The Effect And Mechanism Of Targeting MET And VEGF On Reversing EGFR-TKIs Resistance In EGFR-Mutant NSCLC With Concomitant Aberrant MET Activation

1.Construction and identification of gefitinib resistant EGFR-mutant NSCLC with concomitant aberrant MET activationBackground: EGFR-TKIs are effective in patients with EGFR sensitive mutations in NSCLC.However,drug resistance inevitably occurs during the treatment.Aberrant MET activation in NSCLC,which is associated with a poor prognosis,reportedly mediates primary and acquired resistance to EGFR-TKIs in EGFR-mutant NSCLC.However,the existing molecular mechanisms are not sufficient to fully explain MET induced-malignant biological behaviors and drug resistance.This study aims to construct EGFR-TKIs resistant cell lines and screen the clones with abnormal MET activation,so as to provide experimental objects for further study research on EGFR-TKIs drug-resistance caused by MET.Methods: HCC827 and PC-9 NSCLC cells with EGFR 19 exon deletion mutation were selected.The gefitinib-resistant PC-9GR cells were induced by concentration increase method in vitro.Western blot was used to screen the clones of drug resistant strains with high expression and abnormal activation of MET.In addition,Zhu Feng’s team from Tongji Medical College of Huazhong University of Science and Technology provided a HCC827 GR gefitinib resistant cell line with MET amplification and aberrant MET activation as a gift.CCK8 assay was used to detect the changes of gefitinib resistance index of PC-9GR and HCC827 GR cells.Morphological changes of drug-resistant cells were observed under microscope,EMT related molecules were detedcted.Transwell invasion assay was used to detect the change of invasion ability of drug-resistant cells compared with parental cells.Western blot was used to detect the changes of EGFR,MET and downstream related signaling pathways between gefitinib-treated cells and their parents.Exons 18 to 21 of EGFR were sequenced in drug-resistant cells,and the presence of T790 M mutation was further analyzed.The change of MET gene copy number in drug-resistant cells was detected by q PCR.Results: HCC827 and PC-9 cells with EGFR-sensitive mutations showed sensitivity to lower concentrations of gefitinib compared with EGFR wild-type cells.Gefitinib resistant HCC827 GR cells and PC-9GR cells were induced by increasing gefitinib concentration,and the IC50 of gefitinib resistant cells was significantly higher than that of parental cells.Morphologically,the two drug-resistant cell lines showed fusiform cells,enlarged cell space,and exhibited increased EMT phenotype and stronger invasive abilities.Western blot analysis showed that the activities of EGFR,ERK and AKT in parental cells were significantly inhibited when treated with gefitinib,and the phosphorylation level of MET was slightly inhibited.However,with the same concentration of gefitinib,the EGFR phosphorylation level in the resistant cell lines was significantly inhibited,but the activity of MET and related downstream signaling molecules were maintained.Exon sequencing analysis of EGFR18-21 showed that there was no T790 M mutation in HCC827 cells and PC-9 cells,and no new T790 M mutation occurred in the two drug-resistant cell lines while maintaining the original EGFR mutation.The MET gene copy number of PC-9GR and HCC827 GR cells was higher than that in their parental cells.Conclusion: In this study,gefitinib resistant cell lines with aberrant activation of MET were successfully obtained.The EMT phenotype and invasion ability were increased in resistant cell lines compared with parental cells.MET and its downstream signaling pathway were still significantly activated under the gefitinib treatment.No acquired mutation of EGFR T790 M was found in drug-resistant cell lines,but the copy number of MET gene increased in varying degrees.2.Crosstalk between MET and VEGF/VEGFR2 signaling pathways in NSCLC cellsBackground: Aberrant activation of MET can endow cancer cells with the ability of proliferation,survival,invasion and metastasis.In recent decades,MET has become a widely concerned therapeutic target in NSCLC.The signal transduction of MET involves many molecular events.With the gradual disclosure of the important role of MET in the development of cancer drug resistance,the crosstalk between MET and other RTK signals has also been deeply studied.Anti-angiogenesis drugs targeting VEGF and its receptors have been recognized as an effective anti-tumor treatment strategy.There are complex positive and negative feedback regulation between MET and VEGF signaling pathway and many other RTKs.However,little is known about the potential interaction between MET and VEGF/VEGFRs/NRPs signaling pathway and its effect on tumor growth in NSCLC.In this part,we aimed to explore the crosstalk between MET and VEGF signaling pathways in NSCLC,so as to further explore the potential value of combined inhibition of MET and VEGF in improving the sensitivity to EGFR-TKIs in EGFR mutant NSCLC with aberrant activation of MET.Methods: The expression of VEGFRs and its co-receptors(NRPs)in parental cells and gefitinib resistant cells with aberrant MET activation were analyzed by q PCR.Western blot was used to detect the effects of crizotinib and MET silence,or MET overexpression on the expression and activity of VEGFR2.Bioinformatics analysis and double luciferase reporter gene were used to further clarify the regulation mechanism of MET on VEGFR2 transcription.The expression and activity of MET in NSCLC was detected by Western blot under the stimulation with exogenous VEGF.Immunofluorescence and co immunoprecipitation(co-IP)were used to reveal the specific mechanism of the reverse regulation of VEGF to MET.Results: VEGFR2 expression and its phosphorylation activity were significantly increased in gefitinib-resistant cell lines with abnormal MET activation.Loss function of MET by crizotinib and MET sh RNA could reduce the expression and activity of VEGFR2 in gefitinib resistant cell lines.Overexpression of MET in NSCLC cell lines with low expression of MET could promote the expression and activation of VEGFR2.We confirmed that MET promoted VEGFR2 expression and activation through MEK/ERK signaling pathway by inhibiting MEK/ERK,PI3K/AKT,and JAK/STAT3 signaling pathways.We further revealed that MET promoted ETS-1 expression through ERK signaling and promoted VEGFR2 transcription through ETS-1.On the contrary,VEGF could significantly induce the activation of MET and the downstream signals in NSCLC cell lines,and the activation effect is more significant in cells with high MET expression.Inhibition of VEGFR2 could attenuat the activation of MET induced by VEGF.Co-IP results showed that MET and VEGFR2 interacted in NSCLC,and VEGF stimulation promoted physical interaction between VEGFR2 and MET and phosphorylation of MET.Conclusion: MET up-regulates VEGFR2 expression in a MEK/ERK dependent manner and promotes VEGFR2 transcription through ETS-1.VEGF promotes physical interaction between VEGFR2 and MET,which further promotes phosphorylation of MET.The results suggest that MET forms a positive feedback loop with VEGF/VEGFR2 signaling pathway in NSCLC cells,resulting in continuous downstream signal activation.3.Combined inhibition of MET and VEGF enhances therapeutic efficacy of EGFR-TKIs in EGFR-mutant NSCLC with concomitant aberrant MET activationBackground: In the above study,our results showed that there was positive feedback between MET and VEGF/VEGFR2 signaling pathway.Aberrant activation of MET could further promote the activation of VEGF/VEGFR2 and downstream signal.VEGF/VEGFR2 also promoted MET and downstream signal activation.The purpose of this study was to determine the effect of combined inhibition of METand VEGF/VEGFR2 signaling pathway on the therapeutic sensitivity of EGFR-TKI in gefitinib resistant cell lines with MET aberrantion.Methods: Three clinically approved drugs were used in this study,gefitinib(EGFR inhibitor),crizotinib(Anaplastic Lymphoma Kinase and MET inhibitor)and bevacizumab(anti-VEGF antibody).The effects of different drug combinations on downstream signal activity and apoptotic molecules of gefitinib-resistant cells were detected.In vivo,xenograft tumor model was used to detect the inhibitory effect of different drug combinations on tumor.The clinicopathological data and diagnosis and treatment of a patient with EGFR mutation combined with MET amplification were analyzed retrospectively,and the clinical characteristics and potential treatment strategies of EGFR mutation combined with MET amplification were summarized.Results: Compared with any single or dual drug treatment,the triple therapy of gefitinib,crizotinib and bevacizumab inhibited cell growth more significantly,and this triple inhibition resulted in the significant inhibition of MET and ERK activity and the significant up-regulation of cleaved-PARP expression.In vivo studies showed that gefitinib alone had limited inhibitory effect on the growth of drug-resistant cell tumors,and the tumor growth could be significantly inhibited in combination with crizotinib.Most importantly,the dual inhibition of MET and VEGF significantly increased the antitumor effect of gefitinib in drug-resistant cell tumors.Consistent with the tumor volume data,the expression level of Ki-67 protein in tumor tissue samples of the three-drug combination treatment group was the lowest,TUNEL assay showed the most significant increase in the level of apoptosis.Moreover,the degree of vascularization in tumor tissues was the lowest under the three-drug combination treatment.In addition,in the treatment of a patient with lung adenocarcinoma in our center,we also determined the feasibility of this combination treatment after EGFR-TKI resistance.Conclusion: Combined inhibition of MET and VEGF/VEGFR2 signaling pathway may be beneficial for reversing EGFR TKIs resistance,which can control the progression of NSCLC by inhibiting tumor cell proliferation,promoting tumor cell apoptosis and inhibiting angiogenesis.