Novel Approaches To Overcome HGF-mediated Resistance To Gefitinib In EGFR Mutant Non-small Cell Lung Cancer Cells

Lung cancer is the leading cause of cancer death in the world and the incidence and mortality increased gradually during the past several decades in China. Non-small-cell-lung-cancer (NSCLC) accounts for about80-85%of lung cancer cases. Most of NSCLC patients are recognized in the late stages and lose the opportunity of surgery. Recent therapeutic strategies for NSCLC have focused on the development of molecular targeted drugs. Multiple prospective trials have shown a response rate of70%to80%for patients with tumors harboring epithelial growth factor receptor (EGFR) mutations. However, the overall survival is not significantly improved because most patients who initially respond will eventually develop acquired resistance to TKIs. The over-expression of hepatocyte growth factor (HGF) is an important mechanism of acquired resistance. Therefore, new approaches are needed to overcome this resistance, which may offer an improved survival benefit. Part I Crizotinib overcomes HGF-mediated resistance to gefitinib in EGFR mutant non-small cell lung cancer cells Aims:The over-expression of hepatocyte growth factor (HGF) is an important mechanism of acquired resistance to gefitinib in non-small-cell-lung cancer (NSCLC) patients with epithelial growth factor receptor (EGFR) mutation. We aim to evaluate whether crizotinib can overcome two mechanisms of HGF-mediated resistance to gefitinib in EGFR-mutant NSCLC cells in vitro.Methods:The roles of crizotinib on HGF-induced resistance to gefitinib were assessed in two EGFR mutant lung cancer cell lines HCC827and PC-9. The resistant cell model was established by exogenously adding recombinant HGF protein. CCK-8assays and flow cytomety were used to evaluate the effects of crizotinib on cell proliferation and apoptosis. Western Blot was used to detect the activation of HGF/MET pathway.Results:Both HCC827and PC-9were highly sensitive to gefitinib, but exogenously HGF triggered obvious resistance to gefitinib. Adding gefitinib and crizotinib simultaneously significantly inhibited tumor cells growth in a dose dependent manner even in the presence of HGF in both cells. Moreover, adding crizotinib additional to cells achieved a19.8%accumulation of apoptotic cells compared with that when gefitinib was used singly. Although50nM crizotinib monotherapy had no effect on the proliferation of both cells, the impact of HGF was almost totally reversed by50nM crizotinib. Futhermore, crizotinib prevented the emergence of gefitinib-resistant cells induced by transient exposure to HGF in HCC827cells. At the protein levels, the combined treatment of gefitinib and crizotinib significantly suppressed both the phosphorylation of MET and EGFR, resulting in downstream Akt and ERK1/2inhibition, regardless of the presence of HGF. Conclusinon:Crizotinib, as a potent MET tyrosine kinase inhibitor, can reverse HGF-induced gefitinib resistance in NSCLC. This study supports that crizotinib may be used in a new therapeutic field. Part II MicroRNA-34a overcomes HGF-mediated gefitinib resistance in EGFR mutant lung cancer cells Aims:MicoRNA-34a (miR-34a) is considered as one potent tumor-suppressor. The ectopic expression of miR-34a induces apoptosis, cell-cycle arrest, migration and invasion inhibition in multiple types of cancer including lung cancer. Meanwhile, miR-34a plays important roles in HGF/MET signaling. This study aims to test whether the ectopic introduction of miR-34a could overcome HGF-mediated resistance to gefitinib in EGFR mutant lung cancer by modulating HGF/MET pathway in vitro and in vivo.Methods:The roles of miR-34a on HGF-induced resistance to gefitinib were assessed in EGFR mutant lung cancer cell lines HCC827and PC-9with an exon19deletion. The resistant cell model was established by exogenously adding recombinant HGF protein. To better characterize the role of miR-34a, we conducted gain of function analysis by transfecting cells with chemically synthesized miR-34a mimics. CCK-8assays and flow cytomety were used to evaluate the effects of miR-34a on cell proliferation and vm apoptosis. HCC827cells stably expressing vector alone or miR-34a were mixed with HGF-producing fibroblasts, MRC-5cells, and injected subcutaneously to BALB/c-nude mice to establish the HGF-induced gefitinib-resistant mouse xenograft models. The therapeutic effects of miR-34a plus gefitinib were assayed. Western Blot was used to detect the activation of HGF/MET pathway. Luciferase assay was conducted to evaluate whether miR-34a translationally repressed MET by targeting the essential binding sequences located in3’-UTR of MET.Results:Exogenously adding recombinant HGF induced significantly resistance to gefitinib in HCC827and PC-9. The introduction of miR-34a inhibited the proliferation of resistant cells in a concentration dependent manner. Compared with the negative control (NC) group, miR-34a at40nM caused a28%increased percentage of cell growth inhibition in HCC827and34%in PC-9. Meanwhile, the enforced expression of miR-34a resulted in a significant higher rate of apoptosis than the NC treatment. In vivo, a dramatic tumor growth regression was observed in miR-34a plus gefitinib group compared with control group in HGF-induced gefitinib resistant mouse models. The detection of protein indicated that the over-expression of miR-34a down-regulated the expression of MET, resulting in downstream Akt and ERK1/2pathway inhibition. Futhermore, luciferase assay confirmed that MET was a direct target of miR-34a.Conclusion:This study demonstrates for the first time that miR-34a rescues HGF-induced gefitinib resistance in EGFR mutant NSCLC cells, suggesting a potential therapeutic application of miR-34in the EGFR-TKIs resistance.