Crizotinib Activates Autophagy As A Cytoprotective Response In Human Lung Cancer Cells

Multikinase inhibitor crizotinib has shown a promising prospect against lung cancer in pre-clinical models as well as clinical trials. However, intrinsic or acquired resistance is common and inevitably follows susceptibility. As a novel cytoprotective mechanism for tumor cells to survive under unfavorable conditions, autophagy has been proposed to play an important role in drug resistance of tumor cells. Whether crizotinib can activate autophagy to impair the sensitivity of treated lung cancer cells remains elusive.Objective:To determine whether autophagy is activated by multikinase inhibitor crizotinib, thereby impairing the sensitivity of treated lung cancer cells to this targeted therapy. To further investigate the molecular mechanisms by which crizotinib induces cytoprotective autophagy in targeted lung cancer cells.Methods:Lung cancer cell lines SPC-A1, H2228 and A549 were treated with different concentrations of crizotinib and the level of autophagy were measured by Western blot, indirect immunoflurescence and transmission electron microscopy method.Western blot was performed to investigate whether MET, STAT3, AKT/MTOR and ERK signaling pathway are involved in crizotinib-induced autophagy.MTS assay and FCM were employed to determine whether inhibition of autophagy by pharmacologic inhibitors or shRNA against beclinl can sensitize crizotinib in vitro. Furthermore, a lung cancer xenograft model was established to evaluate the synergistic effects of autophagy inhibitor HCQ on crizotinib in vivo.Results:First, cells were treated with crizotinib (0,0.5,1,2,4μM) for 48h. The proliferation of lung cancer cells were variously inhibited by crizotinib treatment.Second, high level of autophagy was induced by crizotinib in a dose-dependent manner in lung cancer cells.Third, crizotinib treatment suppressed the phosphorylation of MET, STAT3 and AKT/MTOR levels in a dose-dependent manner. MET siRNA transfection reduced the expression of MET and promoted the transition of LC3-I to LC3-II, as well as LC3-II puncta formation under confocal. However, further study revealed that crizotinib induced autophagy by inhibiting cytoplasmic as well as nuclear STAT3 pathway independent of MET activation, followed by increased phosphorylation of EIF2AK2 and EIF2A and decrease of BCL2 in a step-wise manner.Lastly, we observed that co-treatment with crizotinib and CQ or 3-MA significantly suppressed the cell proliferation and enhanced apoptosis, compared with crizotinib treatment alone in vitro. Genetic inhibition of beclinl by shRNA promoted the anti-proliferative effect of crizotinib. Meantime, apoptotic cell death was greatly enhanced in shRNA beclinl cells than control group after crizotinib treatment. Further studies also showed that inhibition of autophagy by HCQ in vivo significantly augmented the anti-proliferative effect of crizotinib in a mouse xenograft lung cancer model. In the tumors tissue, LC3 level was markedly increased after crizotinib treatment. While cleaved caspase-3 were decreased in crizotinib plus HCQ group compared with crizotinib treatment alone. Consistent with the in vitro results, crizotinib suppressed the MET, STAT3 and AKT/MTOR phosphorylation in the xenografte tumors.Conclusions:Crizotinib could induce autophagy in lung cancer cells, promote expression of LC3 and formation of autophagosome which was accompanied by suppression of cytoplasmic and nuclear STAT3 pathways. Autophagy activation plays a cytoprotective role in crizotinib treated lung cancer cells. The disruption of autophagy process facilitates the growth inhibition of crizotinib and enhances apoptotic cell death in lung cancer cells in vitro and in vivo.