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Fig. 3 | Journal of Hematology & Oncology

Fig. 3

From: The role of HGF-MET pathway and CCDC66 cirRNA expression in EGFR resistance and epithelial-to-mesenchymal transition of lung adenocarcinoma cells

Fig. 3

Biological correlations of EGFR with drug resistance, epithelial-to-mesenchymal transition, and metastatic potential of LADC cells. a In vitro, we had shown that the different molecular weights of EGFR in various NSCLC cell lines were closely associated with HGF and c-Met pathways. Molecular weights of EGFR in surgical biopsies of LADC also varied from patient to patient (tumor samples 830, 831, 836, 840, and 844 above 170 kDa; tumor specimens 839, 842, and 843 around 170 kDa). Interestingly, protein levels of mutant EGFR were generally lower than those of wild type. Samples that had higher molecular weights were mostly positive for c-Met and higher serum HGF, supporting our in vitro results that EGFR could be modified through HGF/c-Met pathway. b Using CoCl2 to induce hypoxia-activated c-Met phosphorylation (p-c-Met) increased protein level of EGFR and SAE2, as well as epithelial-to-mesenchymal transition (EMT) markers, e.g., vimentin and paxillin, in a dose-dependent manner in H23 cells. c c-Met phosphorylation and increase of the related proteins were initiated about 30–60 min following CoCl2 treatment of H23 cells. Appearance of HIF-1α became visible about 3–6 h following CoCl2 challenge, indicating that the increase of EMT-related protein expression occurred before traditional hypoxic activation. The hypoxia-mediated protein increases were abolished when SAE2 gene was silenced. Moreover, addition of actinomycin D did not affect CoCl2-induced cell responses. d In addition to the increases of proteins as shown by Western blotting analysis, confocal immunofluorescence micrographs revealed that hypoxia altered protein distribution inside the cells as well. For instance, vimentin was accumulated in the enlarged cytoplasmic vacuole-like structures, but not on the plasma membrane. Intracellular distribution of paxillin was generally similar to that of vimentin, except that some cells started to have nuclear signals of paxillin. Nuclear signals of ATM, however, were markedly reduced, and most of the proteins were retained in the cytoplasm in elongated spherical forms. e Exposure of H23 cells (□, control) to hypoxic condition increased resistance to cisplatin (, hypoxia for 12 h; , hypoxia for 24 h). Using shRNA-lentivirus to silence SAE2 gene expression (by shSAE2-1 [▲] or by shSAE2-2 []) increased sensitivity of LADC cells to cisplatin. f Silencing of SAE2 expression in A549 cells reduced cell mobility across extracellular matrix protein-coated carbonate membrane (the left panel). When H23 cells were exposed to hypoxia prior to the cell mobility examination, the cell migration ability increased significantly (the right panel). The statistical differences were significant (WT A549 vs. shSAE2-1 and shSAE2-2, P < 0.0004 [***], ANOVA; control H23 vs. H23 cells exposed to hypoxia, P < 0.0358 [*], ANOVA). [*P < 0.05; ***P < 0.001]

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