title: DR4 specific TRAIL variants are more efficacious than wild-type TRAIL in pancreatic cancer.
abstract: Current treatment modalities for pancreatic carcinoma afford only modest survival benefits. TRAIL, as a potent and specific inducer of apoptosis in cancer cells, would be a promising new treatment option. However, since not all pancreatic cancer cells respond to TRAIL, further improvements and optimizations are still needed. One strategy to improve the effectiveness of TRAIL-based therapies is to specifically target one of the 2 cell death inducing TRAIL-receptors, TRAIL-R1 or TRAIL-R2 to overcome resistance. To this end, we designed constructs expressing soluble TRAIL (sTRAIL) variants that were rendered specific for either TRAIL-R1 or TRAIL-R2 by amino acid changes in the TRAIL ectodomain. When we expressed these constructs, including wild-type sTRAIL (sTRAIL(wt)), TRAIL-R1 (sTRAIL(DR4)) and TRAIL-R2 (sTRAIL(DR5)) specific variants, in 293 producer cells we found all to be readily expressed and secreted into the supernatant. These supernatants were subsequently transferred onto target cancer cells and apoptosis measured. We found that the TRAIL-R1 specific variant had higher apoptosis-inducing activity in human pancreatic carcinoma Colo357 cells as well as PancTu1 cells that were additionally sensitized by targeting of XIAP. Finally, we tested TRAIL-R1 specific recombinant TRAIL protein (rTRAIL(DR4)) on Colo357 xenografts in nude mice and found them to be more efficacious than rTRAIL(wt). Our results demonstrate the benefits of synthetic biological approaches and show that TRAIL-R1 specific variants can potentially enhance the therapeutic efficacy of TRAIL-based therapies in pancreatic cancer, suggesting that they can possibly become part of individualized and tumor specific combination treatments in the future.
authors.affiliation: a National University of Ireland; Galway; National Centre for Biomedical Engineering Science and Apoptosis Research Centre; Molecular Therapeutics Group ; Galway , Ireland.
authors.forename: Rui
authors.initials: R
authors.lastname: Yu
authors.forename: Stella Maris
authors.initials: SM
authors.lastname: Albarenque
authors.forename: Robbert H
authors.initials: RH
authors.lastname: Cool
authors.forename: Wim J
authors.initials: WJ
authors.lastname: Quax
authors.forename: Andrea
authors.initials: A
authors.lastname: Mohr
authors.forename: Ralf M
authors.initials: RM
authors.lastname: Zwacka
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005260:Female; D015870:Gene Expression; D014644:Genetic Variation; D006801:Humans; D051379:Mice; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000008:administration & dosage / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays
chemical_list: D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D011994:Recombinant Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand
keywords: AML, Acute myeloid leukemia; ANOVA, Analysis of variance between groups; Apoptosis; BSA, Bovine Serum Albumin; Bcl-xL, B-cell lymphoma-extra large; CMV, Cytomegalie virus; CuZnSOD, Copper-Zinc Superoxide Dismutase; DMEM, Dulbecco's modified Eagle's medium; DNA, Deoxyribonucleic acid; DR4 specific TRAIL variant; EGFP, Enhanced green fluorescent protein; ELISA, Enzyme-linked immunosorbent assay; FACS, Fluorescence-activated cell sorting; FADD, Fas-associated protein with death domain; FBS, Fetal bovine serum; FIB, Fibrillin; FLIP, FLICE-like inhibitory protein; Furin CS, Furin cleavage site; IFN-g, Interferon-gamma; ILZ, Isoleucine zipper; MSC, Mesenchymal stem cell; NF-κB, Nuclear factor kappa-light-chain-enhancer of activated B cells; OPG, Osteoprogerin; PBS, Phosphate buffered saline; PCR, Polymerase chain reaction; RANKL, Receptor activator of nuclear factor kappa-B ligand; RNAi, RNA interference; RPMI 1640 medium, Roswell Park Memorial Institute 1640 medium; SDS, Sodium dodecyl sulphate; SDS-PAGE, SDS-Polyacrylamide gel electrophoresis; SEM, Standard error of the mean; TNF, Tumor necrosis factor; TRAIL; TRAIL receptor; TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R1/DR4, TRAIL-receptor 1/Death – receptor 4; TRAIL-R2/DR5, TRAIL-receptor 2/ Death – receptor 5; TRAIL-R3/DcR1, TRAIL-receptor 3/Decoy-receptor 1; TRAIL-R4/DcR2, TRAIL-receptor 4/Decoy-receptor 2; XIAP; XIAP, X-linked Inhibitor of apoptosis protein; pancreatic cancer; rTRAIL, recombinant TNF-related apoptosis-inducing ligand; sTRAIL, soluble TNF-related apoptosis-inducing ligand; sh-sequence, short-hairpin sequence