Robert G. Korneluk

The inhibitors of apoptosis (IAPs) and their emerging role in cancer.

The inhibitor of apoptosis protein family has been characterized over the past 5 years, initially in baculovirus and more recently in metazoans. The IAPs are a widely expressed gene family of apoptotic inhibitors from both phylogenic and physiologic points of view. The diversity of triggers against which the IAPs suppress apoptosis is greater than that observed for any other family of apoptotic inhibitors including the bcl-2 family. The central mechanisms of IAP apoptotic suppression appear to be through direct caspase and pro-caspase inhibition (primarily caspase 3 and 7) and modulation of and by the transcription factor NF-κB. Although evidence for a direct oncogenic role for the IAPs has yet to be delineated, a number of lines of evidence point towards this class of protein playing a role in oncogenesis. The strongest evidence for IAP involvement in cancer is seen in the IAP called survivin. Although not observed in adult differentiated tissue, survivin is present in most transformed cell lines and cancers tested to date. Survivin has been shown to inhibit caspase directly and apoptosis in general, moreover survivin protein levels correlate inversely with 5 year survival rates in colorectal cancer. Recent data has also implicated survivin in cell cycle control. The second line of evidence for IAP involvement in cancer comes from their emerging role as mediators and regulators of the anti-apoptotic activity of v-Rel and NF-κB transcription factor families. The IAPs have been shown to be induced by NF-κB or v-Rel in multiple cell lines and conversely, HIAP1 and HIAP2 have been shown to activate NF-κB possibly forming a positive feed-back loop. Overall a picture consistent with an IAP role in tumour progression rather than tumour initiation is emerging making the IAPs an attractive therapeutic target.

The gene for neuronal apoptosis inhibitory protein is partially deleted in individuals with spinal muscular atrophy

The spinal muscular atrophies (SMAs), characterized by spinal cord motor neuron depletion, are among the most common autosomal recessive disorders. One model of SMA pathogenesis invokes an inappropriate persistence of normally occurring motor neuron apoptosis. Consistent with this hypothesis, the novel gene for neuronal apoptosis inhibitory protein (NAIP) has been mapped to the SMA region of chromosome 5q13.1 and is homologous with baculoviral apoptosis inhibitor proteins. The two first coding exons of this gene are deleted in approximately 67% of type I SMA chromosomes compared with 2% of non-SMA chromosomes. Furthermore, RT-PCR analysis reveals internally deleted and mutated forms of the NAIP transcript in type I SMA individuals and not in unaffected individuals. These findings suggest that mutations in the NAIP locus may lead to a failure of a normally occurring inhibition of motor neuron apoptosis resulting in or contributing to the SMA phenotype.

The inhibitors of apoptosis (IAPs) as cancer targets

Apoptosis has been accepted as a fundamental component in the pathogenesis of cancer, in addition to other human diseases including neurodegeneration, coronary disease and diabetes. The origin of cancer involves deregulated cellular proliferation and the suppression of apoptotic processes, ultimately leading to tumor establishment and growth. Several lines of evidence point toward the IAP family of proteins playing a role in oncogenesis, via their effective suppression of apoptosis. The central mechanisms of IAP apoptotic suppression appear to be through direct caspase and pro-caspase inhibition (primarily caspase 3 and 7) and modulation of, and by, the transcription factor NF-kappaB. Thus, when the IAPs are over-expressed or over-active, as is the case in many cancers, cells are no longer able to die in a physiologically programmed fashion and become increasingly resistant to standard chemo- and radiation therapies. To date several approaches have been taken to target and eliminate IAP function in an attempt to re-establish sensitivity, reduce toxicity, and improve efficacy of cancer treatment. In this review, we address IAP proteins as therapeutic targets for the treatment of cancer and emphasize the importance of novel therapeutic approaches for cancer therapy. Novel targets of IAP function are being identified and include gene therapy strategies and small molecule inhibitors that are based on endogenous IAP antagonists. As well, molecular mechanistic approaches, such as RNAi to deplete IAP expression, are in development.

The inhibitors of apoptosis: there is more to life than Bcl2

The inhibitor of apoptosis (IAP) genes constitute a highly conserved family found in organisms as diverse as insects and mammals. These genes encode proteins that directly bind and inhibit caspases, and thus play a critical role in deciding cell fate. The IAPs are in turn regulated by endogenous proteins (second mitochondrial activator of caspases and Omi) that are released from the mitochondria during apoptosis. Overexpression of the IAPs, particularly the X-chromosome-linked IAP, has been shown to be protective in a variety of experimental animal models of human neurodegenerative diseases. Furthermore, overexpression of one or more of the IAPs in cancer cell lines and primary tumor samples appears to be a frequent event. IAP gene amplification and translocation events provide genetic evidence that further strengthens the case for classifying the IAPs as oncogenes. Therapeutic strategies that interfere with IAP expression or function are under investigation as an adjuvant to conventional chemotherapy- and radiation-based cancer therapy. This paper reviews the structure and function of the IAP family members and their inhibitors, and surveys the available evidence for IAP dysregulation in cancer.

Activation of noncanonical NF-κB requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2, TRAF3 and the kinase NIK

Recent studies suggest that nuclear factor κB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-κB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.

Identification of XAF1 as an antagonist of XIAP anti-Caspase activity.

The inhibitors of apoptosis (IAPs) suppress apoptosis through the inhibition of the caspase cascade and thus are key proteins in the control of cell death. Here we have isolated the protein XIAP-associated factor 1 (XAF1) on the basis of its ability to bind XIAP, a member of the IAP family. XIAP suppresses caspase activation and cell death in vitro, and XAF1 antagonizes these XIAP activities. Expression of XAF1 triggers a redistribution of XIAP from the cytosol to the nucleus. XAF1 is ubiquitously expressed in normal tissues, but is present at low or undetectable levels in many different cancer cell lines. Loss of control over apoptotic signalling is now recognized as a critical event in the development of cancer. Our results indicate that XAF1 may be important in mediating the apoptosis resistance of cancer cells.

IAP-targeted therapies for cancer.

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.

XIAP: Apoptotic brake and promising therapeutic target

The X-linked Inhibitor of Apoptosis, XIAP, is a key member of the newly discovered family of intrinsic inhibitors of apoptosis (IAP) proteins. IAPs block cell death both in vitro and in vivo by virtue of inhibition of distinct caspases. Although other proteins have been identified which inhibit upstream caspases, only the IAPs have been demonstrated to be endogenous repressors of the terminal caspase cascade. In turn, the caspase inhibiting activity of XIAP is negatively regulated by at least two XIAP-interacting proteins, XAF1 and Smac/DIABLO. In addition to the inhibition of caspases, recent discoveries from several laboratories suggest that XIAP is also involved in a number of other biologically significant cellular activities including modulation of receptor-mediated signal transduction and protein ubiquitination. XIAP is also translated by a rare cap-independent mechanism mediated by a specific sequence called IRES (for Internal Ribosome Entry Site) which is found in the XIAP 5′ UTR. XIAP protein is thus synthesized under various conditions of cellular stress such as serum starvation and low dose γ-irradiation induced apoptosis, conditions that lead to the inhibition of cellular protein synthesis. The multiple biological activities of XIAP, its unique translational and post-translational control and the centrality of the caspase cascade make the control of XIAP expression an exceptionally promising molecular target for modulating apoptosis. Therapeutic benefits can be derived from both the suppression of inappropriate cell death such as in neurodegenerative disorders and ischemic injury or in the activation of latent cell death pathways such as in autoimmune disease and cancer where apoptosis induction is the desired outcome.

A new internal-ribosome-entry-site motif potentiates XIAP- mediated cytoprotection

rogrammed cell death (apoptosis) plays a critical part in regulating cell turnover during embryogenesis, metamorphosis, tissue homeostasis and viral infection1. Dysregulation of apoptosis occurs in such pathologies as cancer, autoimmunity, immunodeficiency and neurodegeneration. Proteins of the inhibitor-ofapoptosis (IAP) family are intrinsic cellular suppressors of apoptosis and are represented by highly conserved members found from insect viruses to mammals2‐4. The most potent mammalian IAP is the X-linked IAP, or XIAP5, whose mechanism of action involves direct inhibition of caspases 3 and 7, key proteases of the apoptotic cascade6. Cellular control of XIAP expression should be fundamental to a cell’s ability to modulate its responses to apoptotic stimuli. However, XIAP messenger RNA is expressed in most tissues and cells at fairly constant levels5, indicating that translational control of XIAP levels may be an important regulatory mechanism. Here we characterize the primary genomic structure and function of XIAP, and show that XIAP expression is controlled at the translational level, specifically through an internal ribosome-entry site (IRES). Several features of XIAP mRNA indicate that it may be translationally regulated, including an unusually long 5′ untranslated region (UTR) (>5.5 kilobases (kb) for murine and >1.6 kb for human XIAP transcripts) with predicted complex secondary structure and numerous potential translation start sites upstream of the authentic initiation codon. This UTR would be expected to present a significant obstacle to efficient translation by conventional ribosome scanning7. An alternative mechanism of translation initiation, mediated through the IRES, has been identified in picornaviruses and in a few cellular mRNAs8. Thus we tested whether the 5′ UTR of XIAP mRNA could be involved in translation initiation from reporter-based bicistronic mRNA transcripts encoding β-galactosidase and chloramphenicol aceytyltransferase (CAT) (for example, see ref. 9). (Translation of β-galactosidase is driven by the 5′ mRNA methylguanosine cap.) Both human and mouse XIAP 5′ UTRs directed translation of the second cistron (encoding CAT) at 150fold higher levels than those produced without the 5′ UTR or with the 5′ UTR in reverse orientation, suggesting the presence of an IRES (Fig. 1a). No activity was detected when using the identical DNA segments cloned into a promoterless construct, confirming P

Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus.

We show here that transient forebrain ischemia selectively elevates levels of neuronal apoptosis inhibitory protein (NAIP) in rat neurons that are resistant to the injurious effects of this treatment. This observation suggests that increasing NAIP levels may confer protection against ischemic cell death. Consistent with this proposal, we demonstrate that two other treatments that increase neuronal NAIP levels, systemic administration of the bacterial alkaloid K2S2a and intracerebral injection of an adenovirus vector capable of overexpressing NAIP in vivo, reduce ischemic damage in the rat hippocampus. Taken together, these findings suggest that NAIP may play a key role in conferring resistance to ischemic damage and that treatments that elevate neuronal levels of this antiapoptotic protein may have utility in the treatment of stroke.