Oncogene (1998) 17, 3247 ± 3259 ã 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc The inhibitors of apoptosis (IAPs) and their emerging role in cancer Eric C LaCasse1, Stephen Baird1,2,3, Robert G Korneluk1,2,3 and Alex E MacKenzie*,1,2,3 1Apoptogen Inc, Suite R306, CHEO Research Institute, 401 Smyth Road, Ottawa, Ontario, Canada, K1H 8L1; 2Department of Pediatrics, University of Ottawa, Ontario, Canada and 3The Solange Gauthier Karsh Molecular Genetics Laboratory, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada, K1H 8L1 The inhibitor of apoptosis protein family has been baculovirus, Autographa californica nuclear polyhedro- characterized over the past 5 years, initially in sis virus (AcMNPV) results in an unconstrained host baculovirus and more recently in metazoans. The IAPs lepidopteran SF-21 cell apoptosis. (The natural host are a widely expressed gene family of apoptotic cell apoptotic response to viral infection is usually inhibitors from both phylogenic and physiologic points eectively suppressed by the infecting baculovirus). of view. The diversity of triggers against which the IAPs Employing this assay, the CpGV and OpMNPV suppress apoptosis is greater than that observed for any genomes were screened for loci which would suppress other family of apoptotic inhibitors including the bcl-2 this cell death, resulting in the identi®cation of family. The central mechanisms of IAP apoptotic founding members of a new class of anti-apoptotic suppression appear to be through direct caspase and genes encoding Cp-IAP and Op-IAP, two proteins pro-caspase inhibition (primarily caspase 3 and 7) and which share both homology and the ability to block modulation of and by the transcription factor NF-kB. apoptosis by a wide number of apoptotic triggers Although evidence for a direct oncogenic role for the (Tables 1, 2 and 5). IAPs has yet to be delineated, a number of lines of evidence point towards this class of protein playing a role BIR and RING Zn ®nger domains in oncogenesis. The strongest evidence for IAP involve- ment in cancer is seen in the IAP called survivin. There exists in the baculoviral IAPs two de®ning Although not observed in adult dierentiated tissue, motifs. The ®rst of these is the BIR (for Baculoviral survivin is present in most transformed cell lines and Inhibitor of apoptosis Repeat) domain; a pair of these cancers tested to date. Survivin has been shown to inhibit comprise the amino terminal half of the viral IAPs. caspase directly and apoptosis in general, moreover The 80 amino acid BIR domain contains a possible survivin protein levels correlate inversely with 5 year serine/threonine phosphorylation site (RX2ST/F) fol- survival rates in colorectal cancer. Recent data has also lowed 17 residues downstream by an invariant glycine implicated survivin in cell cycle control. The second line and then DX3CX2C and HX6C consensus sequences of evidence for IAP involvement in cancer comes from (Figure 1). These latter arrays are also likely metal their emerging role as mediators and regulators of the coordination sites although this remains to be formally anti-apoptotic activity of v-Rel and NF-kB transcription documented; the physical structure of the BIR has not factor families. The IAPs have been shown to be induced yet been documented but is clearly a priority. The by NF-kB or v-Rel in multiple cell lines and conversely, importance of the non-conserved residues in the BIR HIAP1 and HIAP2 have been shown to activate NF-kB domain is re¯ected in the fact that the Ac-IAP found in possibly forming a positive feed-back loop. Overall a a third AcMNPV baculovirus, although containing the picture consistent with an IAP role in tumour progres- conserved BIR residues, is inactive with regard to sion rather than tumour initiation is emerging making apoptotic inhibition (Birnbaum et al., 1994). the IAPs an attractive therapeutic target. The second de®ning motif found in the baculoviral IAPs is the COOH terminus RING Zn ®nger (for Keywords: inhibitor of apoptosis; IAP; programmed Really Interesting New Gene, Freemont et al., 1991) cell death; BIR; RING ®nger; CARD; NAIP; survivin; containing a CX2 CXNCXHX2-3CX2 CXMCX2 C motif NF-kB; caspase (metal coordinating sites underlined, Figure 2). NMR based structural analysis has revealed the RING ®nger to assume a spherical cross brace structure with two Zn coordination sites identi®ed; one comprised of the ®rst Part A. The IAP family and third cluster of metal binding residues and the second coordination de®ned by the second and fourth The ®rst Inhibitor of Apoptosis (IAP) proteins were metal binding residue clusters (Barlow et al., 1994). identi®ed in 1993 and 1994 in the baculoviruses Cydia The RING Zn ®nger has been seen in nearly 100 pomonella granulosis virus (CpGV) and Orgyia proteins, is usually localized at the amino terminus of a pseudotsugata nuclear polyhedrosis virus (OpMNPV) protein and has been shown to be involved in DNA by the laboratory of Lois Miller (Crook et al., 1993; and protein interactions (Borden and Freemont, 1996). Birnbaum et al., 1994). The Miller lab utilized an assay The lack of conservation in the non-cys/his residues in which infection by a mutant strain of a third between various RING ®ngers is in keeping with the diverse functions postulated for these domains. The presence of the RING Zn ®nger appears to be critical to the baculoviral IAP anti-apoptotic function while it is dispensable for some (but not all) cellular IAP *Correspondence: AE MacKenzie apoptotic inhibition (Table 5). IAPs and cancer EC LaCasse et al 3248 Table 1 The IAP and BIR* family members (from 1993 ± 1998) Viral IAPs Baculovirus: CpIAP, OpIAP/OpIAP3, AcIAP*, OpBIRP2*, BusuIAP*, EpIAP* Other: ASFV IAP*/ORF 4CL and ORF A224L, CiVIAP* Nematode CeBIR2*, CeBIR1* Yeast SpBIR1*, ScBIR2* Drosophila IAPs 1 DIAP1/Thread Protein 2 DIAP/DIAP2/dILP/DIHA Vertebrate IAPs Human Murine Rat Porcine Chicken 1 NAIP mNAIP**/NAIP-rs rNAIP (partial) (6 genomic copies) 2 HIAPI/cIAP2/MIHC/hITA MIAP1 RIAP1 PIAP ITA/ch-IAP1 3 HIAP2/cIAP1/MIHB MIAP2/mcIAP1/API2 RIAP2/rIAP1 4 XIAP/hILP MIAP3/MIHA/mXIAP/API3 RIAP3 5 Survivin TIAP/msurvivin 6 BRUCE* **Murine NAIP homologs mNaip1 mNaip2 Naip-rs6 mNaip3 Naip-rs5 mNaip4 mNaip5 Naip-rs3 mNaip6 Abbreviations: IAP, inhibitor of apoptosis; BIR, baculoviral IAP repeat. *Contains a BIR domain, however, it has no proven anti-apoptotic function. CpIAP, Cydia pomonella Granulosis Virus IAP (L05494, U53466, P41436) Crook et al., 1993; OpIAP/OpIAP3, Orgyia pseudotsugata Nuclear Polyhedrosis Virus IAP (L 22564, P41437, U75930) Birnbaum et al., 1994; AcIAP, Autographa californica Nuclear Polyhedrosis Virus IAP (M96361) Clem and Miller, 1994; OpBIRP2, Orgyia pseudotsugata Nuclear Polyhedrosis virus BIR-containing protein 2 (U75930) Uren et al., 1998; BusuIAP, Buzura suppressaria Nuclear Polyhedrosis Virus IAP (AF045936), Hu et al., 1998; EpIAP, Epiphyas postvittana Nuclear Polyhedrosis Virus IAP (AF037358); ASFV IAP, African Swine Fever Virus IAP, ORF 4CL (U18466) Neilan et al., 1997 and ORF A234L Chacon et al., 1995; CiVIAP, Chilo iridescent Virus IAP (M81387, AF003534) Uren et al., 1998; CeBIR1, C. elegans BIR containing protein (Z77654) Uren et al., 1998; SpBIR1, S. pombe BIR containing protein 1 (AL 031323, 3451317) Uren et al., 1998; CeBIR2, C. elegans BIR containing protein 2, (U85911) Uren et al., 1998; ScBIR2, C. cerevisiae BIR containing protein 2 (L47993, 101 9708) Uren et al., 1998; DIAP1 Drosophila IAP 1 (L 49440) Hay et al., 1995; DIAP, Drosophila IAP (U45881, M96581) Liston et al., 1996; DIAP2, Drosophila IAP2 (L49441) Hay et al., 1995; dILP, Drosophila IAP-like protein (U32373) Duckett et al., 1996; DIHA, Drosophila IAP homolog A (U38809) Uren et al., 1996; NAIP, neuronal apoptosis inhibitory protein (U19251, U80017) Roy et al., 1995; Chen et al., 1998; mNAIP1-6, mouse NAIP-1 through 6 (AF007769) Yaraghi et al., 1998; NAIP-rs 1 through 6, NAIP related sequence 1 (U66324), 2 (U66325), 3 (U66326), 4 (U66327), 5 (U66328), 6 (U66329) Scharf et al., 1996; rNAIP, Rat NAIP (partial cDNA) Korneluk et al., (unpublished); HIAP1, human IAP1 (U45878) Liston et al., 1996; cIAP2, cellular IAP2 (L49432) Rothe et al., 1995; MIHC, mammalian IAP homolog C (U37546) Uren et al., 1996; hITA, human inhibitor of T-cell apoptosis, Nicholl et al., 1996; MIAP1, mouse IAP1 (U88908) Liston et al., 1997; RIAP1, rat IAP1 Holcik et al., (unpublished); PIAP, porcine IAP (U79142) Stehlik et al., 1998b; ITA, inhibitor of T-cell apoptosis (U27466) Digby et al., 1996; ch-IAP1, chicken IAP1 (AF008592) You et al., 1997; HIAP2, human IAP2 (U45879) Liston et al., 1996; cIAP1, cellular IAP1 (L49431) Rothe et al., 1995; MIHB, mammalian IAP homolog B (U37547) Uren et al., 1996; MIAP2, mouse IAP2 (U88909) Liston et al., 1997; mcIAP1, mouse cellular IAP1 (L49433) Rothe et al., 1995; API2, mouse apoptosis inhibitor 2; RIAP2, rat IAP2 Holcik et al., (unpublished); rIAP1, rat IAP1 (3445577, AF081503) Bradley, Lareu and Dharmarajan (unpublished); XIAP, X-linked IAP (U45880) Liston et al., 1996; hILP, human IAP-like protein (U32974) Duckett et al., 1996; MIAP3, mouse IAP3 (U88990) Farahani et al., 1997; MIHA, mammalian IAP homolog A (U36842) Uren et al., 1996; mXIAP, mouse XIAP; API3, mouse apoptosis inhibitor 3; RIAP3, rat IAP3, Holcik et al., (unpublished); survivin (U75285) Ambrosini et al., 1997; TIAP, thymus/testis-speci®c IAP (murine survivin homolog) (AB013819) Kobayashi et al., (unpublished); msurvivin, mouse survivin, Gibson, Holcik and Korneluk (unpublished); BRUCE, BIR-Repeat containing ubiquitin conjugating enzyme (Y17267) Hauser et al., 1998. NB accession numbers refer to either gene, genome or protein sequence. Table 2 Classi®cation according to homology domain Non-viral IAPs Category Examples The initial baculoviral IAP discovery was followed in 1 BIR survivin, BRUCE*, ASFV IAP*, 1995 by the identi®cation of a series of metazoan IAPs, CeBIR2* the ®rst of these being the NAIP gene.