| Introdution:Lung cancer is the most common malignant tumor with the highest mortality rate in the world.Metastasis is one the main lethal factors of Lung cancer.It is not easy to identify the molecular mechanisms of metastasis,for in involves many factors,such as lymphatic and blood vessel formation,tumor cell migration and invasion,tumor proliferation,metastasis microenvironment and so on.At present,targeted therapy is the most ideal treatment strategy for mutation-positive non-small cell lung cancer(NSCLC)patients with distant metastasis.The third generation EGFR-TKI osimertinib has been reported to be effective against both T790 M mutations and EGFR sensitizing mutations.However,drug resistance of osimertinib would inevitably occur again.EGFR-dependent resistance mechanisms(such as tertiary EGFR mutations,T790 M disappearance,EGFR amplification),bypass pathway activation(such as MET and ERBB2 amplification,RAS-MAPK pathway activation,PTEN deletion)and phenotypic alteration(SCLC transformation,epithelial-mesenchymal transition,EMT)have been reported to confer acquired resistance to osimertinib.In spite of gradually deeper understanding of the resistance mechanisms,there is limited effective treatment strategy to overcome drug resistance in most cases.Therefore,it is necessary to have more comprehensive understanding of lung cancer metastasis and the resistance mechanism of osimertinib,and explore new therapeutic schedules to overcome the drug resistance.Besides the above-mentioned potential resistance mechanisms,tumor microenvironment(TME)may also contribute to tumor metastasis and osimertinib resistance.TME is the general term for the cellular environment factors of tumor growth,including tumor blood,lymphatic vessels,extracellular matrix,cancer-associated fibroblasts(CAFs),vascular cells,infiltrating immune cells and so on.TME has been reported to have close relationship with cancer cell proliferation,apoptosis,migration and EGFR-TKI resistance.For example,CAFs induced erlotinib resistance in PC-9 cells through activation of Hedgehog signaling pathway and induction of the EMT.Strong Programmed Death-Ligand(PD-L1)expression on the tumor cells significantly correlated with poor prognosis to EGFR-TKI therapy.Furthermore,microenvironmental heterogeneity,including differences in drug concentration,oxygen and glucose content,also affected the evolution of gefitinib and erlotinib resistance.Therefore,further understanding of the impact of TME on the TKI resistance may ultimately help to develop more precise therapeutic treatment to enhance EGFR-TKI efficacy.Endogenous electric fields(EFs)play crucial parts in the regulation of cell migration,which is closely associated with embryonic development,wound healing and immune surveillance,and cancer invasion.Moreover,endogenous EFs are important component s of TME and could be important cues for the migration of many types of cancer cells,including glioma,breast,as well as lung cancer cells.For example,EFs directed the electrotaxis of H1975 lung adenocarcinoma cells by activating stretch-activated cation channels.We have also found that EFs determined electrotaxis of H1650 and H1650-M3 human lung cancer cells by caveolin-1-mediated STAT3 activation.In several studies,EFs have been demonstrated to induce cellular responses such as migration and proliferation by activating AKT pathway and promoting EMT.Interestingly,osimertinib inhibited proliferation and induced apoptosis of lung cancer cells by inhibiting AKT-dependent pathway.However,there is no research that explores whether EFs in the TME affect the efficacy of osimertinib.Objectives: The third generation EGFR-TKI osimertinib has been reported to be effective against both T790 M mutations and EGFR sensitizing mutations.However,drug resistance of osimertinib would inevitably occur again.TME may also contribute to osimertinib resistance.As an important component of TME,EFs have been demonstrated to induce cellular responses such as migration and proliferation by activating AKT pathway and promoting EMT.Interestingly,osimertinib inhibited proliferation and induced apoptosis of lung cancer cells by inhibiting AKT-dependent pathway.However,there is no research that explores whether EFs in the TME affect the efficacy of osimertinib.In this study,we want to investigate the impact of EFs on the cell migration and osimertinib cytotoxicity in lung cancer cells.Methods: We established an experimental model to simulate biological electric field exposure that induced directional migration of lung cancer PC-9GR and H1975 cells by constant direct current.Time-lapse images of the cells were acquired using the ImageXpress Micro high-throughput imager.The cell migration was quantified using ImageJ software with MTrackJ and Chemotaxis tool plugins.The effect of electric field exposure on the cell proliferation and apoptosis was examined by Ki67 incorporation assay and TUNEL assay.Immunofluorescence assay and Western blotting were performed to detect the potential mechanisms of EFs on cell migaration and osimertinib-induced cytotoxicity.Results:1.We found a gradually increasing cathodal migration when cells were stimulated with higher EFs,and the migration speeds were also increased with higher EFs stimulation.These results suggest that EFs guide the directional migration of highly-metastatic lung cancer PC-9GR and H1975 cells in a voltage-dependent manner.2.Osimertinib treatment resulted in decreased phosphorylation of AKT and FOXO3 a,and enhanced nuclear translocation of FOXO3 a,which led to upregulation of Bim expression and cytotoxicity.In the presence of EFs,AKT and FOXO3 a were highly activated,and nuclear translocation of FOXO3 a was suppressed,resulting in decreased expression of Bim and suppressed cytotoxicity of osimertinib.Conclusions: Our data provided compelling evidence that endogenous EFs in tumor microenvironment played crucial parts in enhancing the lung cancer cells migration and inhibiting the efficacy of osimertinib via regulationg AKT/FOXO3a/Bim pathway. |
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