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. 2024 Oct 1;13(1):97.
doi: 10.1186/s40164-024-00565-9.

Combined inhibition of MET and VEGF enhances therapeutic efficacy of EGFR TKIs in EGFR-mutant non-small cell lung cancer with concomitant aberrant MET activation

Shanshan Huang #  1 , Yaling Long #  1 , Yuan Gao  1 , Wanling Lin  1 , Lei Wang  1 , Jizong Jiang  1 , Xun Yuan  1 , Yuan Chen  1 , Peng Zhang  2 , Qian Chu  3
Affiliations

Combined inhibition of MET and VEGF enhances therapeutic efficacy of EGFR TKIs in EGFR-mutant non-small cell lung cancer with concomitant aberrant MET activation

Shanshan Huang et al. Exp Hematol Oncol. .

Abstract

Background: Aberrant activation of mesenchymal epithelial transition (MET) has been considered to mediate primary and acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, mechanisms underlying this process are not wholly clear and the effective therapeutic strategy remains to be determined.

Methods: The gefitinib-resistant NSCLC cell lines were induced by concentration increase method in vitro. Western blot and qPCR were used to investigate the relationship between MET and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway. Double luciferase reporter gene and co-immunoprecipitation were used to further reveal the regulation mechanism between MET and VEGF/VEGFR2. The effect of combined inhibition of MET and VEGF/VEGFR2 signaling pathway on the therapeutic sensitivity of EGFR-TKI in gefitinib resistant cell lines with MET aberration was verified ex vivo and in vivo.

Results: We successfully obtained two gefitinib-resistant NSCLC cell lines with EGFR mutation and abnormal activation of MET. We observed that MET formed a positive feedback loop with the VEGF/VEGFR2 signaling, leading to persistent downstream signaling activation. Specifically, MET up-regulated VEGFR2 expression in a MAPK/ERK/ETS1-dependent manner, while VEGF promoted physical interaction between VEGFR2 and MET, thereby facilitating MET phosphorylation. A MET inhibitor, crizotinib, combined with an anti-VEGF antibody, bevacizumab, enhanced the sensitivity of NSCLC cells to gefitinib and synergistically inhibited the activation of downstream signaling in vitro. Dual inhibition of MET and VEGF combined with EGFR TKIs markedly restrained tumor growth in both human NSCLC xenograft models and in an EGFR/MET co-altered case.

Conclusions: Our work reveals a positive feedback loop between MET and VEGF/VEGFR2, resulting in continuous downstream signal activation. Combined inhibition of MET and VEGF/VEGFR2 signaling pathway may be beneficial for reversing EGFR TKIs resistance.

Keywords: EGFR TKIs resistance; MET; Non-small cell lung cancer; VEGF/VEGFR2 signaling.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Construction and identification of gefitinib resistant EGFR-mutant NSCLC with concomitant aberrant MET activation (A) Experimental design to generate the gefitinib-resistant cell clones. (B) (C) Cells treated with increasing concentrations of gefitinib. Cell viability relative to untreated controls was measured by CCK-8 assays after 48 h. (D) DNA sequencing peaks of the cell line shown, with codon changes involving the EGFR T790M sequence marked in red boxes (ACG > ATG). (E) Relative quantitative analysis of copy number of MET gene in the indicated cells. (F) (G) Western blotting analysis of the expression of related signaling molecules in gefitinib-resistant cells and their parent cells. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test.). Graphs show mean ± SD
Fig. 2
Fig. 2
MET up-regulates VEGFR2 expression in a MAPK/ERK/ETS1 dependent manner (A) qPCR analysis of the expression of VEGF-associated receptor or co-receptor in indicated cells. (B) (C) Western blotting analysis of the expression of MET, VEGFR2 and their phosphorylation levels in indicated cells. (D) Western blotting analysis of the expression of MET, VEGFR2 and their phosphorylation levels indicated cells upon treatment with crizotinib at different concentrations for 6 h. (E) Western blotting analysis of the expression of MET, VEGFR2 and their phosphorylation levels in HCC827GR cells subjected to knockdown of MET (shMET). (F) Western blotting analysis of the expression and phosphorylation of VEGFR2 in PC-9/MET cells upon treatment with DMSO, MEK, PI3K, or JAK small molecular inhibitor for 24 h compared with PC-9/Vec cells. (G) Western blotting analysis of the expression and phosphorylation of VEGFR2 in HCC827GR cells upon treatment with U0126 or SCH772984. (H) Western blotting analysis of the expression of ETS1 and VEGFR2 in HCC827GR and PC-9GR cells transfected with ETS1 siRNA. (I) ETS1 mRNA levels were monitored in PC-9 cells transfected with MET alone or treated with U0126. (J) Dual Luciferase reporter assay was performed to check the promoter activities of VEGFR2 in PC-9 cells transfected with MET alone or ETS1-siRNA. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test.). Graphs show mean ± SD
Fig. 3
Fig. 3
VEGF induces MET activation through VEGFR2/MET complex in NSCLC cells (A) (B) Western blotting analysis of the expression of the indicated proteins and their phosphorylation levels in indicated cells upon treatment with VEGF (50ng/ml) for different time. (C) Western blotting analysis of the expression of the indicated proteins and their phosphorylation levels in HCC827GR cells upon treatment with the VEGFR2 inhibitors apatinib or vandetanib for 24 h. (D) (E) Immunoprecipitation assay of MET-VEGFR2 interaction
Fig. 4
Fig. 4
MET-Driven resistance to EGFR TKIs may be overcome by added using of the crizotinib and bevacizumab in EGFR-mutated NSCLC (A-D) HCC827GR cells were incubated with gefitinib, crizotinib, bevacizumab (200 µg/ml) or various combinations. Cell viability relative to untreated controls was measured by CCK-8 assays after 48 h. (E) Western blotting analysis of the expression of the indicated proteins and their phosphorylation levels in HCC827GR cells upon treatment with gefitinib, crizotinib, bevacizumab or various combinations. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test.). Graphs show mean ± SD
Fig. 5
Fig. 5
Triple inhibition of EGFR, MET, and VEGF suppresses growth and angiogenesis in HCC827GR and PC-9GR tumors in vivo (A) (D)NCG mice bearing HCC827GR or PC-9GR tumors (>200 mm3 in size) were administered gefitinib and/or crizotinib orally once daily and/or bevacizumab intraperitoneally each five days. Representative tumor xenografts of each group. (B) (E) Tumor growth curve in each group. (C) (F) Body weight of each group. (G) Tumor proliferating cells were determined by Ki-67 immunohistochemical staining. (F) Tumor vessels were determined by CD31 immunofluorescence staining. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA). Graphs show mean ± SD
Fig. 6
Fig. 6
A lung adenocarcinoma with concomitant EGFR and de novo MET amplification response well to combination of TKI and bevacizumab The patient clinical history is summarized in Fig. 6. 44-year-old male stage IV lung adenocarcinoma with left lung tumor was detected harboring of EGFR-19del and MET amplification using PCR and FISH. The patient was treated with erlotinib + bevacizumab and achieved partial response (PR) with a PFS with 13 months. After PD, NGS performed on both tissue and plasma biopsies revealed that the patients obtained first-generation resistant mutation EGFR-T790M, concomitant with EGFR-19del. The patient was treated with osimertinib + bevacizumab and achieved PR. He developed PD again with a PFS of 10.2 months, and repeated biopsies sequencing identified concomitant EGFR-19del and MET amplification. Then, the patient was treated with crizotinib + bevacizumab and the best curative effect was stable disease. Four months later, he developed PD and the third biopsy still revealed positive EGFR-19del and MET amplification. The patient received osimertinib + crizotinib + bevacizumab and he achieved PR one month after treatment initiation and the PFS is more than nine months
Fig. 7
Fig. 7
Schematic diagram showing the mechanism of the interaction between MET and VEGF/VEGFR2 signaling in EGFR-mutant NSCLC cells with MET overactivation
See this image and copyright information in PMC

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