Receptor-selective Mutants of Apoptosis-inducing Ligand 2/Tumor Necrosis Factor-related Apoptosis-inducing Ligand Reveal a Greater Contribution of Death Receptor (DR) 5 than DR4 to Apoptosis Signaling

Abstract

Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers programmed cell death in various types of cancer cells but not in most normal cells. Apo2L/TRAIL is a homotrimeric protein that interacts with five receptors: death receptor 4 (DR4) and DR5 mediate apoptosis activation, whereas decoy receptor 1 (DcR1), DcR2, and osteoprotegerin counteract this function. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. However, the relative contribution of DR4 and DR5 to ligand-induced apoptosis is unknown. To investigate this question, we generated death receptor-selective Apo2L/TRAIL variants using a novel approach that enables phage display of mutated trimeric proteins. Selective binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective Apo2L/TRAIL variants examined in this study showed a markedly reduced ability to trigger apoptosis, whereas the DR5-selective variants had minimally decreased or slightly increased apoptosis-inducing activity. These results suggest that DR5 may contribute more than DR4 to Apo2L/TRAIL-induced apoptosis in cancer cells that express both death receptors. Apo2L1 (or TRAIL), a member of the tumor necrosis factor (TNF) superfamily, induces apoptosis in a broad spectrum of human cancer cell lines while sparing most normal cells (1). Apo2L/TRAIL triggers apoptosis through binding to the death receptors DR4 (2) and/or DR5 (3, 4). These receptors contain a cytoplasmic death domain that recruits adaptor molecules involved in caspase activation (5). In addition to these two signaling receptors, Apo2L/TRAIL binds to three decoy receptors that inhibit apoptosis induction: DcR1, DcR2, and OPG. DcR1 and OPG lack a cytoplasmic domain, whereasDcR2 has a truncated death domain that is non-functional for apoptosis initiation (1, 6). Like several other members of the TNF superfamily, Apo2L/TRAIL is synthesized as a type II transmembrane protein that can be proteolytically cleaved to release a soluble, homotrimeric molecule. A recombinant version of soluble homotrimeric Apo2L/TRAIL (residues 114 -281) induces apoptosis in various cancer cell lines but not in normal cells (7,8). Administration of soluble Apo2L/TRAIL in mouse xenograft models of human cancer results in marked anti-tumor activity without systemic toxicity (7-9). These results have prompted further evaluation of Apo2L/TRAIL as a potential therapeutic agent for human cancer.Structural studies show that homotrimeric TNF superfamily ligands bind three receptor molecules (10 -12), suggesting that the basic functional signaling unit is trimeric. This notion is further supported by the trimeric structure of certain signaling adaptor molecules that act downstream of the receptors, such as the TNF receptor-associated factors (13,14). Further crosslinking of receptors beyond the trimeric unit in some cases can lead to stronger signal initiation; this can be modeled by ...

Mechanisms of enhancement of TRAIL tumoricidal activity against human cancer cells of different origin by dipyridamole

“…Characterization of the mechanisms underlying this enhancement revealed two novel findings of dipyridamole treatment: (1) upregulation of TRAIL DRs, and (2) downregulation of survivin, which are well-known regulators of TRAIL-triggered apoptotic signals (Azuhata et al, 2006;Carlo-Stella et al, 2007). We have also shown that DR5 contributed preferentially toward dipyridamole-induced TRAIL sensitization, corroborating the findings reported previously (Kelley et al, 2005). …”

Histone deacetylase inhibitors synergistically potentiate death receptor 4-mediated apoptotic cell death of human T-cell acute lymphoblastic leukemia cells

“…1, 3), suggesting that other factors regulate the sensitivity at the downstream signaling of the receptors. However, it seems that some leukemia cells undergo apoptotic cell death through signaling by either pro-apoptotic DR4 or DR5, but not both, as has been found for various solid tumors [25,26]. The substantial sensitivity of HL-60 (AML) and U937 (monoblastic leukemia) cells to TRAIL-mediated cytotoxicity, but not to AY4, suggests that DR5 is the main receptor that transduces apoptotic signaling in the leukemia cells.…”

“…The receptor-specific agonistic mAb studies demonstrated that, while various solid tumors were more sensitive to DR5-mediated apoptosis than DR4-induced apoptosis [25,26], primary CLL cells underwent apoptotic cell death almost exclusively through DR4, not DR5 [23,24,27]. However, there have been limited studies of how DR4-or DR5-mediated signaling alone or in combination with chemotherapeutic agents can contribute to apoptosis for other leukemia cells, such as ALL, AML, and CML cells.…”

Enforced covalent trimerization increases the activity of the TNF ligand family members TRAIL and CD95L

“…The differential effect of noncrosslinked hTRAIL on KB and L929 cells can be explained by earlier findings demonstrating that TRAILR2 is much more better activated by secondary cross-linked trimers of soluble TRAIL than by non-crosslinked molecules, whereas TRAILR1 is stimulated with the same efficiency by crosslinked and non-crosslinked TRAIL. 5,13,14 Thus, in KB cells that express TRAILR1 and TRAILR2, non-crosslinked TRAIL trimers already significantly induce cell death via the TRAILR1, whereas TRAILR1-mediated cell death is not relevant in the murine L929 cell line, because in mice only a homolog of human TRAILR2 exists. 15,16 In fact, in human Jurkat cells, which express only TRAILR2, noncrosslinked Flag-hTRAIL(95-291) is also practically inactive (Figure 2c).…”