A Functional, New Short Isoform of Death Receptor 4 in Ewing's Sarcoma Cell Lines May be Involved in TRAIL Sensitivity/Resistance Mechanisms

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. Mol Cancer Res; 10(3); 336–46. ©2012 AACR.

[1]  O. Delattre,et al.  Preclinical Evidence that Use of TRAIL in Ewing's Sarcoma and Osteosarcoma Therapy Inhibits Tumor Growth, Prevents Osteolysis, and Increases Animal Survival , 2010, Clinical Cancer Research.

[2]  H. Wakelee,et al.  Phase I and pharmacokinetic study of lexatumumab (HGS-ETR2) given every 2 weeks in patients with advanced solid tumors. , 2010, Annals of oncology : official journal of the European Society for Medical Oncology.

[3]  Gen Sheng Wu.  TRAIL as a target in anti-cancer therapy. , 2009, Cancer letters.

[4]  M. von Mehren,et al.  Mapatumumab, an antibody targeting TRAIL-R1, in combination with paclitaxel and carboplatin in patients with advanced solid malignancies: results of a phase I and pharmacokinetic study. , 2009, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[5]  Geoffrey J. Barton,et al.  Jalview Version 2—a multiple sequence alignment editor and analysis workbench , 2009, Bioinform..

[6]  F. Kruyt.  TRAIL and cancer therapy. , 2008, Cancer letters.

[7]  D. Lawrence,et al.  Structural and functional analysis of the interaction between the agonistic monoclonal antibody Apomab and the proapoptotic receptor DR5 , 2008, Cell Death and Differentiation.

[8]  R. Plummer,et al.  Phase 1 and Pharmacokinetic Study of Lexatumumab in Patients with Advanced Cancers , 2007, Clinical Cancer Research.

[9]  Daniel B Longley,et al.  c-FLIP: a key regulator of colorectal cancer cell death. , 2007, Cancer research.

[10]  N. Athanasou,et al.  Cellular and humoral mechanisms of osteoclast formation in Ewing's sarcoma , 2007, British Journal of Cancer.

[11]  J. Eberle,et al.  Resistance of melanoma cells to TRAIL does not result from upregulation of antiapoptotic proteins by NF-κB but is related to downregulation of initiator caspases and DR4 , 2007, Oncogene.

[12]  A. Tolcher,et al.  Phase I pharmacokinetic and biologic correlative study of mapatumumab, a fully human monoclonal antibody with agonist activity to tumor necrosis factor-related apoptosis-inducing ligand receptor-1. , 2007, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[13]  M. Bonneville,et al.  Complex Interplay of Activating and Inhibitory Signals Received by Vγ9Vδ2 T Cells Revealed by Target Cell β2-Microglobulin Knockdown1 , 2006, The Journal of Immunology.

[14]  E. Steliarova-Foucher,et al.  Bone tumours in European children and adolescents, 1978-1997. Report from the Automated Childhood Cancer Information System project. , 2006, European journal of cancer.

[15]  E. Steliarova-Foucher,et al.  Cancer incidence and survival in European adolescents (1978-1997). Report from the Automated Childhood Cancer Information System project. , 2006, European journal of cancer.

[16]  R. Bataille,et al.  Mcl-1L cleavage is involved in TRAIL-R1- and TRAIL-R2-mediated apoptosis induced by HGS-ETR1 and HGS-ETR2 human mAbs in myeloma cells. , 2006, Blood.

[17]  I. Holen,et al.  Role of osteoprotegerin (OPG) in cancer. , 2006, Clinical science.

[18]  W L Allen,et al.  c-FLIP inhibits chemotherapy-induced colorectal cancer cell death , 2006, Oncogene.

[19]  T. Wilson,et al.  Chemotherapy and TRAIL-mediated colon cancer cell death: the roles of p53, TRAIL receptors, and c-FLIP , 2005, Molecular Cancer Therapeutics.

[20]  B. Fang,et al.  Mechanisms of resistance to TRAIL-induced apoptosis in cancer , 2005, Cancer Gene Therapy.

[21]  Wafik S El-Deiry,et al.  TRAIL and apoptosis induction by TNF-family death receptors , 2003, Oncogene.

[22]  D. Baltimore,et al.  Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5 , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[23]  J. Goldblum,et al.  Expression of Fli-1, a Nuclear Transcription Factor, Distinguishes Vascular Neoplasms From Potential Mimics , 2001, The American journal of surgical pathology.

[24]  N. Mitsiades,et al.  Fas-mediated apoptosis in neuroblastoma requires mitochondrial activation and is inhibited by FLICE inhibitor protein and Bcl-2. , 2001, Cancer research.

[25]  O. Delattre,et al.  Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57KIP2 and c-Myc expression , 2001, Oncogene.

[26]  C. Niemeyer,et al.  Sensitivity of Ewing's sarcoma to TRAIL-induced apoptosis , 2001, Cell Death and Differentiation.

[27]  N. Mitsiades,et al.  Ewing's sarcoma family tumors are sensitive to tumor necrosis factor-related apoptosis-inducing ligand and express death receptor 4 and death receptor 5. , 2001, Cancer research.

[28]  A. Craft,et al.  Prognostic factors in Ewing's tumor of bone: analysis of 975 patients from the European Intergroup Cooperative Ewing's Sarcoma Study Group. , 2000, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[29]  D. Stuart,et al.  Structure of the TRAIL–DR5 complex reveals mechanisms conferring specificity in apoptotic initiation , 1999, Nature Structural Biology.

[30]  H. Hense,et al.  Factors associated with tumor volume and primary metastases in Ewing tumors: results from the (EI)CESS studies. , 1999, Annals of oncology : official journal of the European Society for Medical Oncology.

[31]  P. Hersey,et al.  Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma. , 1999, Cancer research.

[32]  Jane Y. Wu,et al.  Alternative splicing and programmed cell death. , 1999, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[33]  S R Sprang,et al.  Modularity in the TNF-receptor family. , 1998, Trends in biochemical sciences.

[34]  J. Bell,et al.  TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL , 1997, Current Biology.

[35]  M. Cohen,et al.  Visual presentation of the staging of pediatric solid tumors. , 1996, Radiographics : a review publication of the Radiological Society of North America, Inc.

[36]  T. Blundell,et al.  Comparative protein modelling by satisfaction of spatial restraints. , 1993, Journal of molecular biology.

[37]  C. Denny,et al.  Ewing sarcoma 11;22 translocation produces a chimeric transcription factor that requires the DNA-binding domain encoded by FLI1 for transformation. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[38]  M. Santamaría,et al.  The neuroectodermal tumor of bone , 1984, The American journal of surgical pathology.

[39]  F. Graham,et al.  Characteristics of a human cell line transformed by DNA from human adenovirus type 5. , 1977, The Journal of general virology.

[40]  J. Ewing.  Diffuse endothelioma of bone , 1972 .

[41]  M. Bonneville,et al.  Complex interplay of activating and inhibitory signals received by Vgamma9Vdelta2 T cells revealed by target cell beta2-microglobulin knockdown. , 2006, Journal of immunology.

[42]  A. Evdokiou,et al.  Death to the bad guys: Targeting cancer via Apo2L/TRAIL , 2005, Apoptosis.

[43]  N. Liabakk,et al.  Monoclonal antibodies against TRAIL. , 2004, Vitamins and hormones.

[44]  中谷 文彦.  Identification of p21WAF1/CIP1 as a direct target of EWS-Fli1 oncogenic fusion protein , 2004 .