title: A functional, new short isoform of death receptor 4 in Ewing's sarcoma cell lines may be involved in TRAIL sensitivity/resistance mechanisms.
abstract: Ewing's sarcoma (ES) is a high-grade neoplasm arising in bones of children and adolescents. Survival rate decreases from greater than 50% to only 20% after 5 years for patients not responding to treatment or presenting metastases at diagnosis. TRAIL, which has strong antitumoral activity, is a promising therapeutic candidate. To address TRAIL sensitivity, 7 human ES cell lines were used. Cell viability experiments [3'[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro-)benzene sulfonic acid hydrate (XTT) assay] showed that 4 of the 7 ES cell lines were resistant to TRAIL. Western blotting and flow cytometry analyses revealed that DR5 was uniformly expressed by all ES cell lines, whereas DR4 levels were higher in sensitive cell lines. In TRAIL-sensitive TC-71 cells, knockdown of TNFRSF10A/DR4 by short hairpin RNA (shRNA) was associated with a loss of sensitivity to TRAIL, in spite of DR5 presence. Interestingly, we identified a new transcript variant that results from an alternative splicing and encodes a 310-amino acid protein which corresponds to the 468 aa of DR4 original isoform but truncated of aa 11 to 168 within the extracellular TRAIL-binding domain. According to modeling studies, the contact of this new DR4 isoform (bDR4) with TRAIL seemed largely preserved. The overexpression of bDR4 in a TRAIL-resistant cell line restored TRAIL sensitivity. TRAIL resensitization was also observed after c-FLIP knockdown by shRNA in two TRAIL-resistant cell lines, as shown by XTT assay and caspase-3 assay. The results presented in this study showed that DR4, both as the complete form or as its new short isoform, is involved in TRAIL sensitivity in ES.
authors.affiliation: 1INSERM, Equipe labellisee LIGUE 2012 UMR 957, Laboratoire de Physiopathologie de la Résorption Osseuse et Thérapie des Tumeurs Osseuses Primitives, EA3822, Nantes, France.
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Picarda
authors.forename: Sylvanie
authors.initials: S
authors.lastname: Surget
authors.forename: Romain
authors.initials: R
authors.lastname: Guiho
authors.forename: Stéphane
authors.initials: S
authors.lastname: Téletchéa
authors.forename: Martine
authors.initials: M
authors.lastname: Berreur
authors.forename: Franck
authors.initials: F
authors.lastname: Tirode
authors.forename: Catherine
authors.initials: C
authors.lastname: Pellat-Deceunynck
authors.forename: Dominique
authors.initials: D
authors.lastname: Heymann
authors.forename: Valérie
authors.initials: V
authors.lastname: Trichet
authors.forename: Françoise
authors.initials: F
authors.lastname: Rédini
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects / Q000235:genetics; D000595:Amino Acid Sequence; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000378:metabolism; D045744:Cell Line, Tumor; D019008:Drug Resistance, Neoplasm / Q000187:drug effects*; D004354:Drug Screening Assays, Antitumor; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000737:chemistry / Q000378:metabolism*; D012512:Sarcoma, Ewing / Q000378:metabolism* / Q000473:pathology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*
chemical_list: D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D050505:Mutant Proteins; D020033:Protein Isoforms; D012333:RNA, Messenger; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand