Cyclosporine A Sensitize Human NSCLC Cells To Crizotinib Through Inhibition Of Ca²⁺/calcineurin/Erk Pathway

Tyrosine kinase inhibitors (TKIs) occupy an important position in the treatment of lung cancer nowdays. However, although TKIs are effective in eradicating the lung cancer with corresponding targets, the exsistence of primary and acquired drug-resistance tend to attenuate the therapeutic effect of them. So it is of great significance to search for safe potentiation strategies. Here we report that Cyclosporine A (CsA) significantly increase the anti-tumor effect of crizotinib, a multitarget tyrosine kinase inhibitor of MET, EML4-ALK and ROS1, in multiple NSCLC cell lines which express c-MET. Through further studying, we figured out although crizotinib could effectively inhibit c-MET and two of its downstream pathways, namely AKT and STAT3 pathway, it activate Erkl/2 rather than inhibit it in a time and concentration-dependent manner via increasing concentration of Ca2+ in cytoplasm. CsA could effectively block the activation of Erkl/2 through inhibition of calcineurin, thus enhance the cytotoxicity of crizotinib against NSCLC in vitro and in vivo. The Erkl/2 inhibitor, PD98059, was capable of enhancing the cytotoxicity of crizotinib against NSCLC in vitro and in vivo exactly like the CsA do. In conclusion, combine with CsA or inhibitors of Erkl/2 are promising and novel strategies to improve clinical efficacy of crizotinib in potential targeted NSCLC patients.Purpose:To verified if CsA was able to enhance the cytotoxicity of crizotinib against NSCLC cells in vitro and in vivo and investigate the potential mechanisms. By this study, we aimed at providing the strategies for rational use of crizotinib and enhancement of its clinical efficacy.Methods:We assess the synergistic effects of CsA to crizotinib in NSCLC cells by MTS assay and plate clone formation experiment; The mechanisms of synergistic effectss of CsA to crizotinib were investigated by flow cytometry apoptosis analysis and flow cytometry cell cycle analysis; Western blot was used to detect the change of apoptosis markers, cell cycle-related proteins and MET signaling-related proteins after corresponding treatment; Knockdown or overexpress Erkl/2 by transfection of siRNA or plasmid; Estimate the synergistic effects of CsA and PD98059 in vivo by xenograft model of SPC-A1 cell; Assess the knockdown efficiency of calcineurin siRNA by RT-PCR; Measure the iCa2+of the lung cancer cells by Furo-3MA and flow cytometry.Results:Cyclosporine A (CsA) significantly increase the anti-tumor effect of crizotinib in multiple NSCLC cell lines which express c-MET by enhancing the apoptosis and G2/M arrest induced by crizotinib.Through further studying, we figured out that MET and two of its downstream pathways, namely the STAT3 and PI3K-AKT-MTOR pathway, are effectively inhibited after treatment of crizotinib. However, RAS-RAF-ERK signaling pathway is hardly blocked, on the contrary, ERK1/2 was significantly activated during specific time period when treated by crizotinib. CsA itself have no influence on MET, STAT3 and AKT, neither can it influence the inhibition efficiency of crizotinib upon these three proteins. However, CsA could not only inhibit the basal level of activated ERK1/2 in NSCLC cells, but could also restrain the activation of ERK1/2 induced by crizotinib effectively. To further verify if CsA fulfill its sensitizing effects via inhibiting Erkl/2, and activation of Erkl/2 the method of NSCLC evade the cytotoxicity of crizotinib, we treat NSCLC cells with crizotinib, PD98059, a MEK1/2 inhibitor, and the combination of them. It proved out PD98059 could sensitize human NSCLC cells to crizotinib through significantly augment the apoptosis and G2/M arrest induced by crizotinib exactly like CsA. Knockdown Erkl/2 by transfection of siRNA could also sensitize human NSCLC cells to crizotinib like CsA and PD98059 do. On the contrary, overexpression of Erkl/2 made SPC-A1 and PC-9 cells more resistant to criotinib. Taken together, we can safely draw the conclusion that activation of Erkl/2 is a method by which NSCLC maintains survival upon treatment with crizotinib, and CsA enhance the cytotoxicity of crizotinib at least partly due to inhibition of Erkl/2.After further study, we discovered that when treated with crizotinib, the iCa2+ of NSCLC cells significantly increased and activate Calcineurin/Erk signaling. CsA block the activation of Erkl/2 through inhibition of calcineurin. Both FK506, another inhibitor of calcineurin, and siRNA of calcineurin could enhance the cytotocixity of crizotinib in NSCLC cells.Finally, we verified that CsA and PD98059 were able to enhance cytotoxicity of crizotinib in vivo by using a xenograft model.Conclusion:Crizotinib could induce the increase of iCa2+ in lung cancer cells and further activate the Calcineurin/Erk signaling, which proved out to be an important mechanism for lung cancer to escape the cytotoxicity of crizotinib. CsA could enhance the cytotoxicity of crizotinib in vitro and in vivo by blocking Calcineurin/Erk signaling, PD98059 was also able to enhance the cytotoxicity of crizotinib in vitro and in vivo by inhibit the activation of Erkl/2.