH positive cells.Cells were treated with hypoxia and gefitinib to study the biological influences on phenotypes of NSCLC cells.Cell growth,cell cycle,Western and other experimental methods were used to observe the changes of cell biological characteristics and the expression of related signaling pathways.Further,RNA-seq technology was adopted to detect the differential transcripts expression between ALDH positive cells and cells under treatment with hypoxia or/and gefitinib.Gene sequencing,fluorescence in situ hybridization(FISH),immunofluorescence,etc.,were used to study the microenvironment related resistance mechanim in EGFR-TKI intrinsic and adaptive resistant NSCLC cells.The results showed that: 1.Cotreatment of NSCLC cells with hypoxia and gefitinib could activate EMT along with increased ratio of ALDH positive cells,and as a result increase the drug resistance.IL-6 played an important role in gefitinib resistance and ALDH positive cells;2.Functional studies indicated that IL-6 could activate JAKSTAT3 and EMT signaling pathways,and promote ALDH expression.High IL-6 expression in NSCLC biosamples was found to be associated with short overall survival in clinical NSCLC patients;3.EMT activation and an increase in ALDH positive CSCs also existed in EGFR-TKI intrinsic and adaptive resistant NSCLC cells.This study revealed that hypoxia and inflammation cytokines constitued-tumor microenvironment play important role in gefitinib adaptive resistance,which is caused by activation of EMT and enrichment of ALDH positive lung cancer stem-like cells.Effective interventive strategies targeting specific mechanism of drug resistance is of great significance for NSCLC treatment.This study provides a new insight into mechanism of TKI resistance and option of NSCLC targeting therapy.