title: EGFR-targeted TRAIL and a Smac mimetic synergize to overcome apoptosis resistance in KRAS mutant colorectal cancer cells.
abstract: TRAIL is a death receptor ligand that induces cell death preferentially in tumor cells. Recombinant soluble TRAIL, however, performs poorly as an anti-cancer therapeutic because oligomerization is required for potent biological activity. We previously generated a diabody format of tumor-targeted TRAIL termed Db(αEGFR-sc)TRAIL, comprising single-stranded TRAIL molecules (scTRAIL) and the variable domains of a humanized variant of the EGFR blocking antibody Cetuximab. Here we define the bioactivity of Db(αEGFR)-scTRAIL with regard to both EGFR inhibition and TRAIL receptor activation in 3D cultures of Caco-2 colorectal cancer cells, which express wild-type K-Ras. Compared with conventional 2D cultures, Caco-2 cells displayed strongly enhanced sensitivity toward Db(αEGFR)-scTRAIL in these 3D cultures. We show that the antibody moiety of Db(αEGFR-sc)TRAIL not only efficiently competed with ligand-induced EGFR function, but also determined the apoptotic response by specifically directing Db(αEGFR)-scTRAIL to EGFR-positive cells. To address how aberrantly activated K-Ras, which leads to Cetuximab resistance, affects Db(αEGFR-sc)TRAIL sensitivity, we generated stable Caco-2tet cells inducibly expressing oncogenic K-Ras(G12V). In the presence of doxycycline, these cells showed increased resistance to Db(αEGFR-sc)TRAIL, associated with the elevated expression of the anti-apoptotic proteins cIAP2, Bcl-xL and FlipS. Co-treatment of cells with the Smac mimetic SM83 restored the Db(αEGFR)-scTRAIL-induced apoptotic response. Importantly, this synergy between Db(αEGFR)-scTRAIL and SM83 also translated to 3D cultures of oncogenic K-Ras expressing HCT-116 and LoVo colorectal cancer cells. Our findings thus support the notion that Db(αEGFR)-scTRAIL therapy in combination with apoptosis-sensitizing agents may be promising for the treatment of EGFR-positive colorectal cancers, independently of their KRAS status.
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Möller
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Siegemund
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Sven
authors.initials: S
authors.lastname: Beyes
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Freiburg, Germany; Faculty of Biology, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.
authors.forename: Ricarda
authors.initials: R
authors.lastname: Herr
authors.affiliation: Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: Lecis
authors.affiliation: Department of Experimental Oncology, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Delia
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Kontermann
authors.affiliation: Institute of Molecular Medicine and Cell Research (IMMZ), Faculty of Medicine, Albert-Ludwigs-University of Freiburg, Freiburg, Germany; Centre for Biological Signalling Studies BIOSS, Albert-Ludwigs-University of Freiburg, Freiburg, Germany.
authors.forename: Tilman
authors.initials: T
authors.lastname: Brummer
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Pfizenmaier
authors.affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.
authors.forename: Monilola A
authors.initials: MA
authors.lastname: Olayioye
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D040761:Biomimetic Materials / Q000494:pharmacology*; D045744:Cell Line, Tumor; D000068818:Cetuximab / Q000494:pharmacology; D015179:Colorectal Neoplasms / Q000473:pathology*; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism; D058990:Molecular Targeted Therapy; D009154:Mutation; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D018631:ras Proteins / Q000235:genetics*
chemical_list: D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; C117307:KRAS protein, human; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D000068818:Cetuximab