The Mechanism Of Cellular FLICE-like Inhibitory Protein(c-FLIP) In Cancer Cell Epithelial-Mesenchymal Transitions(EMT)

c-FLIP(cellular FLICE-like inhibitory protein)is a master anti-apoptotic regulator homologous to caspase-8 with a inactive caspase-like domain.Via its own Death Effective Domains(DEDs),c-FLIP prevents the binding of procaspase-8 to FADD or interfers with the autocatalytic activation of caspase-8 at the death-inducing signaling complex(DISC),leading to apoptosis inhibition.As an important target on anti-tumor therapy,c-FLIP is getting more and more attention.In addition to its anti-apoptotic role,c-FLIP also activates several signaling pathways involved in regulating cell proliferation,cell survival and carcinogenesis through NF-κB ERK/AP-1 and β-catenin pathway.Malignant tumoris characterized by the unrestricted rapidly proliferation,abnormal differentiation and metastasis,especially tumor metastasis is the most difficult task in the treatment of cancer.In the previous experiments,we found that c-FLIP expression was positively correlated to cell migration in six different differentiated lung cancer cells:95C,spcAl,A549,H446,H1299 and PC9.But the role and acting mechanism of c-FLIP involved in migration are still unclear.Starting from the relationship between structure and properties,we established stable expression of c-FLIP and its mutants in A549 cell lines as research platforms to explore the mechanism of c-FLIP in cell migration.Using several biotechnologic methods such as Transwell,Wound-healing and RTCA assay,we detected the effects of c-FLIP or its mutant expression on cell proliferation and migration ability.The results suggested that c-FLIP was involved in cell migration by its caspase-8 like domain.Many literatures have reported that cell migration is closely related to epithelialmesenchymal transition(EMT).Tumor cells acquire abilities of metastasis and invasion principally through the EMT.The disruption of cell polarity is the initial stage of EMT,then tumor cells breakdown the extracellular matrix(ECM)and migrate to the adjacent or distal organs.To figure out the possibility of c-FLIP involved in the regulation of EMT,we tested EMT marker expression(E-cadherin,vimentin,Fibronectin)both in the protein expression level and transcription level by Q-PCR and Western blot.the results showed that c-FLIP and the domain C8-L contributed to the EMT process.In the classical TGF betal induced EMT process,we detected the activation of related signals including Smad dependent pathway and Smad independent pathway.The signal activity was evaluated by luciferase reporter and western blot analysis.Overexpression of c-FLIP or C8-L can activate Smad signal pathway in response to TGFbetal stimulation.Accordingly,c-FLIP siRNAs blocked the process induced by TGF betal.It suggested that the regulation of c-FLIP on EMT is dependent on Smad pathway.