The proapoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, death receptor 4 (DR4, TRAIL-R1) and DR5 (TRAIL-R2), are attractive targets for the development of anti cancer agents because they, selectively induces cell death in cancer cells without cytotoxicity to most normal cells either in vitro or in vivo. In addition to the cognate ligand of recombinant TRAIL, a number of agonistic monoclonal antibodies (mAbs) have been developed by targeting DR4 or DR5, some of which are now under various phases of clinical trials. However, many cancer cells with apoptotic weak points are resistant to TRAIL or agonistic mAbs, limiting their potential as an anti-cancer therapeutics. We developed agonistic human single chain variable fragment (scFv) antibody HW1 specifically targeting DR5 and Kringle domain (KD) variant KD548-Fc targeting both DR5 and DR4, both of which induce cell death in various cancer cells, including TRAIL-resistant cells. In this thesis I show detailed cell death mechanisms of anti-DR5 HW1 and anti-DR4/DR5 KD548-Fc in cancer cells .
In Chapter 1, general introduction was described about TRAIL, its receptors, and the cell death mechanism(s). Also, state-of-the-art of development of agonistic mAbs was described.
In Chaptor 2, the cell death mode of anti-DR5 scFv HW1. which specifically binds to DR5 without competing with TRAIL for the binding, was described. HW1 treatment as a single agent induces autophagic cell death in a variety of both TRAIL-sensitive and TRAIL-resistant cancer cells, but exhibits much less cytotoxicity on normal cells. HW1-mediated autophagic cell death occurs predominantly via the c-Jun NH2-terminal kinase (JNK) pathway in a caspase-independent manner. Analysis of the DR5-associated signaling complex induced by HW1 exhibits the recruitment of TNF receptor？associated death domain (TRADD) and TNF receptor？associated factor 2 (TRAF2), but not Fas-associated death domain (FADD), caspase-8, or receptor-interacting protein 1 (RIP1), which is distinct from that induced by TRAIL that consists of FADD and caspase-8. Hence, our study provides a new strategy for the elimination of cancer cells, including TRAIL-resistant tumors, through nonapoptotic cell death.
In Chaptor 3, the molecular links between JNK activation and autophagic cell death signaling of HW1 were investigated. The DR5-stimulated JNK activation, which was essential for the autophagic cell death of HCT116 cells induced by anti-DR5 HW1 scFv, resulted in upregulation of Beclin-1 expression, Bcl-2 phosphorylation, and p53 phosphorylation, suggesting that these pro-autophagic signaling pathway are involved in the HW1-mediated autophagic cell death.
In Chaptor 4, the cell death mode of KD548-Fc, an Fc-fused DR5/DR4-dual specific kringle domain variant with partial competition with TRAIL for the binding, was described. Stimulation of DR4 and DR5 by KD548-Fc, activated NADPH oxidase 1 (Nox1) to produce superoxide and accumulate intracellular reactive oxygen species (ROS), leading to sustained JNK activation-dependent apoptotic cell death in human HeLa and Jurkat cells. Importantly, Nox1-mediated production of O2- is critical for KD548-Fc induced caspase independent apoptosis because such death is inhibited by downregulation of the Nox1 protein by siRNA. KD548-Fc treatment induces the formation of DR5/DR4-signaling complex containing TRADD, TRAF2, riboflavin kinase (RFK), Nox1, and small GTPase Rac1. Depletion of RFK, but not TRADD and TRAF2, failed to recruit Nox1 and Rac1 to DR5 and DR4, demonstrating that RKF directly binds to DR5/DR4 and plays a role of adaptor to link DR5/DR4 with Nox1. These results reveal a link between DR5/DR4, RFK and Nox1, providing a mechanism of DR5/DR4-mediated ROS accumulation which subsequently triggers sustained JNK activation-dependent apoptotic cell death in tumor cells.
In conclusion, the results shown here suggest that, DR4 and/or DR5 have a capability to transduce diverse cell death signaling by recruiting distinct intracellular proteins, which may be determined by which regions of the receptor are recognized and stimulated by agonists. Accordingly, deep understanding of the diverse molecular signaling mechanisms mediated by DR4 and/or DR5 and their cross-talks leading to a final outcome of the stimulation will be essential to design next-generation agonists targeting DR4 and/or DR5.