title: Upregulation of c-FLIP-short in response to TRAIL promotes survival of NSCLC cells, which could be suppressed by inhibition of Ca2+/calmodulin signaling.
abstract: TNF-related apoptosis-inducing ligand (TRAIL) is a promising cytokine for killing tumor cells. However, a number of studies have demonstrated that different cancer cells resist TRAIL treatment and, moreover, TRAIL can promote invasion and metastasis in resistant cells. Here we report that TRAIL rapidly activates caspase-8 in a panel of non-small-cell lung carcinomas (NSCLCs). Adenocarcinomas derived from the lung in addition to high caspase-8 expression are characterized by increased expression of DR4 compared with adjacent non-neoplastic tissues. Blocking DR4 or lowering caspase-8 expression significantly reduced apoptosis in NSCLC cell lines, indicating the importance of DR4 and signifying that higher levels of caspase-8 in lung adenocarcinomas make them more susceptible to TRAIL treatment. Despite rapid and robust initial responsiveness to TRAIL, surviving cells quickly acquired resistance to the additional TRAIL treatment. The expression of cellular-FLIP-short (c-FLIPS) was significantly increased in surviving cells. Such upregulation of c-FLIPS was rapidly reduced and TRAIL sensitivity was restored by treatment with cycloheximide. Silencing of c-FLIPS, but not c-FLIP-long (c-FLIPL), resulted in a remarkable increase in apoptosis and significant reduction of clonogenic survival. Furthermore, chelation of intracellular Ca(2+) or inhibition of calmodulin caused a rapid proteasomal degradation of c-FLIPS, a significant increase of the two-step processing of procaspase-8, and reduced clonogenicity in response to TRAIL. Thus, our results revealed that the upregulation of DR4 and caspase-8 expression in NSCLC cells make them more susceptible to TRAIL. However, these cells could survive TRAIL treatment via upregulation of c-FLIPS, and it is suggested that blocking c-FLIPS expression by inhibition of Ca(2+)/calmodulin signaling significantly overcomes the acquired resistance of NSCLC cells to TRAIL.
authors.affiliation: Institute of Environmental Medicine, Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: V O
authors.initials: VO
authors.lastname: Kaminskyy
authors.forename: O V
authors.initials: OV
authors.lastname: Surova
authors.forename: T
authors.initials: T
authors.lastname: Piskunova
authors.forename: I B
authors.initials: IB
authors.lastname: Zborovskaya
authors.forename: E M
authors.initials: EM
authors.lastname: Tchevkina
authors.forename: L
authors.initials: L
authors.lastname: Andera
authors.forename: B
authors.initials: B
authors.lastname: Zhivotovsky
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology; D053181:Caspase 8 / Q000378:metabolism; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002147:Calmodulin; D011500:Protein Synthesis Inhibitors; D034741:RNA, Small Interfering; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D003513:Cycloheximide; D053181:Caspase 8; D002118:Calcium