HGF Induces EGFR-TKIs Resistance In Different Genotypes Of NSCLC And Reversal Effect Of C-Met Inhibitors

The incidence and mortality of lung cancer are rising at an alarming rate,and their death rate is now the highest in the.world.Oncologists once predicted that if China cannot control the issue of smoking and air pollution in the next 10 years,it will become the largest country in lung cancer and its annual incidence will exceed 1 million.With the significant improvement of people’s living standards,the current molecular target therapy has been widely concerned,as one of the conventional treatment.Molecular targeted therapy inhibits the growth and survival of tumor cells by preventing mutations and/or aberrant inheritance of oncogenes in the body.the discovery and application of epidermal growth factor receptor tyrosine kinase inhibitors have provided new opportunities for the treatment of lung cancer.As one of the most commonly used molecular targeted therapies,its objective response rate and progression-free survival of sensitive mutant non-small cell lung cancer are significantly better than other treatments including chemotherapy.Nevertheless,EGFR-TKIs including gefitinib and erlotinib also inevitably have acquired resistance,resulting in limited therapeutic effects.However,at present,the mechanism of its drug resistance is not yet fully understood and has become a hot research issue in the world.In recent years,epithelial mesenchymal transition factor and its ligand hepatocyte growth factor have been identified as one of the important targets of many malignant tumors such as non-small cell lung cancer.Studies have shown that a significant proportion of patients with EGFR-mutant lung cancer are associated with high expression of HGF,amplification of MET gene,and secondary mutation of EGFR-T790M,suggesting that HGF/MET may be related to EGFR-TKIs resistance.May be related to its stimulation of c-Met activation caused by phosphorylation.In this study,in vitro and in vivo experiments to explore HGF-induced EGFR-TKIs resistance mechanism and c-Met inhibitor SU11274 its reversal effect.Part I:Study on the Resistance of EGFR-TKIs to Different EGFR Genotypes of NSCLC Induced by HGF and Its MechanismObjective:To investigate the drug resistance of HGF-induced lung cancer cells to EGFR-TKIs.and its mechanism in vitro and in nude mice.Methods:1.In vitro experiments:Human NSCLC cell line PC9(EGFR mutant and sensitive strain),H292(EGFR wild type and sensitive strain),PC9/R(EGFR mutant and acquired drug resistant strain),A549(EGFR wild type,Drug strains).Western blotting was used to detect the expression of protein.Cell proliferation and cell viability were determined by MTT assay.Apoptosis and cell cycle were detected by flow cytometry.The lentiviral packaging interfered RNA-infected cell lines,and the expression of c-Met-mRNA and protein was detected by RT-PCR.The experiment was divided into three groups:C group(control group),H group(HGF treatment group),G group(Gefitinib treatment group),HG group(HGF+gefitinib treatment group).After treatment with the corresponding drugs,the growth of cultured lung cancer cells was observed,and the changes of c-Met and its downstream channels were detected by immunohistochemistry.2.In vivo experiments:NSCLC cell line PC9,H292 and human embryonic lung fibroblast(MRC-5)was selected.The concentration of HGF in MRC-5 culture supernatant was detected by ELISA,the cell proliferation was detected by MTT assay,and the changes of c-Met and its downstream channels were detected by Western blotting.The model of nude mice was established by using PC-9 and H292 cells.C group,H group,G group,HG group,observe the growth of the tumor,weigh the tumor mass,calculate the tumor inhibition rate.Immunohistochemistry was used to detect the changes of c-Met and its downstream channels in nude mice.Results:1.In vitro experiments:1)Effect of HGF on c-Met and its phosphorylated protein expression in NSCLC:After HGF induction,phosphorylation of c-Met and its downstream channel protein is activated.2)The results of HGF-induced Gefitinib resistance in human NSCLC cells:After HGF induction,the inhibitory effect of gefitinib on H292,PC9,and A549 cells was significantly attenuated,but there was no significant effect on the proliferation of PC9/R cells.3)The effect of HGF and gefitinib treatmentalone or in combination on cell apoptosis:The apoptosis rate of PC9,H292 and A549 cells in HG group was significantly lower than that in G group(P<0.05).There was no significant difference in the apoptosis rate of PC9/R cells(P>0.05).4)The effect of HGF and gefitinib treatmentalone or in combination on NSCLC cell cycle:The results showed that the ratio of G0/G1 phase of four cells was significantly higher than that of the control group(P<0.05).The proportion of G0/G1 phase decreased and the S phase increased in HGF treated A549 cells(P<0.05).H292 cells decreased the proportion of G0/G1 phase,S phase and G2/M ratio increased significantly,with statistical significance(P<0.05).5)The effect of HGF and gefitinib treatmentalone or in combination on c-Met and its downstream protein expression in NSCLC cells:All four cells can be stimulated by HGF,resulting in the phosphorylation of c-Met and its downstream channel proteins.HGF can still stimulate the phosphorylation of c-Met,Akt,Stat3 and Erkl/2 in PC9,H292 and A549 cells under gefitinib treatment.In PC9/R cells,although it stimulated the expression of c-Met phosphorylation protein,it did not stimulate the expression of phosphorylated protein downstream of c-Met.HGF could not stimulate the expression of EGFR and ErbB3 phosphoprotein in all four types of cells.EGFR-TKIs could significantly inhibit the expression of EGFR and ErbB3 phosphoprotein regardless of the presence or absence of HGF.6)Test results after interference with c-Met:The above-mentioned effects of HGF after c-Met interference were significantly inhibited.2.In vivo experiments:1)Detection of HGF in cultured lung cancer cells:MRC-5 cells secrete HGF when cultured in vitro.2)The effect of HGF secreted by MRC-5 cells on the proliferation rate of NSCLC cells:HGF secreted by MRC-5 cells can enhance the survival rate of PC9,H292 cells.3)The effect of HGF secreted by MRC-5 cells on c-Met and its downstream channel proteins in NSCLC cells:HGF secreted by MRC-5 cells can activate c-Met in PC9 and H292 cells and increase downstream channel protein expression.4)The effect of HGF on tumor growth of PC9 and H292 cells inhibited by gefitinib:Gefitinib can significantly inhibit the growth of transplanted tumors in nude mice(P<0.01),and HGF secreted by MRC-5 cells can reduce its inhibitory effect.5)Results of c-Met and its downstream channel protein expression in nude mice:HGF secreted by MRC-5 cells can activate c-Met and its downstream channel phosphorylation.Conclusion:HGF induced NSCLC cell lines with different EGFR genotypes to be resistant to gefitinib.The sustained activation of c-Met and downstream signaling channels is an important mechanism for inducing gefitinib resistance in different genotypes of NSCLC cells.Part II:c-Met inhibitors reverse HGF-induced EGFR-TKIs(gefitinib)resistance in NSCLCs with different EGFR genotypesObjective:To investigate the effects of c-Met inhibitor SU11274 in HGF-induced sensitive NSCLC on gefitinib resistance in vitro and in nude mice.Methods:1.In vitro experiments:human NSCLC cell line PC9(EGFR mutant,sensitive strain),H292(EGFRwild type,sensitive strain),PC9/R(EGFR mutant,acquired resistant strain),A549(EGFR wild type,Primary drug-resistant strains).Western blotting was used to detect the protein expression.Cell proliferation and cell viability were determined by MTT assay.Apoptosis was detected by flow cytometry.The experiment was divided into three groups:C group(control group),H group(HGF treatment group),G group(Gefitinib treatment group),HG group(HGF+gefitinib treatment group),HGS group(HGF+gefitinib+SU11274 treatment group),respectively,after treatment with corresponding drugs,observed the growth of cultured lung cancer cells,immunohistochemical detection of c-Met and its downstream channel changes.2.In vivo experiments:NSCLC cell line PC9(EGFR mutant,sensitive strain),H292(EGFR wild type,sensitive strain)and human embryonic lung fibroblasts(MRC-5)were selected and cell proliferation was detected by MTT assay.The model of nude mice xenografted with PC-9 and H292 cells was established.The C group(control group),H group(HGF treatment group),HS group(HGF+SU1127 treatment group),HD group(HGF+0.1%DMSO treatment group),G group(gefitinib treatment group),HG group(HGF+gefitinib treatment group),HGS group(HGF+gefitinib+SU11274 treatment group).Observe the growth of the tumor,draw up the histogram and curve of tumor growth,weigh the tumor weight and calculate the tumor inhibition rate.Immunohistochemistry was used to detect the changes of c-Met and its downstream channels in nude mice.Results:1.In vitro experiments:1)The expression of c-Met and its downstream channels after treated with SU11274 at different concentrations:SU11274 can inhibit the activation of c-Met and its downstream channel protein phosphorylation in PC9,H292,PC9/R and A549 cells induced by HGF.2)The inhibitory effect of c-Met inhibitor on the proliferation of lung cancer cells:SU11274 has almost no inhibitory effect on cell proliferation.3)Effect of gefitinib combined with SU11274 on the growth of lung cancer cells induced by HGF:SU11274 can inhibit the increase of the survival rate of PC9,H292 and A549 cells induced by HGF,but has no obvious inhibitory effect on PC9/R cells.4)Effect of gefitinib combined with SU11274.on HGF-induced apoptosis Results:SU11274 can significantly increase the apoptosis rate of gefitinib to HGF-induced 4 kinds of lung cancer cells.5)Effect of gefitinib combined with SU11274 on the expression of c-Met and its downstream channels in HGF-induced NSCLC cells:HGF can significantly stimulate the phosphorylation of c-Met and phosphorylation of Akt,Stat3 and Erk1/2 downstream channel proteins.In PC9,H292 and A549 cells,HGF could still stimulate the phosphorylation of c-Met,Akt,Stat3 and Erk1/2 after gefitinib treatment,and SU11274 could inhibit its expression.In PC9/R cells,although HGF could stimulate the expression of c-Met phosphorylation protein,it could not stimulate the expression of c-Met phosphorylation downstream pathway,and the inhibitory effect of SU11274 was not obvious.2.In vivo experiments:1)Effect of gefitinib on the growth of tumor in each group after SU11274 was inhibited by c-Met.Results:When gefitinib and SU11274 combined with HGF-induced lung cancer cells,the inhibitory effect on tumor growth was significantly better than gefitinib alone(P<0.05).The tumor volume and weight in HGS group were less than those in HG group(P<0.05).2)SU11274 inhibits c-Met expression in c-Met and its downstream channel proteins in transplanted tumors:Compared with the gefitinib group,the expression of c-Met,Akt,Stat3,Erkl/2 phosphorylation protein is significantly decreased in gefitinib and SU11274 combined with HGF-induced lung cancer cells.Conclusion:The combination of c-Met inhibitor SU11274 and gefitinib reverses HGF-induced gefitinib resistance in NSCLC cells with different EGFR genotypes.The mechanism may be related to inhibition of HGF activated c-Met and its downstream channel phosphorylation proteins expression.