IAP-targeted therapies for cancer

Abstract

DNA damage, chromosomal abnormalities, oncogene activation, viral infection, substrate detachment and hypoxia can all trigger apoptosis in normal cells. However, cancer cells acquire mutations that allow them to survive these threats that are part and parcel of the transformation process or that may affect the growth and dissemination of the tumor. Eventually, cancer cells accumulate further mutations that make them resistant to apoptosis mediated by standard cytotoxic chemotherapy or radiotherapy. The inhibitor of apoptosis (IAP) family members, defined by the presence of a baculovirus IAP repeat (BIR) protein domain, are key regulators of cytokinesis, apoptosis and signal transduction. Specific IAPs regulate either cell division, caspase activity or survival pathways mediated through binding to their BIR domains, and/or through their ubiquitin-ligase RING domain activity. These protein–protein interactions and post-translational modifications are the subject of intense investigations that shed light on how these proteins contribute to oncogenesis and resistance to therapy. In the past several years, we have seen multiple approaches of IAP antagonism enter the clinic, and the rewards of such strategies are about to reap benefit. Significantly, small molecule pan-IAP antagonists that mimic an endogenous inhibitor of the IAPs, called Smac, have demonstrated an unexpected ability to sensitize cancer cells to tumor necrosis factor-α and to promote autocrine or paracrine production of this cytokine by the tumor cell and possibly, other cells too. This review will focus on these and other developmental therapeutics that target the IAPs in cancer.

Introduction

The inhibitor of apoptosis (IAP) gene family regulates the cell's decision to live or die in response to daily stresses and insults. In this review, we provide an overview of the mammalian IAPs, with particular emphasis on their many cellular roles in apoptosis suppression, signal transduction and proliferation. In addition, we address the relationship of the IAPs with cancer, and the various therapeutic modalities targeting these gene products for clinical benefit.

The first discovered cellular, non-viral, IAP is the mammalian gene NAIP (Roy et al., 1995). The human IAP family has rapidly expanded to include seven other members: XIAP, cIAP1, cIAP2 (Rothe et al., 1995; Duckett et al., 1996; Liston et al., 1996; Uren et al., 1996), ILP2 (Lagace et al., 2001; Richter et al., 2001), BRUCE (Hauser et al., 1998; Chen et al., 1999), survivin (Ambrosini et al., 1997) and livin (Vucic et al., 2000; Kasof and Gomes, 2001) (Figure 1 and Table 1). The IAPs, deserving of their name, effectively suppress apoptosis induced by a variety of stimuli, including death receptor activation, growth factor withdrawal, ionizing radiation, viral infection, endoplasmic reticulum stress and genotoxic damage (LaCasse et al., 1998; Liston et al., 2003; Cheung et al., 2006b; Hunter et al., 2007).

Figure 1
figure1

Domain structure of the mammalian inhibitor of apoptosis (IAP) family. The domain organization is shown for the eight mammalian IAPs. The presence of at least one baculovirus IAP repeat (BIR) domain is the defining characteristic of the IAP family. The human IAPs possess either one (survivin, BRUCE, livin and ILP2) or three tandem amino-terminal BIR domains (XIAP, cIAP1, cIAP2 and NAIP). Several IAPs contain an E3 ubiquitin ligase zinc-finger (RING) domain at the carboxy terminus. Both cIAP1 and cIAP2 possess a caspase-recruitment (CARD) domain in the linker region between the BIR and the RING domains. Uniquely, NAIP possesses a nucleotide-binding and oligomerization (NOD) domain, as well as a leucine-rich repeat (LRR) domain seen in other NOD-like receptor (NLR) proteins involved in innate immunity. BRUCE contains an ubiquitin-conjugating (UBC) domain but no RING domain. Survivin contains a coiled-coil (CC) domain that binds chromosomal passenger proteins, INCENP and borealin. The BIR domains shown in green are longer type 2 domains found in the IAPs with roles in mitosis, compared with the type 1 BIR domains, in black, with roles in signal transduction and apoptosis suppression. Note that the drawings are not to scale and that the length of the IAP proteins, and their splice isoforms, is shown in Table 1.

Table 1 Properties of the human (and mouse) IAP genes and proteins

The defining characteristic of the IAPs is a baculovirus IAP repeat (BIR) domain. IAPs contain 1–3 BIR domains of 70–80 amino acids encoding a C2HC-type zinc-finger motif that tetrahedrally chelates one zinc atom, and forms a globular structure consisting of four or five α-helices and a variable number of antiparallel β-pleated sheets (Hinds et al., 1999; Sun et al., 1999). The two human IAPs, survivin and BRUCE, both with a role in cell mitosis (Ruchaud et al., 2007; Pohl and Jentsch, 2008), have an earlier evolutionary origin compared with the type 1 human members with roles in apoptosis and immunity (Robertson et al., 2006).

In addition to BIR domains, IAPs contain various other domains that are summarized in Figure 1. Several mammalian IAP family members contain a carboxy-terminal RING (really interesting new gene) zinc-finger domain. In XIAP, cIAP1 and cIAP2, this RING domain has been shown to possess E3 ubiquitin ligase activity, directly regulating auto- or trans-ubiquitination and protein degradation (Yang et al., 2000; Suzuki et al., 2001; Silke et al., 2005; Cheung et al., 2008).

Within the IAP family, the presence of a CARD domain is unique to cIAP1 and cIAP2 (Figure 1). The role of the CARD domain in the IAPs is unknown but they undoubtedly function in protein–protein interactions (Martin, 2001). Notably, a functional nuclear export signal exists within the CARD domain of cIAP1, which appears to be important for cell differentiation (Plenchette et al., 2004), revealing one such role for the CARD domain. However, other functions likely still exist. NAIP is unique among the IAPs in that it possesses a nucleotide-binding and oligomerization domain as well as leucine-rich repeats that classify this IAP along with other proteins involved in innate immunity, called the nucleotide-binding and oligomerization domain-like receptors (Kanneganti et al., 2007; Mariathasan and Monack, 2007; Ting et al., 2008). BRUCE, the largest of the IAPs, lacks a RING domain, but instead possesses an ubiquitin-conjugating domain capable of performing a similar function. Survivin, the smallest IAP, possesses a coiled-coil domain required for its interaction with chromosomal passenger proteins, INCENP and borealin, and for the maintenance of residency in the nucleus (Chantalat et al., 2000; Engelsma et al., 2007; Jeyaprakash et al., 2007) (Figure 1).

Several alternatively spliced mRNA products for the survivin, livin and cIAP2 genes exist that lead to various protein isoforms of differing size and function (Conway et al., 2000; Ashhab et al., 2001; Crnkovic-Mertens et al., 2006; Mosley and Keri, 2006; Noton et al., 2006; Kappler et al., 2007; Knauer et al., 2007; Mola et al., 2007; Nachmias et al., 2007; Hu et al., 2008) (Table 1). The presence of the two closely related genes, cIAP1 and cIAP2, in mammals is confusing and has led to some misidentification errors in databases and has produced some confusion in the published literature. This gene duplication event is fairly recent, as zebrafish only possess one cIAP gene (Santoro et al., 2007). Ts-IAP/hILP2 gene is present on an autosomal chromosome (Lagace et al., 2001) (Table 1 and Figure 1) and found only in great apes (Richter et al., 2001). Ts-IAP produces a mutated, truncated and unstable version of XIAP that, because of its autosomal location, is not subject to X-chromosome inactivation (Lagace et al., 2001; Shin et al., 2005).

IAP proteins: multifaceted inhibitors of apoptosis

Inhibition of caspase function

All apoptotic signaling pathways converge on the caspases making these proteases the ultimate effectors of apoptotic cell death. Not surprisingly, caspases need to be tightly regulated, as their inappropriate activation can have severe consequences. The first level of caspase regulation is seen in their structure and activation. Caspases are synthesized as inactive zymogens and are only activated through signaling through strictly controlled pathways. A second level of regulation involves the specific inhibition of active caspases by naturally occurring cellular inhibitors.

The IAPs are the only known endogenous proteins that regulate the activity of both initiator and effector caspases. Controlled expression of the IAPs has been shown to influence cell death in a variety of contexts and is believed to have important consequences with respect to human cancer (LaCasse et al., 1998). The mechanism by which the IAPs inhibit apoptosis was first interrogated in the laboratory of John Reed (Deveraux et al., 1997, 1998). In these early studies, XIAP was found to prevent caspase-3 processing in response to caspase-8 activation. Therefore, XIAP was suggested to inhibit the extrinsic apoptotic signaling by blocking the activity of the downstream effector caspases, as opposed to interfering directly with caspase-8 activation (Deveraux et al., 1997, 1998). Supporting this concept, XIAP was shown to specifically bind to caspase-3 and -7, but not to caspase-1, -6, -8 or -10 (Deveraux et al., 1997, 1998). In vitro assays confirmed that XIAP, as well as cIAP1 and cIAP2, could prevent downstream proteolytic processing of pro-caspase-3, -6 and -7 by blocking cytochrome c-induced activation of pro-caspase-9 in the intrinsic pathway (Deveraux et al., 1998).

The role of the IAPs in apoptosis suppression is continuously being evaluated. The Salvesen's laboratory (Eckelman and Salvesen, 2006; Eckelman et al., 2006) suggests that only XIAP is a direct inhibitor of caspases, and that other IAPs simply bind caspases but do not inhibit them. This suggestion is based on the fact that XIAP is relatively stable and exhibits the greatest potency for caspase inhibition compared with the other IAPs.

Importantly, although cIAP1 and cIAP2 can bind to caspases, their ability to inhibit caspases in vitro has been attributed to an artifact of the glutathione S-transferase fusion moiety, which allows aggregation and steric inhibition of the caspases by the cIAP fusions at concentrations that are not achieved physiologically (Eckelman and Salvesen, 2006). Nevertheless, cIAP1 and cIAP2 can be considered true antagonists of caspase function. For example, cIAP1 and cIAP2 antagonize caspase activity when co-expressed in yeast (Wright et al., 2000), rescuing them from cell death. Yeast represents a simple genetic system devoid of many complicating factors (Jin and Reed, 2002) and is more physiologically relevant compared with in vitro enzyme systems. However, this yeast system does not address the issue of ‘direct’ caspase inhibition, as cIAPs may inactivate caspase through the ubiquitin–proteasomal system, or by other direct or indirect means.

In summary, caspase-3 is inhibited exclusively by the linker region between BIR1 and BIR2, whereas caspase-7 inhibition requires both the linker region and the BIR2 domain of XIAP. It is evident that caspase-9 is inhibited in direct response to BIR3 binding, thereby preventing further caspase-9 activation as well as suppressing downstream effector caspase activity. Thus far, none of the IAP BIR1 domains has been shown to have any caspase-inhibiting activity; however, Akt/PKB (protein kinase B)-mediated phosphorylation at serine 87 within BIR1 is suggested to reduce auto-ubiquitination and stabilizes XIAP (Dan et al., 2004; but see Cheung et al., 2008). BIR1 therefore, appears to play a regulatory role rather than directly participating in apoptosis suppression. The BIR1 domains of cIAP1 and cIAP2, for example, are proposed to play a role in tumor necrosis factor (TNF) receptor-associated factor (TRAF) interactions and ubiquitination reactions (Vaux and Silke, 2005; Samuel et al., 2006). Furthermore, the BIR1 domain of XIAP interacts with TAB1 (KD=14.3 μM) and induces nuclear factor-κB (NF-κB) activation through the TAK1 pathway (Lu et al., 2007).

Ubiquitination and RING E3 ligase function of the IAPs

The post-translational modification of proteins with ubiquitin chains is an important regulatory mechanism that can result in their proteasomal degradation or allow signal transduction to occur, depending on the type of ubiquitin linkage used. Typically, a K48 ubiquitin-linkage destines the protein to the proteasome, whereas a polyubiquitin chain based on K63 is recognized for cellular signal transduction (Hochstrasser, 2006; Hunter, 2007). The ubiquitination and subsequent proteasomal degradation or signaling of the IAPs may be a key regulatory event in the apoptotic program. The carboxy-terminal RING domain of the IAPs mediates both ubiquitin-dependent and -independent degradation of the IAPs, as well as that of their substrates (Yang et al., 2000; Suzuki et al., 2001; Silke et al., 2005; Cheung et al., 2008). RING domain-containing proteins possess E3 ubiquitin ligase activity and function as specific adapters by recruiting target proteins to a multi-component complex. Earlier studies on the RING domains of XIAP and cIAP1 show that this domain is involved in the ubiquitination and degradation of the IAPs in response to apoptotic triggers (Yang et al., 2000). Other studies show that XIAP and cIAP1 are able to promote the ubiquitination and proteasomal degradation of caspase-3 and -7, thereby enhancing the antiapoptotic effect of the IAPs (Suzuki et al., 2001). Although the RING domain is involved in the ubiquitination and degradation of the IAPs, it remains unclear whether this activity enhances the antiapoptotic activity of the IAPs, or actually antagonizes the activity. Studies using RING deletion mutants have provided evidence for both scenarios (Yang et al., 2000; Suzuki et al., 2001).

K322 and K328 are the sites of ubiquitination in XIAP, and the role of XIAP ubiquitination has been examined by site-directed mutagenesis (Shin et al., 2003; Cheung et al., 2008). Compared with XIAP wild type, XIAP K322/328R mutant protein in cultured cells does not lead to XIAP accumulation and reveal no differences in their ability to protect against Bax- or Fas-induced apoptosis (Shin et al., 2003). In response to Sindbis virus infection, mammalian cells produce a pro-apoptotic carboxy-terminal fragment of cIAP1 containing the CARD and RING domains (cIAP1-CR) (Clem et al., 2001). Ectopic expression of cIAP1-CR (Cheung et al., 2008), as well as cIAP1-RING domain fused to a carrier (Silke et al., 2005), targets XIAP for degradation. However, the role of XIAP ubiquitination in this processing appears minimal as XIAP mutants that were not ubiquitinated were also readily degraded by cIAP1-CR (Cheung et al., 2008). These findings suggest that ubiquitin-mediated destruction of the XIAP may not be as significant as believed. Similarly, ubiquitination of livin is likewise not a prerequisite for RING-mediated turnover (Cheung et al., 2008). In contrast, for the metabolism of cIAP1 and cIAP2, RING-mediated ubiquitination remains essential (Silke et al., 2005; Cheung et al., 2008). These divergent responses of IAPs to ubiquitination might in part explain the rapid degradation of cIAP1 and cIAP2, but not XIAP, in response to Smac mimetic compounds (SMCs, see below) (Vince et al., 2007; Vucic and Fairbrother, 2007). Taken together, these findings indicate that ubiquitination of XIAP and livin, but not cIAP1 and cIAP2, is uncoupled from the regulation of its abundance, and might yet serve an undetermined role.

Although the fate of the ubiquitinated cIAPs appears certain, notwithstanding de-ubiquitination, the ultimate destination for the destruction of the ubiquitinated species depends on the initial trigger. The landmark study of Yang et al. (2000) has shown that in response to glucocorticoid or etoposide, cIAP1 degradation is proteasome dependent. More recently, Vince et al. (2008) have shown that TWEAK, a ligand for the TNF superfamily receptor FN14, can induce lysosomal degradation of cIAP1 in a complex with TRAF2. Ubiquitin-K29 linkage is known to mediate lysosomal degradation (Chastagner et al., 2006). Whether or not cIAP1 degradation in response to TWEAK is facilitated by K29-type linkage, some other exotic type of mixed linkage or other undefined molecular interactions, remains to be seen. Nonetheless, although ubiquitination of the cIAPs is not necessary as a signal to the proteasome, it does appear to be a common tag for their destruction.

Typically, cIAP2 is present in low abundance in the cell. The levels of cIAP2 are presumably maintained through constitutive ubiquitination and subsequent degradation by cIAP1 (Conze et al., 2005; Cheung et al., 2008; Mahoney et al., 2008). Insight into cIAP1 E3 ligase function came from the generation of the cIAP1-null mouse (Conze et al., 2005). Although these mice appeared to be both viable and fertile, they had significantly elevated cIAP2 protein levels with normal mRNA levels, suggesting that in a normal setting, cIAP1 ubiquitination of cIAP2 leads to low protein levels of cIAP2 (Conze et al., 2005). Furthermore, co-expression and in vitro binding studies of cIAP1 and cIAP2 (along with TRAF1 or TRAF2), demonstrate that the cIAPs and TRAF proteins form a multimeric complex. In this complex, TRAF1 and TRAF2 appear to function as adaptors, bringing cIAP1 or cIAP2 together, allowing cIAP1 to ubiquitinate cIAP2 (Conze et al., 2005). The upregulation of cIAP2 in the absence cIAP1 was similarly seen in mouse embryonic fibroblasts (MEFs) (Mahoney et al., 2008) and certain tumor cells (Cheung et al., 2008) transiently silenced by cIAP1-targeted RNAi. Collectively, these observations support the concept that IAP–IAP or IAP–partner interactions play roles in regulating their relative abundance, through post-translational modifications, to impact on the progression of apoptosis.

Modulation of survival signal-transduction pathways

Over the past year, a series of landmark papers (Gaither et al., 2007; Petersen et al., 2007; Santoro et al., 2007; Varfolomeev et al., 2007; Vince et al., 2007; Bertrand et al., 2008; Mahoney et al., 2008; Wang et al., 2008) have demonstrated that the cellular IAP proteins (cIAP1 and 2) are critical regulators of the NF-κB signal transduction. NF-κB is a pleiotropic signaling pathway involved in a diverse range of biological processes, particularly in innate and adaptive immunity, as well as in proliferation and survival. In response to receptor stimulation by ligands of the TNF family or to various intracellular stressors (for example, DNA damage or viral infection), a series of signaling events are initiated that culminate in the activation of heterodimeric NF-κB transcription factors. On activation, NF-κB heterodimers translocate into the nucleus and transactivate a large number of target genes, which in turn elicit the particular biological response. Depending on how the NF-κB pathway is activated, the response generated can be classified into the so-called classical or alternative NF-κB pathways. Although cross-talk exists, these two pathways generally have distinct functions and exert a unique biological footprint. Typically, the classical pathway is stimulated by TNFα, DNA damage or viruses, and occurs in nearly all cell types, exerting its effect through p65/p50 heterodimers. In contrast, the alternative pathway is often stimulated by CD40-L, B-cell activation factor (BAFF)-L or TWEAK, occurring predominantly in lymphoid cells to elicit function through p52/RelB heterodimers.

A role for cIAP1 and cIAP2 in NF-κB signaling has long been implied, although this has only been conclusively demonstrated recently. The earliest evidence was generated in David Goeddel's laboratory, where cIAP1 and cIAP2 were shown to interact with TRAF2 (Rothe et al., 1995; Shu et al., 1996). TRAF2 is an adapter protein that functions in both the classical and alternative NF-κB pathways, as well as in mitogen-activated protein kinase (MAPK) signaling pathways (that is, p38 and Jun N-terminal kinase (JNK) signaling). Goeddel's group also showed that cIAP1 and cIAP2 get recruited to TNF receptors in response to TNFα, a process that is dependent on their interaction with TRAF2. Accordingly, it was hypothesized that cIAP1 and cIAP2 participate in TNFα-mediated NF-κB activation. Subsequent overexpression studies supported this notion, demonstrating that cIAP2 has the capacity to activate NF-κB (Chu et al., 1997) and that a RING-deficient version of cIAP1 can cooperate in the activation of NF-κB by TRAF2 (Samuel et al., 2006).

Unexpectedly, when the cIAP1 and cIAP2 knockout mice were generated, they were found to be asymptomatic (Conze et al., 2005; Conte et al., 2006). Furthermore, their primary cells displayed normal TNFα-induced NF-κB activation and were not sensitized to TNFα-mediated cell death (Conze et al., 2005). As such, the physiological involvement of cIAP1 and cIAP2 in NF-κB signaling was called into question. However, a confounding variable in both of these knockout mice is the presence of the other highly similar cIAP, which may be able to compensate. Unfortunately, a cIAP1/cIAP2 double knockout mouse has not yet been generated, as the chromosomal proximity of these two genes precludes a traditional cross-breeding strategy. To circumvent this, we (Mahoney et al., 2008) and others (Gaither et al., 2007; Petersen et al., 2007; Santoro et al., 2007; Varfolomeev et al., 2007; Vince et al., 2007; Bertrand et al., 2008; Wang et al., 2008) have combined genetic knockout and siRNA-mediated knockdown methodologies to generate various versions of cIAP1 and cIAP2 double ‘null’ cells. Additionally, these studies have used potent small molecule compounds that induce the specific degradation of cIAP1 and cIAP2, thereby rendering cells ‘null’ for their expression. Using these approaches, cIAP1 and cIAP2 have been conclusively shown to be critical regulators of both classical and alternative NF-κB signal transduction and are functionally redundant in these roles. Importantly, cIAP1 and cIAP2 regulate these pathways in normal as well as cancer cells.

In the classical pathway, the best characterized role of cIAP1 and cIAP2 is as an essential positive regulator of TNFα-mediated activation (Santoro et al., 2007; Bertrand et al., 2008; Mahoney et al., 2008). In the absence of both cIAP1 and cIAP2, TNFα-mediated NF-κB signaling is dramatically attenuated in many cells, including normal primary cells as well as transformed cancer cells. Consequently, the dual loss of cIAP1 and cIAP2 greatly sensitizes cells to TNFα-mediated apoptosis. Mechanistically, TNFα signals through its receptor TNFR1. On TNFα occupancy, TNFR1 rapidly recruits the TNFR-associated death domain (TRADD) protein as well as the receptor-interacting protein (RIP) 1. TRADD binding in turn recruits TRAF2 and cIAP1/cIAP2 to form a large membrane complex. When this occurs, RIP1 gets modified with long K63-linked polyubiquitin chains, which serves as a docking site for the downstream kinases that propagate the NF-κB signal (Ea et al., 2006; Wu et al., 2006) (Figure 2). Originally, the E3 ligase responsible was thought to be TRAF2. TRAF2 is a bona fide E3 ubiquitin ligase, and in its absence TNFα-mediated ubiquitination of RIP1 does not occur (Lee et al., 2004). However, TRAF2 has never been shown to directly target RIP1 for ubiquitination in vitro, which suggests that the loss of TRAF2 indirectly affects RIP1 ubiquitination. Recently, we (Mahoney et al., 2008) have shown that dual loss of cIAP1 and cIAP2 dramatically attenuates TNFα-induced RIP1 ubiquitination, even though both RIP1 and TRAF2 are still recruited to TNFR1. Moreover, Bertrand et al. (2008) have demonstrated that cIAP1 and cIAP2 directly target RIP1 for K63 (and to a lesser extent K48)-linked polyubiquitination in vitro, whereas TRAF2 does neither. Taken together, these data suggest a model in which TRAF2 serves as an adapter protein that bridges the gap between cIAP1/cIAP2 and RIP1 at TNFR1, which enables cIAP1/cIAP2 to target RIP1 for K63-linked polyubiquitination.

Figure 2
figure2

cIAP1 and cIAP2 regulation of classical and alternative nuclear factor-κB (NF-κB) activation pathways. The two major activation pathways for NF-κB are shown. Those tumor necrosis factor (TNF) superfamily receptors that utilize TNF receptor-associated factor 2 (TRAF2) as an adaptor can recruit cIAP1 or cIAP2, which can then positively modulate the classical pathway, or negatively regulate the alternative pathway. In the classical NF-κB pathway, NF-κB dimers (for example, p50/RelA) are retained in the cytoplasm by interaction with IκB. In response to the binding of a ligand to a TNF receptor (TNFR)-related protein, TRAF proteins are recruited to the receptor, and through the ubiquitination of receptor-interacting protein (RIP) kinase (RIP1/RIPK1) suggested to be mediated by the RING domain E3 ligase of cIAP1 and cIAP2, the IKK complex containing IKKα, IKKβ and NEMO (IKKγ) is recruited and activated. The activated IKK complex in turn phosphorylates the inhibitor of κB (IκB), leading to its degradation, thereby releasing NF-κB (p50/RelA) to translocate into the nucleus for transcriptional activation of targeted genes. By contrast, cIAP1 and cIAP2, TRAF2 and TRAF3 represses the alternative NF-κB pathway by promoting the ubiquitination and degradation of NF-κB-inducing kinase (NIK), which is required for phosphorylation of p100 in the p100/RelB complex. Phosphorylated p100 is processed into p52, and NF-κB (that is, the p52/RelB dimer) translocates into the nucleus for transcriptional activation of targeted genes. Hence, the E3 ubiquitin ligase function of cIAP1 or cIAP2, as well as their binding through BIR1 to TRAF2, plays a central role in the regulation of both NF-κB pathways.

Significantly, a recent article by Wang et al. (2008) reported that cIAP1 and cIAP2 negatively regulate RIP1 de-ubiquitination at TNFR1, thereby protecting cells from TNFα-mediated apoptosis. In this study, the small compound-mediated loss of cIAP1 and cIAP2 sensitized cancer cells to TNFα-mediated apoptosis, although oddly there was no effect on NF-κB signaling. This may have been due to the cell lines used, or because the loss of cIAP1 and cIAP2 in these experiments was only partial. Wang et al. (2008) also demonstrated decreased RIP1 ubiquitination at TNFR1 in response to TNFα when cIAP1 and cIAP2 were partially degraded, consistent with a role for cIAP1 and cIAP2 in the ubiquitination of RIP1. However, in contrast to our interpretation (Mahoney et al., 2008) and that of others (Bertrand et al., 2008), these authors suggested that cIAP1 and cIAP2 ‘protect’ RIP1 from de-ubiquitination at TNFR1 rather than promote its ubiquitination. Although further investigation into the role of cIAP1 and cIAP2 in the de-ubiquitination of RIP1 is warranted, the fact that they can directly ubiquitinate RIP1 in vitro strongly suggests that the primary function of the cIAPs is TNFR1 signaling.

In addition to regulating TNFα-mediated NF-κB signaling, recent data have also shown that cIAP1 and cIAP2 also regulate constitutive or ligand-independent classical NF-κB signaling (Varfolomeev et al., 2007; Vince et al., 2007). Intriguingly, in contrast to their role as positive regulators of TNFα-mediated signaling, they act as negative regulators of ligand-independent signaling. Using small molecule cIAP antagonists, both Vince et al. (2007) as well as Varfolomeev et al. (2007) have shown that dual loss of cIAP1 and cIAP2 leads to elevated constitutive classical NF-κB signaling in a variety of cancer cells. Vince et al. (2007) also reported elevated ligand-independent classical NF-κB signaling in MEFs derived from cIAP1−/− mice, which suggested a non-redundant role for cIAP1 in this capacity. However, these MEFs were SV-40 transformed, and we have observed that the expression of cIAP2 is silenced therein (unpublished observation) making them a functional double knockout cell. Mechanistically, preliminary data suggested that loss of cIAP1 and cIAP2 induced the recruitment of RIP1 to TNFR1, although these data were somewhat difficult to interpret. Regardless, it was proposed that cIAP1 and cIAP2 regulate the degree of constitutive NF-κB activation by controlling RIP1 migration to TNFR1. Recent data demonstrate that cIAP1 and cIAP2 constitutively interact with RIP1, lend support to this model (Bertrand et al., 2008; Wang et al., 2008). Clearly, more work is needed to fully substantiate this mechanism, or to propose a new model of how cIAP1 and cIAP2 regulated ligand-independent NF-κB signaling.

As mentioned above, cIAP1 and cIAP2 have also recently been shown to modulate the activity of the alternative NF-κB pathway (Varfolomeev et al., 2007; Vince et al., 2007) (Figure 2). Normally inactive, alternative NF-κB signaling gets activated through the ligation of various TNF receptors such as CD40-R, lymphotoxin β-R and BAFF-R. On receptor ligation, the levels of NF-κB-inducing kinase (NIK) accumulate. NIK is normally co-translationally degraded in a complex containing TRAF2 and the related protein TRAF3. Following sufficient accumulation, NIK in turn phosphorylates downstream kinases that propagate the alternative NF-κB signal. Given that both TRAF2 and TRAF3 proteins have RING zinc-fingers with E3 ligase activity, and that loss of either TRAF2 or TRAF3 results in NIK accumulation and constitutive activation of alternative NF-κB signaling, TRAF2 and/or TRAF3 were believed to target NIK for ubiquitination and proteasomal degradation. Recent studies, however, have revealed that cIAP1 and cIAP2 are the true E3 ligases that constitutively target NIK for K48-linked polyubiquitination and proteasomal degradation (Varfolomeev et al., 2007; Vince et al., 2007). Using small compounds targeting cIAP1 and cIAP2, several groups have now shown that the loss of these IAP proteins leads to the accumulation of NIK and the activation of the alternative pathway. Again, cIAP1 and cIAP2 are functionally redundant in this role, as we have observed that numerous cell lines rendered null for the individual cIAPs using siRNA do not have accumulated levels of NIK, whereas those rendered null for both cIAPs do (unpublished observation). Moreover, both cIAP1 and cIAP2 were shown to directly target NIK for K48-mediated ubiquitination in vitro.

Given the known roles for TRAF2 and TRAF3 in this pathway, a possible model for the control of NIK protein levels is that the TRAF proteins serve as adapters that bring the cIAP proteins into a complex with NIK. Indeed, using a variety of experimental approaches, we have seen that TRAF3 interacts with NIK, whereas TRAF2 interacts with the cIAPs, and that TRAF3 and TRAF2 interact with one another (unpublished observation). These data suggest that TRAF2 and TRAF3 bridge the gap between cIAP1/cIAP2 and NIK, bringing them into a large protein complex (Figure 2). Colocalization by TRAF2 and TRAF3 allows cIAP1 and/or cIAP2 to constitutively target NIK for K48-linked ubiquitination and proteasomal degradation, thereby repressing alternative NF-κB activity.

For the most part, NF-κB signaling strongly favors survival in both the classical and alternative pathways. However, cIAP1 and cIAP2 positively regulate TNFα-mediated and yet negatively regulate ligand-independent classical NF-κB signaling (Santoro et al., 2007; Bertrand et al., 2008; Mahoney et al., 2008), and the alternative pathway (Varfolomeev et al., 2007; Vince et al., 2007). Hence, the intriguing possibility exists that cIAP1 and cIAP2 may have both anti- and pro-survival roles depending on the cell type and context. Indeed, TNFα strongly induces apoptosis in the absence of both cIAP1 and cIAP2 in most cell lines tested to date. In contrast, primary B cells, which rely on alternative NF-κB signaling for survival, undergo less spontaneous apoptosis in the absence of cIAP1 and cIAP2 (unpublished observation).

To complicate matters further, cIAP1 has been shown to facilitate TNFα-mediated apoptosis when TNFR1 and TNFR2 are simultaneously engaged (Li et al., 2002; Wu et al., 2005a). Unlike TNFR1, which is ubiquitously and constitutively expressed, TNFR2 expression is largely restricted to lymphoid cells. Although the precise biological role of TNFR2 remains unclear, co-stimulation of TNFR2 sensitizes cells to TNFα-mediated apoptosis through TNFR1. Elegant work in Jonathan Ashwell's laboratory has demonstrated two mechanisms responsible for such ‘cross-talk’, both involving cIAP1-mediated regulation of signal transduction. The first mechanism is that cIAP1 negatively regulates TNFα-mediated NF-κB signaling when TNFR2 is engaged (Li et al., 2002). How this occurs is somewhat controversial, but it is known that TRAF2 recruits cIAP1 to TNFR2 on exposure to TNFα. Following such recruitment, cIAP1 can target TRAF2 for K48-linked ubiquitination and proteasomal degradation, which likely prevents its own recruitment to TNFR1. As such, the pro-survival NF-κB signaling pathway, which requires cIAP1 or cIAP2 for activation, is not engaged and apoptosis ensues. The second mechanism involves the ability of cIAP1 to target apoptosis signal-regulating kinase 1 (ASK1) for K48-linked ubiquitination and proteasomal degradation (Zhao et al., 2007). ASK1 is a key negative regulator of the stress responsive kinases JNK and p38, which are activated by TNFα but must be de-activated quickly to prevent apoptosis. Primary B cells from cIAP1-null mice revealed that cIAP1 targets ASK1 for K48-linked ubiquitination and degradation in response to TNFR2 ligation. As such, cIAP1 is responsible for regulating the duration of TNFα-induced JNK and p38 signaling in TNFR2-expressing cells (Zhao et al., 2007). In the absence of cIAP1, JNK and p38 signaling are prolonged and cells are sensitized toward apoptosis.

Significantly, XIAP is also known to influence signal-transduction pathways, particularly the TAB1/TAK1 complex through interaction with its BIR1 domain. This TAB1/TAK1 complex impacts on NF-κB signaling pathways, as well as JNK pathways (Wang et al., 2001; Shim et al., 2005). XIAP has been shown to inhibit c-JNK1 (JNK1) activation by transforming growth factor β1 (TGF-β1) through ubiquitin-mediated proteasomal degradation of the TGF-β1-activated kinase 1 (TAK1) (Kaur et al., 2005). XIAP's antiapoptotic activity has been proposed to be achieved by two separate mechanisms: one requiring TAK1-dependent JNK1 activation and the second involving caspase inhibition (Sanna et al., 1998, 2002a, 2002b).

Negative regulation of the IAPs

Multiple proteins play critical roles in maintaining an adequate and fine-tuned balance between too much or too little apoptosis. Intrinsic antagonists of the IAPs exist, which help maintain and regulate this apoptotic balance.

Smac/DIABLO, an IAP antagonist and a caspase activator

Smac/DIABLO (second mitochondria-derived activator of caspases, direct IAP-binding protein with low PI) is a 25-kDa mitochondrial protein that promotes apoptosis through its ability to antagonize IAP-mediated caspase inhibition once released into the cytoplasm. The ability of Smac to prevent the IAP inhibition of caspases makes this protein a functional mammalian equivalent to the Drosophila death proteins, Reaper, Grim and Hid (Du et al., 2000; Verhagen et al., 2000).

Initial mitochondrial targeting of Smac or DIABLO depends on a 53–55 amino-acid leader signal sequence found at its amino terminus. This sequence is proteolytically cleaved within the mitochondria to generate an AVPI-containing amino terminus polypeptide (Burri et al., 2005), sequestered until an apoptogenic stimulus is sensed. On receiving an apoptotic signal through the intrinsic mitochondrial stress pathway, both Smac and cytochrome c are released with similar, but not necessarily identical, kinetics. Although the exact mechanism of Smac release from the mitochondria is not known, one study suggests that, despite their significant size difference, the Smac dimer (100 kDa) and cytochrome c (12 kDa) are released through the same Bax- or Bak-formed membrane pores. This mechanism is supported by the fact that Bcl-2 overexpression can inhibit both cytochrome c and Smac release (Adrain et al., 2001), whereas Bid-induced permeabilization of the outer mitochondrial membrane induces the rapid and complete release of cytochrome c and Smac from the intermembrane space (Madesh et al., 2002).

Other studies, however, suggest that Smac and cytochrome c release is mediated through different mechanisms. Although TRAIL (TNF-related apoptosis-inducing ligand) induces cytochrome c release and apoptosis in wild-type, Bid-null, Bax-null and Bak-null MEFs, in contrast, Bax/Bak double knockout MEFs are resistant (Kandasamy et al., 2003). Moreover, TRAIL-induced mitochondrial Smac release is blocked in all of the single knockout and the double knockout MEFs. Therefore, it is concluded from these experiments that the release of Smac and cytochrome c from mitochondria is, in fact, differentially regulated in receptor-mediated pathways of apoptosis (Kandasamy et al., 2003). Differences in the mechanism of Smac and cytochrome c release occur in the presence of caspase inhibitors. Under these conditions, Smac release is prevented, whereas cytochrome c is permitted, suggesting that Smac efflux from the mitochondria is a caspase-catalysed event (Adrain et al., 2001). It may be that Smac release simply requires further membrane permeability due to increased or persistent mitochondrial damage.

Several Smac isoforms exist and studies using these proteins suggest that the pro-apoptotic function of Smac may be mediated by additional, non-IAP mechanisms. Smacβ, an alternatively spliced cytoplasmic isoform of Smac, lacks the mitochondrial targeting sequence found in full-length Smac. In vitro experiments show that Smacβ interacts with purified XIAP protein; however, in intact cells, XIAP binding is not observed (Roberts et al., 2001). Smacβ is still considered to be pro-apoptotic due to its ability to potentiate apoptosis following death receptor and chemical stimuli (Roberts et al., 2001). Unlike Smacβ, the Smac3 isoform contains both an amino-terminal mitochondrial targeting sequence and an IBM sequence. Following an apoptotic stress, Smac3, similar to Smac, is released from the mitochondria into the cytoplasm where it interacts with the BIR2 and BIR3 domains of XIAP. There are some reports that Smac3 is unique in that it is able to induce the acceleration of XIAP auto-ubiquitination and destruction, whereas Smac only seems to have this effect on cIAP1 and cIAP2 (Fu et al., 2003; Yang and Du, 2004). However, another study describes the ability of Smac to antagonize both XIAP auto-ubiquitination and XIAP-dependent ubiquitination of caspase-7 (Creagh et al., 2004). By disrupting IAP–caspase interactions and repressing the ubiquitin ligase activities of the IAPs, Smac may effectively prevent caspase demise through ubiquitination (Creagh et al., 2004). Although Smac and its isoforms may differ in how they potentiate apoptosis, the involvement of all Smac proteins with the IAPs, at multiple levels, is an effective measure to ensure that the apoptotic program can proceed.

Smac/DIABLO appears to function as a general IAP inhibitor in that it is shown to bind to XIAP, cIAP1, cIAP2, survivin, livin and BRUCE, but not NAIP (Du et al., 2000; Verhagen et al., 2000; Vucic et al., 2002; Davoodi et al., 2004; Hao et al., 2004; Qiu and Goldberg, 2005). The first four amino-terminal residues of mature Smac/DIABLO, Ala-Val-Pro-Ile, are required for Smac/DIABLO function, and deletion of this sequence abolishes Smac/DIABLO–IAP interaction (Chai et al., 2000; Liu et al., 2000; Wu et al., 2000). The crystal structure of Smac/DIABLO indicates that it consists of three extended α-helices bundled together to form an arc-shaped structure, exposing an unstructured amino terminus. Smac/DIABLO homodimerizes through an extensive hydrophobic interface, which is essential for its activity (Chai et al., 2000), forming an extremely stable protein dimer (Goncalves et al., 2008). The newly generated amino terminus in mature Smac/DIABLO makes contacts with XIAP BIR3 and mediates XIAP inhibition. The co-crystal structure of XIAP BIR3 and Smac/DIABLO indicates that the amino-terminal four residues AVPI in Smac/DIABLO recognize a surface groove on BIR3, with the alanyl residue bound within a hydrophobic pocket (Wu et al., 2000). The amino-terminal four residues of the caspase-9 linker peptide (Ala316-Thr-Pro-Phe) share significant homology to the amino-terminal tetrapeptide in mature Smac/DIABLO and Drosophila death proteins. Initially, it was believed that binding of the caspase-9 linker peptide and Smac/DIABLO to the BIR3 domain of XIAP is mutually exclusive, suggesting a competition model in which Smac/DIABLO displaces XIAP from caspase-9 (Srinivasula et al., 2001). Smac/DIABLO is also predicted to bind to XIAP BIR2 and disrupt caspase-3 and -7 inhibition, possibly by steric hindrance (Chai et al., 2000). More recent experiments suggest that Smac/DIABLO is unable to remove caspase-3 and -7 inhibition by the linker-BIR2 domains of XIAP and inefficiently relieves caspase-9 inhibition by BIR3. However, when constructs expressing the XIAP-BIR2 and -BIR3 domains in tandem, Smac/DIABLO effectively prevents IAP inhibition of both initiator and effector caspases. Furthermore, the affinities of the BIR2 and BIR3 domains of XIAP for Smac/DIABLO were shown to be almost identical with each Smac dimer interacting with the BIR2 and BIR3 domains of one XIAP molecule to form a 2:1 stoichiometric complex (Huang et al., 2003). Although individual BIR domains of XIAP are adequate for inhibiting in vitro caspase activity, these findings suggest that both BIR2 and BIR3 are required not only for XIAP-Smac/DIABLO interaction but also for subsequent liberation of caspase inhibition (Huang et al., 2003).

The structural analysis of Smac binding to XIAP indicates that the amino-terminal tetrapeptide recognizes a surface groove on the BIR3 domain, implying that peptides or small molecules modeled on this binding motif might serve as prototypical drugs the activity of which might complement that of Smac (Kipp et al., 2002). When Smac peptides composed of the first 4–8 amino-terminal residues are delivered into MCF-7 breast cancer cells, they are capable of interacting with XIAP, cIAP1 (Arnt et al., 2002; Fulda et al., 2002) and the single BIR domain of livin (Vucic et al., 2002). These peptides enhance apoptosis and long-term antiproliferative effects of a range of chemotherapeutics, including paclitaxel, etoposide and doxorubicin (Arnt et al., 2002; Fulda et al., 2002).

Smac-like peptides delivered in combination with cancer therapeutics such as Apo2 L/TRAIL appears to be a promising method to reduce tumor burden (Arnt et al., 2002; Fulda et al., 2002). The in vivo delivery of Smac peptide, in conjunction with Apo2 L/TRAIL, completely eradicates established intracranial malignant glioma xenograft tumors and increases the survival time of treated mice (Fulda et al., 2002). Apo2 L/TRAIL induces apoptosis through the extrinsic/death receptor pathway by binding to the transmembrane receptors TRAIL-R1/DR4 and TRAIL-R2/DR5. The delivery of Smac peptides in the presence of Apo2 L/TRAIL appears to have a significant impact on sensitizing otherwise resistant cells to apoptosis (Fulda et al., 2002; Guo et al., 2002; Ng and Bonavida, 2002).

Some studies have shown that amino-terminal Smac tetrapeptides bind only to the BIR3 domain of XIAP with low affinity, are sensitive to proteolytic degradation and have a poor capacity to penetrate cells (Li et al., 2004). Efforts to circumvent the limitations of Smac peptides led to the development of small molecule Smac mimetics, compounds that inhibit the IAPs with higher affinities than Smac peptides (Li et al., 2004). Such submicromolar, small molecule, non-peptidic antagonists are reported by Park et al. (2005). These compounds are created by individually replacing several amino-acid residues while still maintaining the essential interactions necessary for binding in the Smac-binding site on XIAP BIR3 (Park et al., 2005). Another identified class of compounds is a series of proteolytically stable, capped tripeptides consisting of unnatural amino acids that bind to XIAP BIR3 with high nanomolar affinities. These compounds are cytotoxic in cancer cells and delay tumor growth in breast cancer xenograft models (Oost et al., 2004).

An additional compound class, a modified oxazoline molecule, has been shown after several modifications to bind to the IAPs with a higher affinity than Smac tetrapeptides, and block IAP interaction with caspase-9. This compound also acts synergistically with Apo2 L/TRAIL and various chemotherapeutic agents (Li et al., 2004). Further studies using a high IAP-expressing breast cancer cell line MDA-MB-231 show that at low nanomolar concentrations, the Smac mimetic compound significantly sensitizes these cells to both Apo2 L/TRAIL- and etoposide-induced apoptosis through caspase-3 activation (Bockbrader et al., 2005). The effectiveness of this dimeric compound is due to its ability to mimic the dimeric structure of Smac protein, which, as mentioned previously, acts at the IAP BIR2 and BIR3 regions to liberate caspase-3, -7 and -9 (Li et al., 2004; Bockbrader et al., 2005). Alternatively, as recently reported for several different SMCs, their mechanism of action may be due to the induction of auto- or trans-ubiquitination of the IAPs, primarily cIAP1 and cIAP2, which results in their rapid loss from the cell within minutes of compound addition and sensitization to TNFα-induced killing (Varfolomeev et al., 2007; Vince et al., 2007).

Other IBM-containing proteins as potential IAP antagonists

Following the identification of Smac, another mitochondrial IAP-binding protein called Omi, or HtrA2, was discovered (Hegde et al., 2002; Martins et al., 2002; Verhagen et al., 2002). Omi/HtrA2 is a mammalian homolog of the Escherichia coli bacterium heat-inducible serine protease, known as HtrA. Another identified IBM-containing protein is an isoform of the polypeptide chain-releasing factor GSPT1/eRF3 protein that is localized to the endoplasmic reticulum (Hegde et al., 2003). Checkpoint kinase 1 (Chk1), a dual function kinase that is active during the S- to –M-phase transition of the cell cycle, regulating Cdc25A function, and prevents mitotic progression in the presence of DNA damage, contains an IBM sequence (Galvan et al., 2004). More IBM-containing proteins, all mitochondria-derived, exist (Verhagen et al., 2007), and potentially more are yet to be found. If we extrapolate from the situation in Drosophila, with four known IAPs (DIAP1/thread, DIAP2, deterin and dBruce) versus eight human IAPs, and five known Drosophila IBM-containing antagonists (reaper, hid, grim, sickle and jafrac), then we would predict that a number of more mammalian IBM-containing proteins remain to be discovered. It is suggested that of all the mitochondrial apoptogenic proteins, Smac (an IBM-containing protein) and cytochrome c remain as the only likely mammalian ‘killer’ proteins (Ekert and Vaux, 2005).

XAF1 is an interferon-inducible IAP antagonist, and a candidate tumor suppressor

Using a yeast two-hybrid screen, our laboratory identified XAF1 (XIAP-associated factor 1) as a XIAP-binding partner (Liston et al., 2001). The ability of XAF1 to bind to XIAP is confirmed by an in vitro expression system (Liston et al., 2001), as well as by immunoprecipitation of endogenous proteins (Arora et al., 2007). XAF1 directly associates with XIAP to antagonize XIAP-mediated caspase-3 inhibition, thereby reversing XIAP-mediated protection against chemotherapeutic drugs such as cisplatin and etoposide. Although the exact molecular interaction between XAF1 and XIAP remains unknown, it appears to be multipartite based on domain analysis (unpublished observations). In contrast to the original report, two groups failed to detect any interaction between XAF1 and XIAP under the conditions tested (Verhagen et al., 2000; Xia et al., 2006). Unexpectedly, one of these studies demonstrated that XAF1 exerted pro-apoptotic effects in cells devoid of XIAP (Xia et al., 2006). In response to TNFα, the induction of XAF1 can dramatically increase the level of apoptosis regardless of the status of XIAP. This is in contrast to the view that XAF1 antagonism of XIAP is its quintessential pro-apoptotic function. However, these ostensibly contradictory findings may be reconciled by the fact that XAF1 also binds, and perhaps antagonizes, cIAP1 and cIAP2 (and other IAPs as well), with at least equal, if not greater affinity than XIAP (Arora et al., 2007). Given that cIAP1 and cIAP2 are critical negative regulators of TNFα-mediated cell death (Mahoney et al., 2008), it should not be surprising at all that the potential antagonism of these proteins by XAF1 can impact on cellular survival, in the absence of XIAP.

Although XAF1 appears to have an affinity toward many IAPs, XAF1 does not bind to survivin directly. Interestingly, despite the lack of direct interaction, XAF1 can destabilize survivin through their mutual intermediate, XIAP (Arora et al., 2007). Dohi et al. (2004) have shown that the binding of survivin to XIAP increases the stability of latter, and reinforces the antiapoptotic activity of this IAP-IAP complex. We have, in turn, demonstrated that the expression of XAF1 can activate the RING E3 ligase of XIAP, subjecting survivin to the ubiquitin–proteasomal pathway for degradation (Arora et al., 2007). In effect, XAF1's tumor suppressor role may be due to its antagonism of the potential tumorigenic function of the XIAP-survivin complex.

XAF1 is ubiquitously expressed in normal tissues, but is absent or detected at only extremely low levels in the majority of the NCI 60 cell line panel of cancer cells (Fong et al., 2000), human colorectal cancer cell lines and primary carcinomas (Zou et al., 2006; Chung et al., 2007). The loss of heterozygosity of xaf1 is a frequent occurrence in tumors (Fong et al., 2000; Liston et al., 2001; Chung et al., 2007). In addition, a recent study shows that XAF1 is a predictive and prognostic factor in bladder cancer patients (Pinho et al., 2008). The downregulation or loss of xaf1 expression in cancer cells may indeed contribute to apoptosis suppression and tumorigenesis through unrestricted IAP activity. Accumulating evidence indicates that restoring XAF1 expression in cancer cells increases their sensitivity to apoptotic triggers (reviewed in Plenchette et al., 2007). A strategy for inducing XAF1 is by exposing tumor cells to interferon-β (IFN-β) (Leaman et al., 2002; Wang et al., 2006b; Arora et al., 2007; Micali et al., 2007). Melanoma cell lines that are responsive to IFN-β-mediated XAF1 upregulation are predisposed to Apo2 L/TRAIL-induced apoptosis in a manner dependent on the XAF1 induction (Leaman et al., 2002). These studies suggest that the re-expression of XAF1 strongly influences cellular sensitivity to cell death initiated by the extrinsic pathway.

In the past few years, we have made some appreciable progress in determining the mechanism controlling XAF1 expression levels. In both cell lines and tumor tissues, the XAF1 promoter is primarily silenced by DNA hypermethylation at the CpG sties (reviewed in Plenchette et al., 2007), and also by HSF1-mediated transcriptional repression (Wang et al., 2006a). XAF1 expression can be restored experimentally on inhibition of DNA methylation, as well as with the addition of IFN as discussed above (Reu et al., 2006; Zou et al., 2006; Arora et al., 2007; Micali et al., 2007). Expectedly, because DNA methyltransferases (DNMTs) are involved in the epigenetic silencing of various genes (Rhee et al., 2002), the inhibition of DNMT1 by either oligonucleotide antisense or methylation inhibitor 5′aza-dC can lead to XAF1 induction (Reu et al., 2006). Importantly, this enhanced induction of XAF1 is a crucial factor for apoptosis mediated by IFN in melanoma cells (Reu et al., 2006). Interestingly, although methylation appears to play a role in the regulation of XAF1 expression, IFN reactivation of XAF1 in certain tumor cells can occur in spite of a persistently hypermethylated promoter (Micali et al., 2007), suggesting that additional regulatory mechanisms are involved.

For tumor cells, XAF1 can be a pro-apoptotic stimulus either by itself, or in combination with additional triggers (Liston et al., 2001; Leaman et al., 2002; Qi et al., 2006). In xenograft nude mouse models in which gastric and colon cancers are established, XAF1 overexpression alone suppresses tumor formation. Further studies show that adenovirus encoding xaf1 delivered by intratumoral injections in combination with Apo2 L/TRAIL or chemotherapeutics enhances apoptosis leads to the eventual complete eradication of established tumors (Qi et al., 2006). Significantly, apoptosis is not induced in normal cells surrounding the tumor (Qi et al., 2006), suggesting that tumor cells have a lower tolerance for XAF1 than normal tissues. XAF1 also shows selective killing potential in other human tumor cells, including pancreatic and breast cancer cell lines. When these human tumor cell lines and normal cell lines are transduced with adenoviral xaf1, approximately 80% of the tumor cells underwent apoptosis compared with 5–10% in the normal cell lines (Yang et al., 2003). The ability of XAF1 to selectively induce apoptosis in tumor cells while leaving healthy cells unaffected makes this candidate tumor suppressor potentially effective for cancer therapy.

The IAPs in cancer and treatment

Convincing data for the involvement of the IAPs in cancer and other human diseases now exist. Direct genetic evidence, as demonstrated by IAP gene amplifications, gene mutations or deletions, chromosomal translocations, indicates causality in cancer and proliferative autoimmune disorders (Table 2). These findings are supported by animal studies and cell culture experiments, demonstrating the immunomodulatory and oncogenic potential of the IAPs, as well as identifying their normal physiological roles.

Table 2 Summary of IAP mutations and deletions in mammals leading to proliferative disorders or cancer

Pathological roles of the IAPs in cancer

A variety of cancer cell lines and primary tumor biopsy samples show elevated IAP expression levels (Ambrosini et al., 1997; LaCasse et al., 1998; Fong et al., 2000; Tamm et al., 2000; Vucic et al., 2000; Li et al., 2001). The most dramatic example of IAP overexpression in tumors is seen with survivin. Survivin expression is limited to embryonic tissues and many different tumor types, but is absent in most adult differentiated tissues (Ambrosini et al., 1997). Furthermore, the presence of survivin in patient tumor biopsy samples correlates with poor prognosis, increased rates of treatment failure and relapse (Mesri et al., 2001). The prognostic significance of IAP overexpression is less clear for some of the other IAPs. For example, XIAP protein levels correlate with disease severity and prognosis in acute myelogenous leukemia (AML) (Tamm et al., 2000) and renal cell carcinoma (Ramp et al., 2004; Yan et al., 2004; Mizutani et al., 2007), but not in non-small cell lung carcinoma (Ferreira et al., 2001a, 2001b) or cervical carcinoma (Liu et al., 2001). XIAP levels have also been shown to increase in leukocytes with the transformation of myelodysplastic syndromes to overt AML (Yamamoto et al., 2004). High expression of XIAP or cIAP2 is associated with shorter overall survival, and lower complete response rates for AML (Hess et al., 2007). Other cross-validation and forward selection studies reveal a three-gene signature, consisting of cIAP2, Bax(l) and BMF, to optimally predict overall survival in AML (Hess et al., 2007). Furthermore, an immunocytochemical survey of tumor tissue shows that XIAP immunostaining patterns allow for the ready distinction of malignant from benign populations in effusion washes (Wu et al., 2005b). In this survey, strong XIAP staining was most prevalent in ovarian carcinoma effusions and less prevalent in mammary carcinoma effusions. XIAP expression increases with advancing stage in ovarian carcinoma (Mao et al., 2007). XIAP is also identified as part of a progression signature in prostate cancer using an immunoblot approach to characterize proteomic alterations in prostate tumors. XIAP protein is seen to increase in expression between benign prostatic tissue, and clinically localized prostate cancer, or metastatic prostate cancer (Varambally et al., 2005). In addition, XIAP protein expression is a strong predictor of prostate cancer recurrence (Seligson et al., 2007). Collectively, these results suggest that the expression levels of the IAPs could be expected to have a significant impact in the development and maintenance of cancer due to their central role in the regulation of apoptosis.

There is direct genetic evidence for the IAPs as proto-oncogenes (Table 2). This is seen in cases with chromosomal amplification of the 11q21–q23 region, encompassing both cIAP1 and cIAP2, which is observed in a variety of malignancies, including medulloblastomas, renal cell carcinomas, glioblastomas, gastric carcinomas and non-small cell lung carcinomas. Esophageal squamous cell carcinomas frequently display this amplification, and transcriptional profiling has identified cIAP1 as the sole target that is consistently overexpressed in these tumors (Imoto et al., 2001). More recent findings show amplification of 11q22 in primary tumors of non-small cell lung and small cell lung cancers. Both cIAP1 and cIAP2 are overexpressed in these primary carcinomas (Dai et al., 2003). Comparison of human and mouse tumors reveal that cIAP1 and cIAP2 are also amplified in the corresponding 11q22 synteneic region in mouse at 9qA1 (Zender et al., 2006). More importantly, when cIAP1 and another gene from 9qA1, YAP, are introduced into murine ES cells and injected into mice, they form hepatomas (Zender et al., 2006). In addition, cIAP2 could also transform p53-null hepatoblasts in combination with c-Myc overexpression. These examples provide concrete proof of the role of cIAP1 as an oncogene (Zender et al., 2006). Furthermore, cIAP1 cooperates with the Myc oncogene in transformation, by acting as a ubiquitin ligase for Mad1, an antagonist of Myc (Xu et al., 2007). A recent report also suggests that cIAP1 and cIAP2 promote cancer cell survival by ubiquitinating RIP1, leading to constitutive RIP1 and NF-κB activity (Bertrand et al., 2008).

Further genetic evidence implicating the IAPs as potential oncogenes is found in the generation of extranodal marginal zone mucosa-associated lymphoid tissue (MALT) B-cell lymphomas. The translocation events t(1;14)(p22;q32) and t(11;18)(q21;q21) are well documented in MALT lymphomas and involve both NF-κB activation and cIAP2 expression. The less frequently observed translocation t(1;14)(p22;q32) results in Bcl-10 overexpression (Vega and Medeiros, 2001). A common feature of these MALT lymphomas is the increased nuclear expression of Bcl-10. Under normal circumstances, cIAP2 exerts immunoregulatory effects on anigen receptor signaling by ubiquitinating and targeting Bcl-10 for degradation (Hu et al., 2006a, 2006b). Bcl-10 forms a ‘signalosome’ complex with CARMA1 and MALT1 that triggers NF-κB activation in response to T-cell and B-cell receptor signaling (reviewed in Schulze-Luehrmann and Ghosh, 2006; Wegener and Krappmann, 2007). Thus, it appears that cIAP family members function as ubiquitin protein ligases and are capable of regulating not only one another, but components of the TNFR and signalosome signaling complexes as well.

A more common translocation event, present in approximately 50% of extranodal MALT lymphomas, involves a gene rearrangement of the MALT1 locus 18q21 with the cIAP2 gene (also known as API2, located at 11q22) resulting in the cIAP2-MALT fusion product t(11;18)(q21;q21) (Dierlamm et al., 1999; Baens et al., 2000; Remstein et al., 2000; Hosokawa, 2005). The encoded chimeric protein consists of the amino-terminal cIAP2 BIR1–3 domains (excluding the RING domain) fused in-frame to the carboxy terminus of MALT1 (Uren et al., 2000) (Figure 3). Typically, gastric MALT lymphomas are associated with chronic inflammation due to Helicobacter pylori infection, and can be treated with antibiotic therapy. Significantly, the majority of these lymphomas that do not respond to antibiotics exhibit the cIAP2-MALT1 translocation. The cIAP2-MALT translocation establishes a positive feedback loop for cIAP2 by activating NF-κB. This in turn, transcriptionally activates the promoter for cIAP2 (Chu et al., 1997; Erl et al., 1999; Hong et al., 2000). In this way, the cIAP2-MALT1 fusion induces NF-κB activation and contributes to an antiapoptotic phenomenon by upregulating transcription of antiapoptotic genes such as cIAP2, cFlip and MnSOD. As a result, the lymphoma cells are no longer dependent on the inflammatory mechanism of NF-κB activation and eliminating the H. pylori is no longer sufficient to shut down inappropriate cell proliferation.

Figure 3
figure3

cIAP2 is translocated and involved in mucosa-associated lymphoid tissue (MALT) lymphomagenesis. The t(11;18)(q21;q21) MALT lymphoma translocation disrupts the cIAP2 gene, also called api2, on chromosome 11q22 and the MALT1 gene, mlt, on chromosome 18q21. The resulting novel cIAP2-MALT1 fusion proteins possess the cIAP2 baculovirus IAP repeat (BIR) domains (prior to the indicated triangles) linked to the carboxy terminus of MALT1 at one of four different points (indicated by triangles on the protein diagram). This schematic diagram represents two such resulting fusion proteins; more variants exist based on the location of the chromosomal breakpoints. The breakpoint frequencies (in percentage) are given for the two affected genes based on a review by Du (2007). The many functions and binding partners attributed to the various domains of both parental proteins are shown to help understand the properties of the resulting fusions. MALT1 possesses a death domain (DD) of unknown function, as well as immunoglobulin (Ig) or Ig-like domains. MALT1 also possesses a caspase-like domain, referred to as a paracaspase, with protease activity. These fusion proteins are both antiapoptotic and nuclear factor-κB (NF-κB) activating either through TRAF6 or TRAF2 binding, Bcl-10 stabilization and nuclear localization, and/or A20 cleavage. A positive feedback amplification loop is established in MALT lymphoma in which the fused gene is expressed under the control of the ciap2 promoter, which contains several NF-κB-responsive sites (not shown).

The exact mechanism of NF-κB activation by the cIAP2-MALT1 fusion is debated, and the subject of much interest and investigation. The cIAP2-MALT1 fusion targets NEMO, the regulatory subunit of IKK, for polyubiquitination and activates NF-κB (Zhou et al., 2005). The E3 ligase function responsible for this activity is unclear as the cIAP2-MALT1 fusion lacks the cIAP2 E3 function, and the direct role of two other candidates, Bcl-10 and TRAF6, was dismissed although not ruled out completely. TRAF6 represents the most likely candidate as a fusion binder with E3 ligase activity and NF-κB-activating properties. This is shown by Noels et al. (2007), and explains why TRAF1 and TRAF2 are not necessary for this NF-κB activation pathway (Varfolomeev et al., 2006). However, TRAF2 binding of BIR1 for fusion contributes also to NF-κB activation (Lucas et al., 2007; Noels et al., 2007). Bcl-10 lacks E3 ligase activity and serves as an adaptor in the CARMA1-Bcl-10-MALT1 signaling complex. It has been shown that cIAP2 acts as an ubiquitin ligase for Bcl-10 (Hu et al., 2006b). This activity is lost in the cIAP2-MALT1 fusion, and concomitantly the Bcl-10 protein is stabilized in these MALT lymphomas. Furthermore, Bcl-10 and cIAP2-MALT1 synergistically activate NF-κB (Hu et al., 2006b). In another twist to this story, it has been recently shown that the paracaspase domain of MALT1 (a caspase-like domain long thought to have protease activity but never proven until now) can cleave the NF-κB inhibitor A20 (Coornaert et al., 2008), as well as Bcl-10 (Rebeaud et al., 2008). The cIAP2-MALT fusion can also cleave A20 providing another means of sustained NF-κB activity (Coornaert et al., 2008). Some of the discrepancies in defining the mode of action of the cIAP2-MALT1 fusions may be due to differences in the fusion constructs used in experiments based on the different patient cases (see Du (2007) and Figure 3 for translocation breakpoints and frequencies). These different fusions incorporate different domains, such as the immunoglobulin domains, that may allow for differential binding of Bcl-10 or other factors.

The oncogenic potential of the cIAP2-MALT1 fusion has been further revealed with the generation of an Eμ-API2-MALT1 transgenic mouse, which also exemplifies the effect of NF-κB activation due to the cIAP2-MALT fusion protein. In this study, results with the Eμ-API2-MALT1 transgenic model show that the expression of the cIAP2-MALT1 fusion protein is not adequate for the formation of lymphomas during the lifespan of the mouse (Baens et al., 2006), unless chronic antigenic stimulation is provided (Sagaert et al., 2006). Expression of the MALT1 fusion did affect B-cell maturation in the bone marrow and triggered the expansion of splenic marginal zone B cells. The survival of B cells in this transgenic model, therefore, is thought to be promoted through enhanced NF-κB activation (Baens et al., 2006). It is noteworthy that the cIAP2-MALT1 fusion in the transgenic model is under control of the Eμ promoter and not the NF-κB-responsive promoter of API2/cIAP2. This may explain the need for continued antigen stimulation for lymphomagenesis to occur in this transgenic model.

Surprisingly, cIAP1 and cIAP2 play a tumor suppressor role in multiple myeloma (a plasma B-cell neoplasm), as deletions of cIAP1/2 in these cancer cells, or mutations in other genes regulating the alternative NF-κB pathway, are commonly found (Annunziata et al., 2007; Keats et al., 2007) (Table 2). The deletion of cIAP1 and cIAP2 allows for NIK stabilization and activation of NF-κB through the alternative pathway, and presumably aids in tumor cell transformation and/or increased cell survival. Thus, based on the positive role of cIAP1 and cIAP2 in classical NF-κB signaling, and their negative role in alternative NF-κB pathways, the cIAPs can be considered to be either oncogenes or tumor suppressors, depending on the cell type and context of NF-κB activation. However, in all cases involving deregulated cIAP1 or cIAP2 expression (such as carcinoma, MALT lymphoma and other non-Hodgkin's lymphoma, and multiple myeloma), NF-κB activation is increased and this is associated with carcinogenesis and increased tumor cell survival.

Targeting the IAPs for cancer therapy

The acquired resistance of cancer cells to apoptosis is one of the defining hallmarks of cancer (Hanahan and Weinberg, 2000), necessitated in part to suppress cell death induced by oncogene activation (Evan and Vousden, 2001). The issue of primary or acquired resistance to current chemotherapeutic-based treatments is a major impediment to effective cancer therapy. Although there are many genetic and biochemical alterations that occur in cancer cells, in vitro experiments demonstrate that the upregulation of IAP expression increases resistance to chemotherapeutic and radiation resistance. The fundamental role of the IAPs in apoptosis regulation suggests that there is value in exploiting the inhibition of IAP expression and function as a direct therapeutic strategy for cancer, and possibly other proliferative disorders as well.

Antisense oligonucleotides to XIAP or survivin as therapeutics

Applications of single-stranded antisense oligodeoxynucleotides (AS ODNs) as selective inhibitors of gene expression are being studied for efficacy in treating particular genetic disorders. In addition, many AS ODNs are being assessed both preclinically and in early clinical trials for several cancer types. The AS ODNs are short stretches of synthetic DNA, approximately 12–30 nucleotides long and are complementary to a specific mRNA strand. Hybridization of the AS ODNs to the mRNA by Watson–Crick base pairing prevents the target gene from being translated into protein, thereby blocking the action of the gene, and resulting in the degradation of the target mRNA (Galderisi et al., 1999; Gleave et al., 2002; Jansen and Zangemeister-Wittke, 2002). The specificity in the AS ODN approach is based on the fact that any sequence of approximately 13 bases in RNA and 17 bases in DNA is estimated to be represented only once in the human genome. A multitude of candidate genes, involved in apoptosis regulation, represents potential targets for antisense-based therapies.

Additional proof-of-principle studies performed with newer double-stranded RNAi-based technologies show that downregulating XIAP gene expression through siRNA increases apoptosis in cultured MCF-7 breast cancer cells and subsequently enhances the killing effects of etoposide and doxorubicin (Lima et al., 2004). Another study using short-hairpin RNAs as an RNAi approach directed against XIAP shows that XIAP mRNA can be reduced by as much as 85% in some breast carcinoma cell lines. This reduction in XIAP dramatically sensitizes these cell lines to TRAIL- and to taxane-induced killing (McManus et al., 2004).

Survivin is expressed in fetal tissues, becomes restricted during development, and is absent in most healthy, differentiated adult tissues (Adida et al., 1998), with some notable exceptions, such as stem cells, thymus, testes, regenerating hepatocytes and endothelial cells (Kobayashi et al., 1999; reviewed in Fukuda and Pelus, 2006). Significantly, survivin is re-expressed during malignant transformation and is found in nearly all tumor types, including neuroblastomas (Islam et al., 2000), pancreatic, prostate, gastric, colorectal, hepatocellular and breast carcinomas, as well as lung and bladder cancers, melanomas, B-cell lymphomas and esophageal cancer (Altieri, 2001). The expression of survivin in cancer is a predictor of both poor prognosis and decreased survival time, and is implicated in conferring chemo- and radio-resistance phenotypes to tumor cells. Numerous studies have addressed the diagnostic and prognostic potential of survivin expression, as well as that of its nuclear versus cytoplasmic localization, phosphorylation status and the presence of splice isoforms (Li, 2005; Span et al., 2006; Yin et al., 2006; Dohi et al., 2007; Kleinberg et al., 2007; Pannone et al., 2007; Piras et al., 2007; Stauber et al., 2007; Brennan et al., 2008; Bria et al., 2008).

On account of the ubiquitous nature of survivin expression in human cancer, one therapeutic focus is the targeting of this IAP in the attempt to downregulate and eliminate its expression. Interestingly, EPR-1 (effector cell protease receptor-1) and survivin are encoded by mRNAs transcribed from opposite strands of the same chromosome locus (Ambrosini et al., 1998). When epr-1 cDNA is overexpressed in HeLa cells, survivin is downregulated, the rate of spontaneous apoptosis increases and cell proliferation is inhibited (Ambrosini et al., 1998). These experiments indicate that downregulating survivin expression can be beneficial. AS ODNs targeting survivin expression in human lung adenocarcinoma cell lines decrease survivin protein levels in a dose-dependent manner, induce apoptosis, stimulate higher levels of caspase-3 activation, and increase the sensitivity of cells to chemotherapeutics (Olie et al., 2000; Jiang et al., 2001). Inhibition of survivin expression, using different AS ODNs spanning the entire survivin gene, induces cell death through either a caspase-dependent or -independent pathway depending on the neuronal tumor type (Shankar et al., 2001). In vivo models showing the effect of survivin downregulation identify this approach as a viable strategy to inhibit tumor growth (Tu et al., 2003; Ansell et al., 2004; Cao et al., 2004). Current phase II clinical trials of a survivin AS ODN are underway by Eli Lilly and Company (Table 3).

Table 3 IAP small molecule antagonists as research tools or clinical development candidates

In contrast to survivin, the importance of XIAP in cancer prognosis is less clear; however, XIAP is arguably the most potent of the IAPs with respect to its ability to inhibit caspase activation and to suppress apoptosis. Survivin, on the other hand, is a weak apoptotic inhibitor and its death-suppressing effects may be mediated by XIAP stabilization (Dohi et al., 2004). Furthermore, XIAP is highly overexpressed in many tumor cell lines of the NCI panel (LaCasse et al., 1998) and its expression has been correlated with poor prognosis or advancing stage in AML and other cancers, as noted earlier. These factors indicate that XIAP is an attractive target for improving treatment responses for a variety of tumor types.

XIAP AS ODNs effectively downregulate both specific mRNA and protein levels in human non-small cell lung cancer growth both in vitro and in vivo. XIAP AS ODNs effectively induce apoptosis on their own and sensitize the tumor cells to the cytotoxic effects of several chemotherapeutics, including Taxol, etoposide and doxorubicin (Hu et al., 2003). Furthermore, the administration of XIAP AS ODNs in a xenograft model of human non-small cell lung cancer results in a significant downregulation of XIAP protein. Tumor establishment is delayed when AS ODNs are combined with vinorelbine treatments (Hu et al., 2003). Other studies show that XIAP AS ODNs enhance tumor regression in conjunction with radiotherapy in a mouse model of lung cancer (Cao et al., 2004). When XIAP is downregulated in the pancreatic carcinoma cell line, Panc-1, using a second generation, mixed backbone AS ODN (AEG35156/GEM640), the treated cells are sensitive to TRAIL-induced killing (McManus et al., 2004). Similarly, TRAIL, in combination with another XIAP AS ODN (in phosphorodiamidate morpholino chemistry), potentiates both cisplatin sensitivity and TRAIL killing in androgen-refractory prostate cancer cells (Amantana et al., 2004). Overall, these promising data indicate that when XIAP protein expression levels are reduced through RNAi or antisense approaches, a decreased apoptotic threshold to an array of chemotherapeutics can be achieved (LaCasse et al., 2005). Notably, a XIAP AS ODN (AEG35156) is being evaluated for cancer treatment (Cummings et al., 2006; Cheung et al., 2006a) and is in multiple phase I or II clinical trials in the United Kingdom, Canada and the United States for solid tumors, lymphoma and leukemia (AML) (Table 3). Initial AEG35156 trial results appear promising (Cheung et al., 2006a; Danson et al., 2007; Dean et al., 2007).

SMCs as promising antagonists of XIAP, livin, cIAP1 and cIAP2

Small molecule IAP antagonists have been designed to mimic the interaction between XIAP and its antagonist Smac, and these show great promise for the treatment of cancer (Varfolomeev et al., 2007). SMCs were intended to de-repress XIAP-mediated inhibition of apoptosis in cancer cells. However, a number of groups (Gaither et al., 2007; Petersen et al., 2007; Varfolomeev et al., 2007; Vince et al., 2007; Bertrand et al., 2008; Wang et al., 2008) have now demonstrated quite unexpectedly that the ability of SMCs to kill cancer cells is due to cIAP1 and cIAP2 degradation rather than XIAP antagonism. Mechanistically, SMCs induce auto-ubiquitination and degradation of cIAP1 and cIAP2, which in turn relieves the repression by cIAP1 and cIAP2 on ligand-independent, classical and alternative NF-κB signaling. Consequently, these NF-κB pathways are activated, which leads to the production of TNFα in a subset of the cancer cells. The newly synthesized TNFα is subsequently secreted to activate TNF receptors through autocrine or paracrine pathways. This TNFα signaling loop strongly induces caspase-8-dependent apoptosis in the absence of cIAP1 and cIAP2, although the mechanism of action is somewhat controversial. On the one hand, our recent data (Mahoney et al., 2008) as well as others (Santoro et al., 2007; Bertrand et al., 2008), have demonstrated that cIAP1 and cIAP2 are critical positive regulators of TNFα-mediated NF-κB activation. Accordingly, in the absence of both cIAP1 and cIAP2, TNFα induces apoptosis because the pro-survival NF-κB pathway is blunted. On the other hand, recent data show that cIAP1 and cIAP2 regulate RIP1 ubiquitination in response to TNFα, by targeting RIP1 directly or acting as a ‘brake’ on its de-ubiquitination. This suggests that cIAP1 and cIAP2 may regulate TNFα-mediated apoptosis in an NF-κB-independent manner (O’Donnell et al., 2007). The dual loss of cIAP1 and cIAP2, therefore, appears to sensitize cells to TNFα-mediated apoptosis by attenuating RIP1 ubiquitination, with unmodified RIP1 having the capacity to interact with caspase-8 and induce apoptosis (Wang et al., 2008). It is noteworthy that although normal cells do not die in response to dual cIAP1 and cIAP2 knockdown (presumably because they do not generate TNFα), they are greatly sensitized to apoptosis on exposure to exogenous TNFα (Mahoney et al., 2008). As such, the pursuit of this very promising new treatment strategy should nevertheless proceed cautiously, to better define the therapeutic window and delineate the systemic and localized levels of TNFα and the tissue-specific toxic effects.

Several biotechnological and pharmaceutical companies now have active programs developing SMCs as anticancer agents (Table 3). Two such compounds have already entered the clinic with more SMCs not far behind. Dimeric SMCs have been found to exhibit a 2- to 3-log increase in potency over their monomeric counterparts (Li et al., 2004). This finding suggests that anchoring of one side of an SMC dimer into BIR3 will favor its interaction of its other half with the lower affinity BIR2 site, and/or that the dual inhibition of BIR2 and BIR3 results in greater caspase activation by simultaneously blocking and releasing caspase-3 or -7, and -9. Alternatively, the SMC dimer may induce ligation of BIR2 and BIR3 within an IAP, or the ligation of BIR3–BIR3 domains between two IAPs, resulting in greater stimulation of auto- or trans-ubiquitination and enhanced protein degradation through the proteasome (Figure 4). Therefore, not unexpectedly, dimeric SMCs are far more potent than monomeric SMCs, as they faithfully reproduce the action of endogenous Smac polypeptides that antagonize the IAPs as homodimer complexes.

Figure 4
figure4

Mechanism of action for dimeric Smac mimetic compounds (SMCs): intramolecular versus intermolecular bridging models. Two models are shown to explain the increased potency of dimeric SMCs versus monomeric SMCs. The small red barbell structure represents a dimeric SMC. The model either involves internal ligation of baculovirus inhibitor of apoptosis repeat (BIR3)–BIR2 domains within the same IAP (intramolecular bridging, shown for example for XIAP) versus ligation of BIR3 domains (the higher affinity site) between two separate IAPs (intermolecular bridging, shown for example for cIAP1). Livin, with a single BIR domain, is not capable of internal ligation and thus only SMC-induced protein dimers are possible. The bivalent binding of a dimeric SMC increases its affinity compared with a monovalent binding of a monomeric SMC. In addition, the forced dimerization or internal ligation of an IAP produced by the dimeric SMCs enhances auto- or trans-ubiquitination leading to IAP degradation, or effectively blocks caspase-3, -7 and -9 entry simultaneously (not shown). The two models are not mutually exclusive within the same cell, and other configurations can be envisaged. That is, head-to-head or head-to-tail protein dimers are possible, allowing for additional interactions besides BIR3–BIR3 pairings, such as BIR2–BIR2 or BIR3–BIR2. In addition, the polarity of the interaction may place the RING domain in a more favorable ubiquitination context closer to its target residues. Much remains to be determined for the mechanism of action of these dimeric SMCs and how they stimulate tumor necrosis factor-α (TNFα) production and subsequent cell death.

Mechanistically, SMCs are thought to be apoptosis sensitizers, not apoptosis inducers, and therefore are postulated to require an additional stimulus such as chemotherapy. However, several cancer cell lines are killed outright by SMCs at low nanomolar concentrations; but the additional stimulus is likely the autocrine TNFα loop. Interestingly, cancer cells that are resistant to single-agent SMC treatment may be sensitized to TRAIL or chemotherapy, increasing the usefulness of these compounds. However, caveats and concerns surrounding SMCs are already being raised with the few studies done so far. For example, SMC treatment may not be appropriate for B- or T-cell neoplasms, such as non-Hodgkin's lymphoma, Hodgkin's disease or multiple myeloma, in which activation of the alternative pathway is common and leads to a survival signal. That is, the SMC-induced loss of cIAP1 and cIAP2 may not lead to cell death by TNFα in these cancers but rather stimulate tumor cell survival. Moreover, the activation of the alternative NF-κB pathway may lead to autoimmune disease, in a manner similar to cells that receive a stimulus from CD40-L, CD30-L, BAFF, LIGHT or lymphotoxin-β, members of the TNFR superfamily of ligand that are capable of providing a survival signal in lymphocytes. Additionally, depending on dosing, tumor burden and tumor sites, coupled with the local and systemic levels of TNFα produced in response to SMCs, the normal surrounding or distant tissue(s) may be negatively affected, such as that seen with cancer cachexia, anemia or sepsis. Therefore, patients treated with SMCs should be monitored for such possibilities and related adverse events. Clearly, many specific aspects of SMC mechanism of action remain to be determined to maximize their impact in the clinic.

Additional XIAP small molecule antagonists

Other small molecule approaches targeting XIAP have lead to the identification of novel compounds (Table 3). For example, a series of polyphenylurea-based compounds, now called Xantags, were identified that lead to caspase-3/-7 activation prior to the activation of upstream caspase-8 and -9 (Schimmer et al., 2004). Specifically, these Xantags dissociated caspase-3 from XIAP in vitro, but not caspase-9, in contrast to SMCs (Wang et al., 2004). These XIAP antagonists demonstrate anticancer activity in several different models (Schimmer et al., 2004; Wang et al., 2004; Carter et al., 2005; Karikari et al., 2007; Cillessen et al., 2008). Similarly, Wu et al. (2003) undertook a high-throughput biochemical screen of a combinatorial chemical library that led to the discovery of a novel non-peptidic small molecule, TWX006, that has the ability to disrupt the XIAP–caspase-3 interaction.

A structure-based computational screening of a natural product structure database identified embelin, from the Japanese Ardisia herb, as a small molecule inhibitor that binds to XIAP BIR3 and leads to caspase-9 activation in cancer cells with high XIAP and nominal effects in normal cells with low XIAP (Nikolovska-Coleska et al., 2004). Derivatives of embelin have been designed with higher affinity for XIAP BIR3 (for example, compound 6 g, Ki=180 nM) to provide leads for further optimization (Chen et al., 2006). Nuclear magnetic resonance and molecular docking analyses confirmed that embelin interacts with several residues in the XIAP BIR3 domain with which Smac and caspase-9 bind (Nikolovska-Coleska et al., 2004; Obiol-Pardo et al., 2008). It remains to be determined whether embelin and its derivatives possess properties similar to SMCs in their ability to produce TNFα, or sensitize cancer cells to exogenous TNFα, and result in the proteosomal loss of cIAP1 and cIAP2.

Additional cIAP1 and cIAP2 modulators

Another class of cIAP1 protein destabilizers exists in the form of analogs of bestatin (Ubenimex), an aminopeptidase inhibitor used in the treatment of leukemia in Japan (Bauvois and Dauzonne, 2006) (Table 3). The bestatin analogs degrade cIAP1 in the micromolar range, and may, in fact, be Smac mimetics as they bind to BIR3 of cIAP1 (Sato et al., 2008). They are referred to as degradation promoters of cIAP1, and they were not rationally designed on the AVPI tetrapeptide structure. Therefore, it is unclear whether they are bona fide Smac mimetics. Ultimately, co-crystallization studies should reveal whether they bind to the same pocket (IBM groove) on the IAPs as do the Smac mimetics.

Another small molecule inhibitor of NF-κB activation, Ro106-9920, that was identified in a cell-free system as an inhibitor of IκBα ubiquitination (Swinney et al., 2002) requires cIAP2 or a cIAP2-associated protein for activity. However, it has not been definitively demonstrated that cIAP2 E3 ligase is directly inhibited by Ro106-9920 and mechanism remains speculative.

Survivin small molecule antagonists

Several small molecule antagonists of survivin are under development, or in the clinic, all with very different mechanisms of action and chemistries (Table 3). YM155 is a small molecule identified to suppress the activity of the survivin promoter (Nakahara et al., 2007). YM155 is currently undergoing phase II clinical evaluation in patients with cancer (Altieri, 2008). Terameprocol (EM-1421, M4N), a semisynthetic derivative of a naturally occurring plant lignan, is another small molecule that suppresses survivin gene expression (Chang et al., 2004). However, it is known that Terameprocol affects global Sp1-dependent gene expression, and therefore not only targets survivin but other genes as well, such as the cell cycle regulator Cdc2 (Chang et al., 2004; Huang et al., 2006). Previously, the molecular chaperone protein, hsp90, was demonstrated to interact with and stabilize survivin (Fortugno et al., 2003). Global suppression of hsp90 chaperone function, or antibody-mediated disruption of the survivin-hsp90 complex, results in proteasomal degradation of survivin, mitochondrial-dependent apoptosis and cell cycle arrest with mitotic defects (Fortugno et al., 2003). On the basis of these findings, a small molecule peptidomimetic, shepherdin, was rationally designed to disrupt the hsp90–survivin interaction (Plescia et al., 2005). Shepherdin not only affects survivin expression but also destabilizes other hsp90 client proteins. Newer, non-peptidic, small molecules such as AICAR, similar in structure to shepherdin (Meli et al., 2006), are being evaluated. AICAR acts as an AMPK agonist, and thus affects other pathways such as protein translation and glucose metabolism (Koh et al., 2008). AICAR is being investigated primarily as a treatment for type II diabetes (Towler and Hardie, 2007) and its effects may pertain primarily to AMPK activation. AICAR and shepherdin do not show any deleterious effects in mice; however, AMPK activation by AICAR is reported to produce an acute drop in plasma potassium levels (Zheng et al., 2008). Both AICAR and shepherdin appear to have anticancer effects (Buzzai et al., 2007; Guan et al., 2007; Isakovic et al., 2007), including cell death by apoptotic and non-apoptotic means. However, there is still cause for concern with survivin antagonism. Importantly, cell cycle disruption effects and polyploidy are observed with some of the survivin antagonists. This is reminiscent of the multitude of survivin ablation studies performed with antisense or RNAi, which have not only lead to the stimulation of apoptosis but also an increase in multinucleated cells with aberrant chromosome numbers due presumably to cytokinesis defects resulting from survivin's loss (Li et al., 1999; Yang et al., 2004). Survivin is a well-established chromosomal passenger protein involved in chromosome alignment and segregation during mitosis and cytokinesis (Ruchaud et al., 2007). This function of survivin is conserved throughout evolution. Hence, there is concern that survivin antagonism may increase the aggressiveness of the surviving tumor cells by altering or increasing their ploidy, leading to increased dosage of oncogenes or reductions in tumor suppressor gene number. Additionally, specialized pools of normal survivin-expressing cells, such as stem cells, may be affected by these antagonists in ways that are not initially apparent. Further studies will be needed to address these concerns.

Adenoviral expression of dominant-negative mutants of survivin

Adenoviral vectors targeting regulators of both cell cycle and/or apoptosis are being examined for potential applications in cancer gene therapy alone, either as stand-alone treatment or in combination with chemotherapy drugs (Mesri et al., 2001). One such application is the intratumor adenoviral delivery of wild-type p53 in patients with non-small cell lung cancer, thereby restoring checkpoint functions of apoptosis and/or cell cycle arrest (Swisher et al., 1999). Targeting the survivin pathway through dominant-negative mutants in an adenoviral gene therapy approach may also be beneficial for cancer treatment. Transfection of a survivin dominant-negative mutant, carrying a cysteine 84-alanine mutation in the BIR domain or a Thr34-Ala mutation, into melanoma cell lines significantly increases the number of apoptotic cells. The Thr34-Ala mutation destroys a phosphorylation site for the cyclin-dependent kinase p34cdc2, required for survivin's association with pro-caspase-9 and inhibition of apoptosis (O’Connor et al., 2000). Conditional expression of the Thr34-Ala survivin mutant has been shown to prevent tumor formation in a subcutaneous xenograft model of melanoma (Grossman et al., 2001).

A replication-deficient adenovirus (pAd-T34A), used to overexpress the Thr34-Ala survivin dominant mutant, causes spontaneous apoptosis in breast, cervical, prostate, lung and colorectal cancer cell lines. In contrast, pAd-T34A has no observable effects on the proliferation or viability of healthy cells that do not express survivin (Mesri et al., 2001). Mitochondrial release of cytochrome c, cleavage of caspase-9 and -3, and the catalytic enhancement of caspase-3 activity are observed in the apoptotic pAd-T34A-infected cells. In combination with chemotherapeutics such as Taxol, the extent of apoptosis in HeLa and MCF-7 cells was also found to be increased (Mesri et al., 2001). Experiments using the MCF-7 human breast cancer xenograft model shows that pAd-T34A transduction suppresses de novo tumor formation, inhibits the growth of pre-established tumors, reduces intraperitoneal tumor dissemination and induces massive apoptosis in all transduced cells (Mesri et al., 2001). Thus, viral targeting of the survivin pathway using dominant-negative mutants may be a powerful tool for selective cancer gene therapy.

Survivin immunotherapy

Cancer vaccines targeting cancer-restricted epitopes represent a novel approach to eradicate tumors through stimulation of the host's immune system. Survivin is a shared tumor-associated antigen expressed in a variety of malignancies. Sera from cancer patients contain antibodies and cytolytic T-cells (CTLs) against survivin. Approaches targeting survivin epitopes are currently undergoing preclinical and clinical evaluation (reviewed in Altieri, 2008). Several phase I trials, involving either administration of survivin peptides or survivin-directed autologous CTLs, have concluded and progressed on to larger phase II trials.

Conclusions

Apoptosis is controlled at multiple intracellular nodes, each of which is influenced by pro- and antiapoptotic proteins. The equilibrium between the cell death that induces the caspase cascade and inhibition of that process by the IAPs constitutes a fundamental decision point. The recurrent deregulation of IAP activity in cancer cell lines and tumors indicates that this decision point is important in determining cell fate. The IAPs not only control cell death but also influence signal-transduction pathways, differentiation, protein turnover and progression through the cell cycle. Still, many aspects of IAP function in all these processes remain to be elucidated. With the recognition of apoptosis suppression as a fundamental aspect of human cancer, the IAPs and other antiapoptotic proteins are acknowledged as being outstanding therapeutic targets with several small molecules now undergoing intense clinical investigation.

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Acknowledgements

RGK is a Howard Hughes Medical Institute (HHMI) International Research Scholar and a Fellow of the Royal Society of Canada. RGK is supported by funds from the Canadian Institutes of Health Research (CIHR), and the HHMI. HHC is the recipient of a Michael Cuccione Foundation and CIHR Institute of Cancer Research postdoctoral fellowship. SP and DJM are recipients of a Natural Sciences and Engineering Research Council of Canada postdoctoral fellowships. ECL is supported by funds from the Optimist Club's Campaign against Childhood Cancers.

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Correspondence to E C LaCasse.

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LaCasse, E., Mahoney, D., Cheung, H. et al. IAP-targeted therapies for cancer. Oncogene 27, 6252–6275 (2008). https://doi.org/10.1038/onc.2008.302

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Keywords

  • XIAP
  • cIAP1
  • cIAP2
  • survivin
  • livin
  • birc

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