암세포 표면의 세포사멸수용체 5 및 4를 표적으로 작용하는 인간항체와 크링글 도메인의 세포사멸 기작 연구
Cell death mechanisms of agonistic human antibody and Kringle domain variants targeting the death receptor 5 and/or 4
초록/요약
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.
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CONTENTS
ABSTRACT 8
LIST OF TABLES 11
LIST OF FIGURES 12
1. General introduction16
1.1. TRAIL and TRAIL receptors 16
1.2. TRAIL-induced apoptosis and mechanisms of cell death resistance 18
1.3. Other signal transduction pathways mediated through TRAIL receptors 20
1.4. Clinical application of agonistic monoclonal antibodies against TRAIL receptors 21
1.5. Programmed cell death (PCD) 24
2. A human scFv antibody against DR5 induces autophagic cell death in both TRAIL-sensitive and TRAIL-resistant cancer cells 27
2.1. Summary 27
2.2. Introduction 28
2.3. Materials and Methods 30
2.3.1. Cell lines and reagents 30
2.3.2. Cell surface binding assay 30
2.3.3. Transmission electron microscopy 31
2.3.4. Light and fluorescence microscopy 31
2.3.5. Western blotting 32
2.3.6. RNA interference 32
2.3.7. Immunoprecipitations 33
2.4. Results 34
2.4.1. HW1 specifically binds to DR5 without competition with TRAIL for the binding 34
2.4.2. HW1 induces cell death in multiple TRAIL-sensitive and TRAIL-resistant tumor cells with much less cytotoxicity on normal cells 38
2.4.3. HW1 induces autophagy in both TRAIL-sensitive and TRAIL-resistant tumor cells 45
2.4.4. HW1-induced autophagic cell death occurs mainly through the JNK pathway 49
2.4.5. TRADD and TRAF2, but not FADD, caspase-8, or RIP, are recruited to the HW1-DR5 signaling complex 55
2.5. Discussion 57
3. Upregulation of Beclin-1 expression and phosphorylation of Bcl-2 and p53 are involved in the JNK-mediated autophagic cell death 60
3.1. Summary 60
3.2. Introduction 61
3.3. Materials and Methods 63
3.3.1. Cell lines and reagents 63
3.3.2. Cell viability assays 63
3.3.3. Western blotting analyses and immunoprecipitations64
3.4. Results 65
3.4.1. Constitutive JNK activation enhanced HW1-mediated cell death 65
3.4.2. HW1-induced JNK activation mediated upregulation of Beclin-1 expression and Bcl-2 phosphorylation 69
3.4.3. HW1-induced JNK activation phosphorylated p53, which in turn inhibited mTOR activity and upregulated DRAM expression 72
3.5. Discussion 75
4. TRAIL receptor 1 and 2 can generate reactive oxygen species by activating Nox1 NADPH oxidase 76
4.1. Summary 76
4.2. Introduction 77
4.3. Materials and Methods 79
4.3.1. Cell lines and reagents 79
4.3.2. Cell viability assays 79
4.3.3. Transmission electron microscopy 80
4.3.4. Confocal fluorescence microscopy 80
4.3.5. DNA fragmentation assay 81
4.3.6. Caspase-8 and -3 activation assay 81
4.3.7. Western blotting 81
4.3.8. Immunoprecipitations 82
4.3.9. Measurement of mitochondrial membrane potential83
4.3.10. NADPH Oxidase Assay 83
4.3.11. Measurement of ROS production 84
4.3.12. Protein purification 84
4.3.13. ELISA 86
4.3.14. Competition ELISA 86
4.3.15. Annexin V and PI staining 87
4.3.16. RNA interference 88
4.3.17. Surface Plasmon resonance (SPR) 89
4.4. Results 89
4.4.1. KD548-Fc specifically binds to both DR4 and DR5 without cross-reactivity with other death receptors 89
4.4.2. KD548-Fc induces apoptotic cell death in various cancer cell lines
94
4.4.3. KD548-Fc induces caspase-independent, apoptosis cell death through JNK activation 97
4.4.4. KD548-Fc induces ROS accumulation and prolonged JNK activation.
99
4.4.5. KD548-Fc induces a distinct signaling complex composed of DR4/5, TRADD, TRAF2, RFK, Nox1 and Rac1, but not caspase-8, FADD or RIP1 102
4.4.6. Nox1 NADPH oxidase is responsible for KD548-Fc induced O2- generation, cell death and JNK activation 107
4.4.7. Mapping of DR4-RFK and DR5-RFK interacting domains 112
4.5. Discussion 117
5. Conclusions 119
6. References 121
7. Abstract in Korean 130
