The Role And Mechanism Of TGF-β/IL-11/STAT3 Axis In EGFR TKI Resistant Lung Adenocarcinomas

Lung cancer remains the leading cause of cancer incidence and mortality worldwide,which seriously affects human survival and health.Lung adenocarcinoma is the most common subtype of lung cancer and approximately 30% of patients with lung adenocarcinoma carry activating mutations in the epidermal growth factor receptor(EGFR).Although EGFR tyrosine kinase inhibitors(TKIs)developed for EGFR activating mutations have achieved good clinical results,most lung cancer patients eventually develop resistance to EGFR TKIs.Therefore,it is very crucial to study the mechanism of EGFR TKIs resistance in lung adenocarcinoma.The main objective of this study was to discover novel mechanisms of acquired resistance to EGFR TKIs treatment,and to identify signal pathway changes and key genes that may cause drug resistance.To this end,we treated the EGFR-activated lung adenocarcinoma cell line PC9 with increasing erlotinib concentration in vitro and established an erlotinib-resistant cell lines PC9-ER.Firstly,we excluded the known mechanisms of EGFR TKIs resistance in PC9-ER cells,including EGFR mutation,EGFR amplification,MET gene amplification,AXL activation,etc.Then we performed RNA sequencing(RNA-seq)on PC9 and PC9-ER cells.Differential gene analysis revealed that the expression of cytokine interleukin-11(IL11)was significantly upregulated in PC9-ER cells.Gene set enrichment analysis(GSEA)further showed that JAK/STAT3 signaling pathway was also activated in PC9-ER cells.To investigate the function of the IL11/STAT3 signaling pathway on EGFR TKIs resistance of PC9-ER cells,we constructed stable cell lines with knockdown of IL11 or its receptor IL11 RA.It was found that IL11 or IL11 RA knockdown can restore sensitivity to erlotinib in PC9-ER cells.Besides,the combination of erlotinib and JAK/STAT3 signaling pathway inhibitors,ruxolitinib or S3I-201,can also reverse PC9-ER cells resistance in different extents.In addition,we explored the changes in IL11 expression after erlotinib treatment of PC9 cells under different conditions and found that the expression of IL11 was significantly decreased when PC9 cells are stimulated in high concentration of erlotinib for short periods,but it was gradually increased in indicated time points during the process of drug resistance in PC9-ER cells.These results suggested that the resistance mechanism conferred by increasing IL11 expression was a long-term effect.Furthermore,we found that TGF-β signaling pathway which can regulate IL11 expression was also activated in PC9-ER cells,and the combination of erlotinib with TGF-β signaling pathway inhibitor SB431542 can also reverse the drug resistance in PC9-ER cells.In summary,we established a drug-resistant cell line PC9-ER that does not contain the common EGFR TKIs resistance mechanisms and first discovered that activation of the TGF-β/IL11/STAT3 signaling pathway can confer EGFR TKIs resistance.Also,the combinations of erlotinib and JAK/STAT3 signaling pathways or TGF-β signaling pathway inhibitors can effectively reverse EGFR TKIs resistance of PC9-ER cells,suggesting that STAT3 may be a potential target for EGFR TKIs resistance.The findings further improved our understanding of the molecular mechanism of EGFR TKIs resistance in lung adenocarcinoma,and may provide theoretical basis and new therapeutic strategies for delaying the occurrence of EGFR-TKIs resistance and further treatment of lung cancer patients with undefined EGFR-TKIs resistant mechanism.