Studies On The Loss Of Histone Lysine Methyltransferase EZH2 Confers Resistance To EGFR-TKI In Non-small Cell Lung Cancer

Lung cancer have developed one of the most common cancers and the leading cause of cancer deaths,which accounts for about 18.4% of total cancer deaths around worldwide.Cancer target-therapy,such as Epidermal growth factor receptor(EGFR)-tyrosine kinase inhibitors(TKI)has been widely used in the clinical treatment of nonsmall cell lung cancer(NSCLC)patients.The primary indicator of EGFR-TKI as firstline therapy in patients with lung cancer is activation mutation on EGFR.Unfortunately,EGFR-TKI drug resistance inevitably occurs on over 50% patients,The tumor progressed and deteriorated.Activation of MET kinase,such as by the ways of ligand hepatocyte growth factor(HGF)stimulation and MET amplification,has long been known to confer resistance to EGFR-TKIs.In addition,more and more evidence demonstrated that the epigenetic heterogeneity plays critial roles in cancer drug resistance.However,whether epigenetic heterogeneity can drive EGFR-TKI resistance by influencing MET expression remains obscure unknown.Purpose:To reveal whether histone methyltransferase EZH2 can mediate EGFR-TKI resistance in non-small cell lung cancer,and elucidate the mechanism of action of EZH2 in EGFR-TKI resistance.and to explore appropriate solutions to reverse drug resistance and provide the possibility of precision treatment for lung cancer patients,ultimately providing clinical guidance for the solution of drug resistance in non-small cell lung cancer.Methods: In this paper,through the construction of Gefitinib resistant strain model of non-small cell lung cancer,after 6 months of Gefitinib drug pressure screening,HCC827/HCC827 GR and PC9/PC9 GR resistant strains were obtained.First,the level of EZH2 in resistant strains was detected,and then sh EZH2 knockdown cell lines were constructed in sensitive strains to explore the mechanism of EZH2 in Gefitinib resistance.EZH2 inhibitor GSK343 was used to detect changes in related protein levels and EGFR-TKI sensitivity for NSCLC cells.The molecular changes of EZH2 and MET were detected by PI3K-Akt inhibitor combined with Gefitinib treatment to further exploring the role of the negative feedback axis of "Met-AKT-EZH2" in drug resistance.At the same time,AKT inhibitor combined with Gefitinib was given to tumor-bearing mice to determine whether generating synergistic anti-tumor effects.Clinical samples of NSCLC patients were collected to detect changes in the levels of EZH2 and MET,and to comprehensively analyze whether EZH2 is associated with EGFR-TKI resistance.Results: We found that the level of EZH2 in Gefitinib sensitive HCC827 and PC9 cells was significantly lower than that in drug-resistant HCC872 GR and PC9 GR cells.We established the stable cell lines with knockdown of EZH2 in HCC827 and PC9 cells,and then we discovered that knock-down of EZH2 can mediate the Gefitinib resistance,increase the proliferation rate and enhance the capability of colony formation.Meanwhile,we also found that increasing MET expression and phosphorylation can be observed in sh EZH2 cells,The MET expression and phosphorylation was further raised under Gefitinib administration.The results of CHIP assay show that EZH2 and H3K27m3 were enrichment in the promoter region of MET gene.Furthermore,the EZH2 inhibitor,GSK343,can enhance the Gefitinib resistance in sensitive and resistant cells.Inhibition of MET or PI3K/AKT pathway can up-regulated EZH2 levels and restored sensitivity to EGFR-TKI.On the contrary,over-expression of MET or HGF stimulation can activate the MET-AKT pathway,then we observed down-regulation of EZH2 and increased phosphorylation of EZH2.We also demonstrated that AKT can phosphorylated EZH2 on the site of Serine 21.Furthermore,the combination therapy of PI3K/AKT inhibition and Gefitinib,which interrupts the loop,can enhanced the tumorsuppressive effect.In this way,the drug resistance of tumor can be reversed.This result has been verified in both cell experiment and animal experiment.We found that EZH2 was inversely associated with MET expression levels in clinical patients and was associated with EGFR-TKI resistance.Conclusions: Comprehensive these results,we can safely draw the conclusion that EZH2 was involved in the process of EGFR-TKI resistant by negatively regulating the MET protein.,"MET-AKT-EZH2" negative feedback loop can enhance EGFR-TKI resistance state.EGFR-TKI sensitivity can be restored by blocking the "MET-AKTEZH2" axis.This conclusion was also verified by the tumor-burdened experiments in mice,AKT inhibitors combined EGFR-TKI can reversed drug resistance,which play a synergistic antitumor effect.Clinically,EZH2 can serve as a biomarker for EGFR-TKI sensitivity potentially.Innovative points: This paper exploratively found that histone methyltransferase EZH2 can negatively regulate the expression of MET kinase,thereby promoting EGFR-TKI resistance of cells.At the same time,we point out that the safety of EZH2 inhibitor should be carefully evaluated in the clinical application of anti-tumor.It is proposed for the first time that the MET-AKT-EZH2 loop can enhance the stability of EGFR-TKI resistance in tumors.AKT inhibitor GDC0941 combined with gefitinib can exert synergistic anti-tumor effects,and the combination method has potential guiding significance for clinical treatment.