Cytoprotective effects of IAPs revealed by a small molecule antagonist.

Homeostatic regulation of metazoan cell number is dependent on a tightly regulated balance between the proliferation and death of cells [1, 2]. Deregulation of this balance is a hallmark of many disease states, and the activities of key components of both the proliferative and apoptotic (programmed cell death) machinery are altered in a wide variety of disorders including neurodegenerative, autoimmune and neoplastic diseases [3–5], and factors that influence this balance are of great significance in treating a host of diseases. One important group of factors that regulate apoptosis is the TNF receptor superfamily which, following engagement with its cognate ligands, can elicit pro-survival and/or pro-apoptotic responses [6]. Several TNF receptor family members have attracted much attention as therapeutic targets for the treatment of cancers and immunological disorders. For example, cells from a range of malignancies, including cancers of prostate, colon and hepatic origin, appear highly sensitive to the pro-apoptotic ligand, TRAIL [7–9], and this has led to the development of TRAIL ligands and agonists as potential therapeutic tools for cancer treatment. The central effectors of apoptosis are caspases, a family of intracellular cysteine proteases with specificity for aspartate-containing residues [10, 11]. Caspases function in a hierarchical manner; they are initially synthesized as inactive zymogens and, following activation, upstream or initiator caspases such as Caspases-8 and -9 can become activated by oligomerization, through a process referred to as the induced proximity model [12], which subsequently leads to the cleavage and activation of effector caspases, such as Caspases-3 and -7. IAP (inhibitor of apoptosis) proteins are a group of intracellular proteins, several of which have been shown to directly inhibit caspases [13]. IAPs are characterized by the presence of one or more Baculoviral IAP repeat (BIR) domains, which bind directly to caspases [14]. The most intensively studied IAP protein is X-linked IAP (XIAP), which contains three BIRs. The most carboxy terminal BIR (BIR3) is necessary and sufficient for binding and inhibition of Caspase-9 by XIAP, while BIR2, together with a short proximal amino terminal domain, is involved in binding and inhibition of Caspases-3 and -7 [15, 16]. Interestingly, XIAP has been shown to be elevated in several malignancies [17–19], and so has become a promising target in anti-cancer therapies. The caspase-inhibitory property of XIAP can be neutralized by Smac/DIABLO, a nuclear-encoded, mitochondrial protein that is released into the cytosol following mitochondrial permeabilization [20, 21]. Binding of Smac to XIAP can displace the XIAP:caspase interaction, releasing the caspase and lowering the cellular apoptotic threshold. Many seminal studies have revealed the nature of the Smac:XIAP interaction [20–23], and a number of synthetic compounds have been developed that resemble the XIAP-interacting interface in Smac. Interestingly, several of these compounds have also been found to interact with at least two other IAPs, c-IAP1 and c-IAP2, even though the experimental strategies leading to their discovery were based on the XIAP-Smac/DIABLO interaction paradigm. The data suggest that targeting c-IAP1 and c-IAP2 with Smac mimetics results in a lowering of the apoptotic threshold indirectly, by affecting the activation of the NF-κB transcriptional network, rather than directly relieving the constraints on caspases [24–26]. Much less clear, however, is the relative contribution of XIAP to the pro-apoptotic effects of these compounds, and how suitable XIAP may be as a future molecular target for drug design. In this study we examine the role of XIAP in TRAIL-induced apoptosis, by comparing apoptotic signaling in an XIAP-expressing cell line with a derivative line in which XIAP was ablated by homologous recombination. Using this approach, we compared the effects of combined treatment of TRAIL or TNF with a recently identified IAP inhibitor, {"type":"entrez-protein","attrs":{"text":"AEG40730","term_id":"333957922","term_text":"AEG40730"}}AEG40730. We find that {"type":"entrez-protein","attrs":{"text":"AEG40730","term_id":"333957922","term_text":"AEG40730"}}AEG40730 can induce the degradation of the c-IAPs, and this is likely to be of central importance when compounds such as {"type":"entrez-protein","attrs":{"text":"AEG40730","term_id":"333957922","term_text":"AEG40730"}}AEG40730 are used to sensitize cells to the cytotoxic effects of TNF. Interestingly, in addition to targeting c-IAPs, {"type":"entrez-protein","attrs":{"text":"AEG40730","term_id":"333957922","term_text":"AEG40730"}}AEG40730 also triggered the degradation of XIAP, and this combined effect was found to significantly augment cell killing when utilized in combination with TRAIL or TNF. These findings reveal that Smac antagonists such as {"type":"entrez-protein","attrs":{"text":"AEG40730","term_id":"333957922","term_text":"AEG40730"}}AEG40730 target multiple cellular IAPs, including XIAP, to potentiate apoptosis, and underscore the therapeutic potential of these compounds in combination with different primary apoptotic inducers.