The Inhibitors of Apoptosis (Iaps) and Their Emerging Role in Cancer

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 e€ectively 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 di€erentiated 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. NAIP is alone 1 BIR+1 RING CiV IAP* 2 BIRs CeBIR1*, SpBIR1*, ScBIR2* in the BIR containing proteins insofar as it has three 2 BIRs+1 RING OpIAP, CpIAP, DIAP1 BIRs but no RING Zn ®nger (The A and I are 2 BIRs+1 CARD+1 RING PIAP interposed to distinguish it from the more classic IAPs 3 BIRS NAIP which contain not only the BIR domain but the RING 3 BIRs+1 RING DIAP2, XIAP Zn ®nger as well). This gene was found by virtue of it's 3 BIRs+1 CARD+1 RING HIAP1, HIAP2, ITA absence from the majority of infants with the most Examples of IAPs and *BIR-containing proteins categorized according to the presence of BIR, zinc ring ®nger (RING) or severe form of the pediatric neurodegenerative disorder CARD (CAspase Recruitment Domain) domains. Vertebrate known as spinal muscular atrophy (SMA, Roy et al., members are italicized. 1995). This autosomal recessive condition is caused by IAPs and cancer EC LaCasse et al 3249

Figure 1 Alignment of IAP BIR domains. Alignment of all vertebrate IAP BIR amino acid sequences, plus some other non- vertebrate member sequences, was performed using the PILEUP program of GCG (Genetics Computer Group, Wisconsin). The exact BIR repeat for those IAPs with multiple repeats is listed in order of their appearance in the protein from N-terminus to C- terminus as 1 ± 3 (indicated by the ®nal hyphenated number after the name). The start position for each repeat is indicated for each protein on the left-hand side of the sequence. Please refer to Table 1 for abbreviated names and accession numbers. Alignment of identical residues are highlighted in black while conservative residues are grey. The consensus sequence given is for amino acids that are present in over 70% of the proteins shown and is indicated in upper case if present in 100% of proteins

In the period since the cloning of NAIP, four additional BIR containing human genes have been identi®ed. Three of these (XIAP, HIAP1 and HIAP2) contain NH2 BIR domains and COOH RING ®nger domains as is the case with the baculoviral IAPs (Rothe et al., 1995; Liston et al., 1996; Duckett et al., 1996; Uren et al., 1996). There are two chief features which distinguish these four human IAPs from the viral IAPs; they have three (instead of two) BIR domains and the section between BIR and RING ®nger is longer than that seen in the baculovirus, and contains a CARD (for caspase recruitment domain, Hofmann et al., 1997; see Figure 3) motif in the case of Figure 2 Alignment of IAP RING zinc ®ngers. Alignment of RING ®nger domain amino acid sequences for those IAPs which HIAP1 and HIAP2 (Table 2). The CARD motif, which possess the domain is shown, including the sequence for TRAF6 is predicted to consist of six alpha helices, is commonly (a non-IAP protein) for comparisons sake found in proteins involved in apoptotic signalling, including the caspases (e.g. caspase 1) as well as some of the IAPs. A fourth human gene, survivin contains a a severe loss of motor neurons which results in fatal solitary BIR domain and shows strongest upregulation ¯accid paralysis (Dubowitz, 1995). While the contig- in tumours (Ambrosini et al., 1997). A potential sixth uous SMN gene has been found to be causative of human IAP expressed solely in testes has been SMA (Lefebvre et al., 1995), the tissue expression of identi®ed (Figures 1 and 2). The TIAP (not to be NAIP (Xu et al., 1997a) combined with its strong in confused with murine survivin also called TIAP) is a vitro (Liston et al., 1996) and in vivo (Xu et al., 1997b) transcribed processed pseudogene; whether the TIAP neuroprotective e€ect make it a likely candidate for transcript is also translated is currently unknown modi®cation of the SMA phenotype. It is postulated (Lagace and Korneluk, personal communication). In that motor neurons missing NAIP withstand the low addition to these genes, porcine, chicken, Drosophila, cellular levels of SMN protein poorly and dying earlier rat, murine, yeast and nematode IAPs have all been than they would have otherwise resulting in more found (Table 1). These genes contain a number of severe disease (MacKenzie, 1998). permutations and combinations of BIR, RING Zn IAPs and cancer EC LaCasse et al 3250 ®ngers and other domains (Table 2). Many of the hold true for another. Nonetheless, the conservation of vertebrate IAPs have had their chromosomal localiza- both sequence and function between baculoviral and tion determined (Table 3). One of the more intriguing cellular IAPs suggest strongly that they interact with proteins is a large murine ubiquitin conjugating conserved components of the apoptotic mechanism, an enzyme, BRUCE, which contains both a BIR domain observation supported by the ability of baculoviral and a UBC motif, possibly re¯ecting an intersection of IAPs to inhibit mammalian cell death from a number apoptotic and ubiquitination pathways (Hauser et al., of triggers (Hacker et al., 1996; Dorstyn and Kumar, 1998). 1997; Hawkins et al., 1996, 1998; Table 5). Despite this The two IAPs in Drosophila were identi®ed by apparent conservation of biological e€ect, a mechan- means of screening for suppressors of Reaper induced istic divergence is apparent on close examination. This ocular cell death (Hay et al., 1995). This work was the is seen most clearly in the requirement for the full ®rst to point to the BIR domains as being the main length baculoviral IAP in virtually all arthropod cell arbiters of apoptotic inhibition in metazoan systems; (a death paradigms studied while the BIR domains alone truncated IAP containing solely the BIR domains are sucient for apoptotic inhibition in many systems showed enhanced eye cell rescue). It was also the ®rst using the cellular IAPs. (and still only) to unequivocally show a phenotype associated with loss of IAP function; this consisted of IAPs interact with and inhibit the caspases branching defects in the aristae located on Drosophila antennae and an increase of Reaper induced death. In broad terms it appears that the majority of IAPs inhibit the cysteine proteases known as caspases. While there has been some con¯icting data, all metazoan IAP interacting proteins and mechanisms of action IAPs tested to date have been shown by at least one As is frequently the case with in vitro recapitulation of group to inhibit this class of pro-apoptotic proteases biological systems, IAP mediated anti-apoptosis (Table 4). appears to be a highly trigger and cell type speci®c Speci®cally, IAPs have been shown to directly inhibit phenomenon; what is observed in one system may not activated caspase 3 and 7 (Deveraux et al., 1997; Roy et

Figure 3 Alignment of IAP CARD domains. Alignment of CARD amino acid sequences for all the IAPs which possess a CARD domain, along with sequences from several other (non-IAP) CARD-containing proteins (reviewed in Hofmann et al., 1997) is shown

Table 3 Chromosomal location of vertebrate IAPs and molecular characterization Species IAP Genomic locus #aa Protein size (kDa) mRNA size (kb) References Human NAIP 5q13.1 1403 156 6.5 Roy et al., 1995; Chen et al., 1998; Rajcan- (paralogous Separovic et al., 1996a; Banyer et al., 1998 region 6p21.3) HIAP1 11q22-23 604 68 6.5/8.7 Liston et al., 1996; Young et al. (in press); Rajcan-Separovic et al., 1996b; Nicholl et al., 1996 HIAP2 11q22-23 618 70 4.5 Liston et al., 1996; Rajcan-Separovic et al., 1996b survivin 17q25 142 16.5 1.9 Ambrosini et al., 1997; Ambrosini et al., 1998 XIAP Xq24-25 497 55 9.0 Liston et al., 1996; Rajcan-Separovic et al., 1996b Murine mNAIP1 13 D1-3 1403 159 5.4 Yaraghi et al., 1998; Scharf et al., 1996 mNAIP2 13 D1-3 1447 164 5.5 Yaraghi et al., 1998; Scharf et al., 1996 MIAP1 9A2 600 66 2.9 Liston et al., 1997 MIAP2 9A2 612 68 4.0 Liston et al., 1997 msurvivin 140 16 Gibson, Holcik and Korneluk (unpub- lished); Kobayashi et al. (unpublished) MIAP3 XA3-5 496 55 8 Farahani et al., 1997 Rat RIAP1 602 67 2.5 Holcik and Korneluk (unpublished) RIAP2 589 66 3.5 Holcik and Korneluk (unpublished) RIAP3 497 56 8 Holcik and Korneluk (unpublished) Porcine PIAP 358 41 Stehlik et al., 1998b Avian ITA 611 69 4.2 Digby et al., 1996 ch-IAP1 610 68 3.3 You et al., 1997 IAPs and cancer EC LaCasse et al 3251 Table 4 List of mechanisms of action and binding interactions for IAPs (P=partial activity) Mechanism Functional IAP Non-functional IAP References Caspase-3 direct inhibitor XIAP, HIAP1, HIAP2, NAIP Deveraux et al., 1997; Roy et al., 1997; XIAP-BIR2, survivin, Takahashi et al., 1998; Tamm et al. (in press) (NAIP-BIR*, survivin-BIR*) *Maier, Nicholson, Roy and MacKenzie (un- published) Caspase-7 direct inhibitor XIAP, HIAP1, HIAP2, NAIP Deveraux et al., 1997; Roy et al., 1997; XIAP-BIR2, survivin Takahashi et al., 1998; Tamm et al. (in press) Suppressor of caspase-8 HIAP1+HIAP2+TRAFs Wang et al., 1998 activation Inhibitor of Cytochrome-C XIAP, HIAP1, HIAP2, Deveraux et al., 1998; Takahashi et al., 1998 activation of pro-caspase 9 XIAP-BIR2 TRAF-1 binding protein HIAP1, HIAP2 XIAP, OpIAP, NAIP, Rothe et al., 1995; Uren et al., 1996; Duckett et CpIAP, AcIAP, DIAP1, al., 1998; Roy et al., 1997; Hawkins et al., 1998 DIAP2 TRAF-2 binding protein HIAP1, HIAP2 XIAP, NAIP, survivin, Rothe et al., 1995; Uren et al., 1996; Shu et al., CpIAP, OpIAP, AcIAP, 1996; Duckett et al., 1998; Roy et al., 1997; DIAP1, DIAP2 Hawkins et al., 1998 RIP2/RICK/CARDIAK HIAP2 HIAP1 McCarthy et al., 1998 binding protein NF-kB activator HIAP1, XIAP** Chu et al., 1997; Wang et al., 1998; **Tanner (personal communication) JNK1 activator XIAP, HIAP2(P) HIAP1 Sanna et al., 1998 Reaper, Grim, HID binding HIAP2, OpIAP, CpIAP, McCarthy and Dixit, 1998; Vucic et al., 1997a, protein DIAP1, DIAP2 1998 Reaper, Grim, HID inhibitor DIAP1, DIAP2, HIAP1, Hay et al., 1995; McCarthy and Dixit, 1998; HIAP2, OpIAP, CpIAP Vucic et al., 1997a,b, 1998; Seshagiri and Miller, 1997 Doom binding protein OpIAP, CpIAP Harvey et al., 1997a Doom inhibitor OpIAP, CpIAP, DIAP1, Harvey et al., 1997a,b DIAP2 (P) Thick veins (Tkv) binding DIAP1, DIAP2, DIAP1- Oeda et al., 1998 protein RING TAB1 binding protein XIAP, XIAP-BIR HIAP1, HIAP2 Yamaguchi et al. (in press) al., 1997; Tamm et al., in press). The caspases exist as proteins have yet to be identi®ed. A number of studies inactive pro-caspases which require proteolytic activa- have shown that the mammalian IAPs inhibit tion; in this regard the IAPs have also been shown to apoptosis by non-caspase interactions chie¯y involving inhibit the activation of pro-caspase 9 (Deveraux et al., NF-kB and in one case, JNK1. Two of the ®rst cellular 1998). There is also some evidence for an indirect IAPs (HIAP1 and HIAP2) identi®ed were isolated by inhibition of caspase 1 (ICE) and caspase 2 by virtue of their interaction with the TNFa receptor baculoviral IAPs (Hawkins et al., 1998). Moreover, associated factor 2 (TRAF2; Rothe et al., 1995). despite the presence of the CARD domain in some IAPs, Although no clear cytoprotection devolving from the this inhibitory activity maps to the BIR domains with, in interaction could be documented in this study, it raised at least one case, a single XIAP BIR being both sucient the possibility that IAP apoptotic suppression might be and necessary for the observed inhibition (Takahashi et mediated in part through modulation of the TNFa al., 1998). The case for arthropod caspase inhibition is signal transduction pathway. In this regard, TNFR1 less clear for baculoviral systems, the number of caspases and 2 can trigger both apoptotic and anti-apoptotic to test are far fewer than those identi®ed in mammalian responses in the cell; the latter being mediated chie¯y systems. In contrast to mammalian systems, no e€ect on by the transcription factor NF-kB (Rothe et al., 1995; activated insect caspase by Op-IAP is observed although Wang et al., 1996; Beg and Baltimore, 1996; Van inhibition of the activation of pro-caspases has been Antwerp et al., 1996). TNFa has been shown in Jurkat documented (Manji et al., 1997; Seshagiri and Miller cells to upregulate HIAP1 through NF-kB, conversely 1997). HIAP1 activates NF-kB. Moreover, in one study, HIAP1 protection from TNFa killing was found to be dependent upon the presence of NF- B underlining the Non-caspase inhibitory mechanisms of the IAPs k centrality of this mechanism to IAP protection in There appears to exist with the IAPs the counter- Jurkat cells (Chu et al., 1997). A recent analysis of intuitive (but not unprecedented as has been seen with primary endothelial cells revealed a signi®cant upregu- Bcl-2) situation wherein a single protein possesses lation of HIAP1, HIAP2 and XIAP following exposure biochemically distinct mechanisms directed to the to TNFa (Stehlik et al., 1998a). This stimulation same end. Thus IAPs can also inhibit apoptosis appears to be downstream of NF-kB given the fact through non-caspase mechanisms. The evidence for that overexpression of the NF-kB inhibitor, IkB, non caspase interactions is stronger for Drosophila totally suppressed IAP induction and sensitizes cells IAPs; although baculoviral and human IAPs have also to apoptotic stimuli and, conversely, overexpression of been shown to interact with and inhibit the pro- XIAP reconstitutes cytoprotection, abrogating the IkB apoptotic Drosophila proteins Hid, Reaper, Grim and inhibition. Similarly, a di€erential display analysis of Doom (Hay et al., 1995 ; Vucic et al., 1997a,b; Harvey porcine aortic endothelial cells revealed an inducible et al., 1997a,b; McCarthy and Dixit, 1998; Seshagiri porcine IAP with 73.9% amino acid homology to and Miller, 1997). Mammalian homologs of these HIAP1 (Stehlik et al., 1998b). Speci®cally, LPS, TNFa IAPs and cancer EC LaCasse et al 3252 Table 5 Cell death pathways blocked or not by the IAPs (P=partial protection) Apoptotic trigger Cell type Protecting IAP Non-protecting IAP Reference

TNFa HUVEC (+IkB), XIAP Stehlik et al., 1998a HT 1080 I (IkB-DN), HIAP1+HIAP2+ HIAP1, HIAP2, Wang et al., 1998 embryonic ®broblasts TRAF-1 and -2, HIAP1+HIAP2+ (p65 null) HIAP1+HIAP2+ TRAF3 TRAF1, HIAP1+TRAF1 (P) MCF7 OpIAP Hawkins et al., 1998 HeLa HIAP1 HIAP1-BIR Chu et al., 1997 MCF7F XIAP, XIAP-BIR Duckett et al., 1998 TNF-a+IkB-DN HeLa HIAP1 Chu et al., 1997 TNF-a+cycloheximide HeLa NAIP OpIAP Hacker et al., 1996; Liston et al., 1996 TNF+HIAP1 BIR HeLa HIAP1 Chu et al., 1997 Fas 293 survivin (P), XIAP, Takahashi et al., 1998; Tamm et al., XIAP-BIR2 (P), HIAP1, in press; Roy et al., 1997; Deveraux HIAP2 et al., 1998 CD95L/Fas ligand LN-18, LN-229 XIAP HIAP1, HIAP2 Weller, personal communication CD95L+cycloheximide LN-18, LN-229 XIAP HIAP1, HIAP2 Weller, personal communication; Anti-CD95 antibody HeLa OpIAP Hawkins et al., 1998 MCF7 XIAP Duckett et al., 1998 Anti-CD95+Actinomycin D Hep G2 XIAP Suzuki et al., 1998 FADD/MORT1 HeLa OpIAP (P) MIAP3, HIAP2, Uren et al., 1996; Hawkins et al., XIAP, HIAP1, 1996; Hacker et al., 1996 OpIAP-BIR CHO OpIAP Hawkins et al., 1996 SF-21 OpIAP (P) Vucic et al., 1997b RIP MCF-7 HIAP1, HIAP2 McCarthy et al., 1998 RIP2/RICK/CARDIAK MCF-7 HIAP1, HIAP2 McCarthy et al., 1998 ts v-Rel Chicken spleen cells ch-IAP1 You et al., 1997 ICE/caspase-1 HeLa HIAP1, HIAP2, MIAP3, OpIAP-BIR Uren et al., 1996 OpIAP 293 OpIAP, XIAP Duckett et al., 1996 NIH3T3 MIAP3, HIAP1, Dorstyn and Kumar, 1997 OpIAP, HIAP2 HeLa DIAP1, DIAP2, AcIAP, DIAP1-BIR Hawkins et al., 1998 DIAP2-BIR, OpIAP, CpIAP HeLa, CHO OpIAP OpIAP-BIR Hawkins et al., 1996 293 XIAP XIAP-BIR (P) Sanna et al., 1998 293 OpIAP, XIAP, ch- chIAP-BIR You et al., 1997 IAP1 HeLa OpIAP OpIAP-BIR Hacker et al., 1996 SF-21 OpIAP, CpIAP, Seshagiri and Miller, 1997 AcIAP Caspase 1 (in vitro) HIAP1, HIAP2, Roy et al., 1997 XIAP, NAIP Caspase-2 NIH3T3 HIAP1 (P), MIAP3 (P) Dorstyn and Kumar, 1997 HIAP2 (P), OpIAP (P) HeLa OpIAP, CpIAP AcIAP Hawkins et al., 1998 HeLa, CHO OpIAP Hawkins et al., 1996 MCF7F XIAP Duckett et al., 1998 Caspase-3 NIH3T3 MIAP3, OpIAP HIAP1, HIAP2 Dorstyn and Kumar, 1997 293 survivin, XIAP Tamm et al., in press Active Caspase-3 SF-21 OpIAP, CpIAP, Seshagiri and Miller, 1997 AcIAP Caspase-3 (in vitro) XIAP, HIAP1, NAIP Roy et al., 1997; Deveraux et al., HIAP2, HIAP1-BIR, 1997 HIAP2-BIR XIAP-BIR, NAIP-BIR Maier, Nicholson, Roy and Survivin-BIR MacKenzie (unpublished)

Capase-6 (in vitro) HIAP1, HIAP2, Roy et al., 1997 XIAP, NAIP Caspase-7 293T, CHO OpIAP Hawkins et al., 1996 293 survivin, XIAP Tamm et al., in press Caspase-7 (in vitro) HIAP1, HIAP2, NAIP Roy et al., 1997; Deveraux et al., XIAP, HIAP1-BIR, 1997 HIAP2-BIR Caspase-8 MCF7F XIAP Duckett et al., 1998 Caspase-8 (in vitro) survivin Tamm et al., in press Cytosol+HeLa XIAP Deveraux et al., 1997 Nuclei (data not shown) Active SF Caspase SF-21 OpIAP, CpIAP, Seshagiri and Miller, 1997 AcIAP continued IAPs and cancer EC LaCasse et al 3253 Table 5 Continued Apoptotic trigger Cell type Protecting IAP Non-protecting IAP Reference pro-SF-Caspase+wt AcMNPV SF-21 OpIAP, CpIAP Seshagiri and Miller, 1997 Active SF caspase SF-21 cytosol OpIAP Manji et al., 1997 Sindbis virus infection BHK, N18 OpIAP, XIAP Duckett et al., 1996 Baculovirus AcMNPV (p357) SF-21 OpIAP, CpIAP AcIAP Manji et al., 1997; Crook et al., infection 1993; Birnbaum et al., 1994; Clem and Miller, 1994 ASFV infection Porcine ASFV IAP (4CL) Neilan et al., 1997 Macrophages In¯uenza infection CHO HIAP1, HIAP2 Brown and Bailly (personal communication) E1A Primary rat kidney CpIAP White (personal communication in cells Clem et al., 1996) IE1 SF-21 CpIAP AcIAP Prikhod'ko and Miller, 1996 Serum withdrawal CHO NAIP, XIAP, Liston et al., 1996 HIAP1, HIAP2 NIH3T3 MIAP3, HIAP2 HIAP1, Op-IAP Dorstyn and Kumar, 1997 PC12 OpIAP Hawkins et al., 1998 IL-3 withdrawal FDC-P1 OpIAP Hawkins et al., 1998 BaF3 survivin Ambrosini et al., 1997 K+ withdrawal Primary rat NAIP (P), XIAP Simons et al., (in press) cerebellar granule (P), HIAP1 (P), neurons HIAP2 (P) Reaper Drosophila eye DIAP1, DIAP2, HIAP2, DIAP1- Hay et al., 1995 (in vivo) HIAP2-BIR (P) RING MCF7 HIAP1, HIAP2 McCarthy and Dixit, 1998 SF-21 OpIAP, DIAP2, Vucic et al., 1997a,b; Seshagiri and CpIAP Miller, 1997 (data not shown) Grim MCF7 HIAP2, HIAP1 McCarthy and Dixit, 1998 SF-21 OpIAP, CpIAP, DIAP1 Vucic et al., 1998 (P), DIAP1-BIR, DIAP2 (P) HID SF-21 OpIAP, CpIAP, Vucic et al., 1998 DIAP1, DIAP1-BIR, DIAP2 (P) Drosophila eye DIAP1 (P), DIAP2 Hay et al., 1995 (in vivo) (P), DIAP1-BIR Doom SF-21 OpIAP, CpIAP, DIAP1-RING, Harvey et al., 1997a,b DIAP1, DIAP1-BIR(P) DIAP2-BIR DIAP2 (P) OpIAP-BIR, CpIAP-BIR OpIAP-RING, CpIAP-RING BIK MCF7 HIAP1, HIAP2 Orth and Dixit, 1997 BAK MCF7 HIAP1, HIAP2 Orth and Dixit, 1997 BAX 293T XIAP, XIAP-BIR XIAP-RING Deveraux et al., 1997 293 survivin (P), XIAP Tamm et al., in press Cytochrome C 293 XIAP Duckett et al., 1998 Cytochrome C (in vitro) 293 Extract XIAP, HIAP1, HIAP2, Roy et al., 1997 HIAP1-BIR, HIAP2-BIR survivin Tamm et al., in press 293, 293T, or Jurkat XIAP Deveraux et al., 1997 extracts, HeLa nuclei Etoposide HT1080I HIAP1, HIAP2 Wang et al., 1998 293T HIAP1, HIAP2, Roy et al., 1997 HIAP1-BIR, HIAP2-BIR 293 survivin (P), XIAP Tamm et al., in press SHSY-5Y XIAP, NAIP (P) Craig and Cherton-Horvat (personal communication) Adriamycin SHSY-5Y XIAP, NAIP (P) Craig and Cherton-Horvat (personal communication) Cisplatin MCF7F XIAP Duckett et al., 1998 SHSY-5Y XIAP NAIP Craig and Cherton-Horvat (personal communication) Taxol NIH3T3 survivin, XIAP survivin-BIR Li et al., in press Actinomycin D SF-21 OpIAP, CpIAP, DIAP2, DIAP1- Harvey et al., 1997b DIAP1 BIR, OpIAP-BIR, CpIAP-RING, CpIAP- BIR, CpIAP-RING SF-21 CpIAP Crook et al., 1993 SF-21 CpIAP, OpIAP AcIAP Clem and Miller, 1994 SF-21 OpIAP McLachlin and Miller, 1997 Amanitin SF-21 CpIAP AcIAP Clem and Miller, 1994 5,6-dichlorobenzimidazole SF-21 CpIAP AcIAP Clem and Miller, 1994 riboside continued IAPs and cancer EC LaCasse et al 3254 Table 5 Continued Apoptotic trigger Cell type Protecting IAP Non-protecting IAP Reference Staurosporine Rat-1 XIAP, HIAP1, Liston et al., 1996 HIAP2 SHSY-5Y XIAP, NAIP Craig and Cherton-Horvat (personal communication) Menadione CHO, Rat-1 NAIP, XIAP, Liston et al., 1996 HIAP1, HIAP2 6-Hydroxy-dopamine Rat brain XIAP Crocker et al., in press Dopaminergic neurons (in vivo) Vincristine NIH3T3 survivin Li et al. (in press) Nocodazole NIH3T3 survivin Li et al. (in press) Gamma-radiation (30 Gy) NIH3T3 MIAP3 HIAP1, HIAP2, Dorstyn and Kumar, 1997 Op-IAP X-radiation (80 Gy) Drosophila eye DIAP1-BIR Hay et al., 1995 (in vivo) UV-radiation MCF7F XIAP Duckett et al., 1998 SF-21 OpIAP Manji et al., 1997 Ischemia (global) Rat brain CA-1 NAIP Xu et al., 1997b Hippocampal Neurons (in vivo) XIAP Crocker et al. (in press) TAB1+TAK1 Xenopus embryo XIAP Yamaguchi et al. (in press) Glutamate excitotoxicity Primary rat HIAP1, HIAP2 Simons et al. (in press) cerebellar granule (P), NAIP, XIAP neurons PHA/phytohemagglutinin Jurkat XIAP Vitte-Mony et al., 1997 Zac/LOT1 SaOs-2 HIAP1 Ho€man and Spengler (personal communication) viral-IAP-BIR SF-21 OpIAP, CpIAP Harvey et al., 1997b CpIAP-RING SF-21 OpIAP, CpIAP Harvey et al., 1997b DIAP1-RING SF-21 DIAP1-BIR (P) Harvey et al., 1997b

and IL-Ib were found to upregulate this gene and this prerequisite for the most e€ective apoptotic suppres- induction was suppressed by IkB suggesting a direct sion at least in this model (Li et al., in press). The role for NF-kB stimulation of these cells. A more Drosophila IAPs have been demonstrated to interact detailed discussion of the role of NF-kB in IAP with Thick Veins, a type I serine/threonine kinase function is given in part B. receptor for Decapentaplegic (a member of the BMP/ TGF superfamily; Oeda et al., 1998). Interestingly, this interaction is mediated by the RING ®nger of DIAP1, Other IAP interactions the only function attributed to this domain so far. A number of other non-caspase mechanisms of Furthermore, XIAP has just been shown to interact apoptotic inhibition have been proposed including a with TAB1 and BMP receptors in humans (Yamaguchi recent co-transfection study of cos-7 and 293 cells et al., in press). TAB1 binds the TGF-activated which revealed an XIAP mediated inhibition of kinase 1, TAK1, which is a member of the MAPKKK caspase 1 by a selective activation of the c-Jun N- family. Overexpression of XIAP stimulated ventraliza- terminal kinase (Sanna et al., 1998). This activation, tion in Xenopus embryos in a TAB1-TAK1-dependent which mapped to the BIR domains and the contiguous manner. Moreover, an interaction between XIAP and spacer region, was essential to the observed apoptotic BMP (for bone morphogenic protein) receptor in suppression. A recent demonstration of the interaction mammalian cells, mediated through the RING ®nger, of HIAP1 (but not HIAP2) with the kinase RIP2 in a has been demonstrated (Yamaguchi et al., in press). A MCF7 breast carcinoma cell line raises the possibility more detailed list of all IAP interactions is given in that the inhibition of this pro-apoptotic kinase directly Table 4. contributes to the suppression of apoptosis (McCarthy et al., 1998). In contrast to previous work showing the The IAPs protect against a broad spectrum of apoptotic cytoprotective e€ect of NF- B, RIP2 is both pro- k triggers apoptotic and an activator of NF-kB underlining the importance of cellular context in IAP function. More IAPs have shown a remarkable ability to block recently, the single BIR containing survivin has been apoptosis induced by a wide spectrum of non-related shown to be expressed in cell cycle dependent manner, apoptotic triggers. The signi®cant majority of apopto- apparently interacting with spindle microtubules. tic triggers are blocked by at least one IAP (Table 5) Disruption both of this binding, which is mediated with very few exceptions. They block, for example, a by the survivin carboxyl terminal coiled coil domain, broader spectrum of triggers than does Bcl-2 possibly and mutation of the BIR domain abrogates the re¯ecting a site of activity further downstream than apoptotic inhibition. This suggests that the inhibition that of the Bcl-2 family (see Table 5 for references). of caspases in situ on the spindle apparatus is a IAPs protect against apoptosis induced by TNFa, Fas IAPs and cancer EC LaCasse et al 3255 Table 6 Clinico-pathological data for survivin expression Cancer type Number of positive cases per stage Correlative data and comments Reference Lung (n=15), colon (n=6), All cases of adenocarcinoma and . No expression seen in Ambrosini et al., 1997 breast (n=7), prostate carcinoma examined low-grade lymphoma (n=7) (n=5), pancreas (n=2) (small number of cases studied), 50% and high grade of high-grade lymphomas lymphoma (n=38) Neuroblastoma (n=72) 47% (34/72) all stages . Correlation with worsening Adida et al., 1998b 36% (14/39) stages I and II prognosis, unfavourable 60% (15/25) stages III and IV histology or disease staging, 74% (17/23) on unfavourable histology . 35% (17/49) favourable histology . Survivin positive, stage I and II were also BCL-2 positive, 82% (14/17) Gastric carcinoma (n=174) 34% (60/174) all stages . Correlation with nuclear p53, Lu et al., 1998 42% (20/48) Stage I 56% (46/82) and BCL-2 35% (18/52) Stage II expression, 69% (9/13) 30% (22/74) Stage III . Correlation with decreased apoptosis, apoptotic index for survivin positive tumors was 0.62%, and 0.97% for negative tumors . No correlation with worsening stage or unfavourable histology Colorectal carcinoma 53% (91/171) all stages . No correlation with nuclear p53 Kawasaki et al., in press (n=171) 53% (52/98) Stages O ± II . Correlation with BCL-2 expression, 53% (39/73) Stages IIIa ± IV 72% (71/98) of BCL-2+ cases were survivin+ while 27% (20/73) of BCL-27 cases were survivin positive . Does not correlate with staging . Correlation with low apoptotic index and shorter survival for survivin7 tumors . Apoptotic index for survivin+ tumors was 0.76% and 1.7% for survivin7 tumors . Survivin+ patients have 10% lower 5- year survival rates, 72% (66/91) vs 82% (66/80), while low apoptotic index pa- tients have a 20% lower 5-year survival rate than those with a high apoptotic index, 69% (72/104) vs 89% (60/67) ligand and transducers of the TNF receptor super- is stronger than that observed by Fas activation alone. family (RIP, RIP2/RICK/CARDIAK, FADD, v-Rel), Increased IAP expression also correlates with in- pro-apoptotic members of the Bcl-2 family (BIK, creased tumour survival (see Part B) and inhibition BAK, BAX) and cytochrome C, chemotherapeutic of follicular atresia in maturing ovarian follicles (Li et drugs (etoposide, cisplatin, taxol, actinomycin D, al., 1998; Johnson et al., 1998). HIAP1 is also induced Adriamycin), ionizing or UV radiation, oxidative by dexamethasone and interferon gamma in a lung stress (menadione, 6-OH dopamine), ischemia, potas- carcinoma cell line under circumstances of dexametha- sium withdrawal, viral infections (baculovirus, Sindbis) sone induced protection against IFNg and Fas or viral proteins (E1A, IE1), growth factor withdrawal mediated apoptosis (Wen et al., 1997). (serum, IL-3), caspases (1, 2, 3, 7, 8 and SF caspase), Drosophila death proteins (Reaper, GRIM, HID, DOOM) and several other triggers that do not fall Part B. Emerging role of the IAPs in cancer into one of the above categories. The only exceptions to this list are glutamate excitotoxicity (which induces Increased apoptosis resistance promotes tumour a necrotic form of cell death), staurosporine which development and resistance to drug and radiation therapy may kill by a secondary mechanism not involving caspase-3 or DNA cleavage (Krohn et al., 1998), and Apoptosis is the chief means by which radio- and vincristine or nocodazole (Li et al., in press). chemotherapy modalities kill cancer cells. The dis- However, only survivin has been tested in the latter covery that the bcl-2 oncogene, characteristic of case; it remains to be seen if other IAPs can protect follicular lymphomas bearing the t(14;18) transloca- against microtubule disruption caused by drugs such tion, is an inhibitor of apoptosis has revolutionized our as vincristine and nocodazole. It is also noteworthy thinking of tumour initiation and progression, identify- that FADD induced cell death was only partially ing apoptotic inhibition as a mechanism of cancer protected by IAPs even though IAPs can protect well formation, progression and resistance to therapy. The against TNFa or Fas ligand induced cell death, bcl-2 family of proteins has grown dramatically to implying that the signal from overexpressed FADD include not only anti-apoptotic members, but also pro- IAPs and cancer EC LaCasse et al 3256 apoptotic members such as Bax. Bax is a candidate induced by IL-3 withdrawal in IL-3 dependent pre-B tumour suppressor and is regulated by p53 (Selvaku- cells (Ambrosini et al., 1997), against taxol induced maran et al., 1994; Yin et al., 1997); loss of p53 activity apoptosis in NIH3T3 cells (Li et al., in press) and against results in loss of Bax activity. Bax is also mutated in Fas, Bax and etoposide in 293 cells (Tamm et al., in colon cancer possibly playing a role in tumour press). Moreover, the use of a survivin antisense results progression (Rampino et al., 1997). A theme is now in increased apoptosis and inhibits cell proliferation emerging that inhibition of apoptosis is a common (Ambrosini et al., 1998; Li et al., in press). Survivin has property for cancer cells, increasing their survival and recently been demonstrated to also inhibit caspase-3 facilitating their escape from immune surveillance and activity directly (Tamm et al., in press; Maier, Nicholson, cytotoxic therapies. Another example of this is the Roy, and MacKenzie, unpublished). Immunohistochem- increased expression of FLIP in melanoma leading to ical analysis of biopsy material has revealed that 60% of an increased resistance to Fas mediated cell killing stage 3 ± 4 neuroblastomas are survivin positive (Adida (Irmler et al., 1997). Similarly, v-Rel prevents apoptosis et al., 1998b), while 53% of colon cancers (all stages) in transformed lymphoid cells (Zong et al., 1997). The (Kawasaki et al., in press) and 35% of gastric cancers (all transforming activity of v-Rel is dependent on its gene stages) are positive (Lu et al., 1998) (see Table 6). regulatory activities and is thought to be mediated, in Survivin correlates with worsening stage or unfavourable part, by its anti-apoptotic e€ects. histology for neuroblastoma (Adida et al., 1998b), and Many and probably most apoptotic processes are with decreased apoptotic index in gastric cancer (Lu et ultimately channeled through the e€ector proteases, al., 1998) or colon cancer (Kawasaki et al., in press). caspases, which are another potential source of cancer More importantly, survivin expression is associated with resistance. This has been demonstrated experimentally shortened 5-year disease survival rates (a 10% decrease by introducing caspase inhibiting viral proteins, such as compared to survivin negative patients, although not p35 or CrmA into cancer cells and observing increased statistically signi®cant). Survivin expression also corre- resistance to cell killing by chemotherapeutics (Antoku lates with decreased apoptotic indices and bcl-2 et al., 1997; Qi et al., 1997; Barge et al., 1997; Los et expression in colon cancer, moreover patient 5-year al., 1997; Srikanth and Kraft, 1998). Data correlating disease survival rates are 20% lower in the patients with a lack of caspase activity with increased cellular low apoptotic index (statistically signi®cant). Although apoptotic resistance is also emerging (Eichholtz-Wirth the apoptotic index is most likely the result of several et al., 1997; Martinez-Lorenzo et al., 1998; Fulda et al., genes in addition to bcl-2 and survivin, it is nonetheless 1998). Recently, the baculoviral caspase inhibitor p35 noteworthy that survivin expression correlates with the has been shown to have transforming activity in apoptotic index in two di€erent cancers and that this has cooperation with IGF-1 signalling (Resnico€ et al., been shown to be clinically relevant for colon cancer. 1998). These ®ndings set the stage for a potential Recently, a possible link between cell cycle progres- oncogenic role for other inhibitors of apoptosis. The sion and apoptosis has emerged. Survivin has been IAPs are prime candidates in this regard because of shown to associate with mitotic spindles through a their caspase inhibitory activity and TNF and Fas coiled-coil interaction involving the C-terminal end of receptor modulatory activities. To date, the two survivin (Li et al., in press). Interestingly, survivin strongest pieces of evidence for IAP involvement in shows cell cycle speci®c expression in G2/M which is cancer are (1) the cancer speci®c expression of an IAP mediated by G1 transcriptional repressor elements in called survivin which is not normally expressed in the survivin promoter. This suggests speci®c dual di€erentiated tissue, and (2) the emerging role of the control of the mitotic spindle checkpoint and IAPs in the anti-apoptotic e€ect mediated through the apoptosis by survivin (Li et al., in press). Survivin NF-kB transcription factor. protects against taxol (a microtubule stabilizer) induced cell death but fails to protect against microtubule disruption (e.g. vincristine and nocodazole) induced The fetal IAP, survivin, is expressed in most cancers and cell death. It will be important to see if other IAPs correlates with shorter disease survival associate with microtubules and if they can protect Survivin is the smallest IAP cloned to date, consisting against vincristine or nocodazole induced cell death. only of 142 amino acids, with only a single N-terminal BIR domain (Ambrosini et al., 1997). Survivin shows IAPs as mediators and regulators of the anti apoptotic markedly di€erent tissue expression when compared activity of v-Rel and NF- B family members with the other IAPs. Data from our laboratory shows k that XIAP, HIAP1 and HIAP2 are comparatively The role the Rel/NF-kB/IkB family members in cancer ubiquitously expressed, while NAIP is more restricted is becoming increasingly evident (reviewed in Gilmore (e.g. brain, liver, macrophage). In contrast, survivin is et al., 1996; Luque and Gelinas, 1997). Besides the well expressed fetally and not in adult di€erentiated tissue known transforming properties of v-Rel in birds, or (Ambrosini et al., 1997; Adida et al., 1998a). bcl-3 (mutant IkB) in human chronic lymphocytic Remarkably, survivin is expressed in most cancers leukemias/lymphomas, ampli®cations and rearrange- tested (lung, colon, breast, prostate, pancreas, high- ments involving Rel/NF-kB members occur in many grade lymphomas, neuroblastomas, gastric) with the solid and haematological malignancies (Gilmore et al., exception of low-grade lymphomas (Ambrosini et al., 1996; Luque and Gelinas, 1997). It is also becoming 1997; Adida et al., 1998b; Lu et al., 1998; Kawasaki et more evident that NF-kB exerts anti-apoptotic e€ects, al., in press) (Table 6). inhibiting TNF induced cell death (Beg and Baltimore, Although the argument for a role of survivin in cancer 1996; Van Antwerp et al., 1996, 1998) and protecting progression or drug resistance is correlative so far, the cells from ionizing radiation and chemotherapeutic protein has been shown to protect against apoptosis drugs. NF-kB activation is required to suppress IAPs and cancer EC LaCasse et al 3257 apoptosis induced by oncogenic ras (Mayo et al., 1997) expression reinforcing the anti-apoptotic e€ects of and to aid in Bcr-Abl-mediated transformation HIAP1. It is interesting to note the zinc ®nger (Reuther et al., 1998). Constitutive activation of NF- apoptosis inhibitor A20 which is induced by NF-kB kB is seen during progression of breast cancer is, in contrast to the HIAPs, a negative regulator of (Nakshatri et al., 1997; Sovak et al., 1997) and is NF-kB activation (Song et al., 1996), as is MnSOD required for proliferation and survival of Hodgkin's (Manna et al., 1998). Also relevant to this discussion is disease tumour cells (Bargou et al., 1997). Interestingly, the possible induction of IAPs through NF-kB inhibition of NF-kB activity attenuates apoptosis following EBV infection. It is postulated that the host resistance in lymphoid cells (Jeremias et al., 1998) cell surface late antigen LMP1 acts as a constitutively and potentiates TNF and cancer therapy-induced active receptor molecule triggering NF-kB activation, apoptosis in a ®brosarcoma cell line (Wang et al., responsible in part for B-cell transformation (Kaye et 1996). Therefore, the identi®cation of anti-apoptotic al., 1996; Izumi et al., 1997; Gires et al., 1997). This genes or survival factor genes induced by NF-kBisof may explain the high level of HIAP1 expression seen in paramount interest. Recently, HIAP1 and PIAP or ch- the Reed-Sternberg cells found in Hodgkin's disease; IAP1 have been shown to be induced by NF-kBorv- LMP1 is present in Reed-Sternberg cells in 70% of Rel, respectively, in multiple cell lines (Chu et al., 1997; such malignancies (Messineo et al., 1998). LMP1 also Wang et al., 1998; Stehlik et al., 1998a,b; You et al., induces A20 (Fries et al., 1996) and bcl-2 (Henderson 1997). There is also evidence for HIAP2 induction by et al., 1991). NF-kB is some systems (Wang et al., 1998; Stehlik et It is therefore interesting to speculate that the al., 1998a) and for XIAP only in endothelial cells induction of IAPs by NF-kB activation is responsible (Stehlik et al., 1998a). CD40L is also able to induce in part or whole for the anti-apoptotic e€ects of NF- HIAP1, presumably through the CD40 receptor, kB which lead to tumour cell survival, B-cell another member of the TNF receptor superfamily, transformation, radiation and drug resistance. Inhibi- which is well known to provide survival signals in B- tion of HIAP function, such as through a knock-out cells (Craxton et al., 1998). Inhibition of IAP induction mouse model, will demonstrate the protective e€ects of by IkB dominant negatives lead to cell death and over IAPs in immune function and cancer and distinguish expression of these IAPs leads to cell survival in these the IAPs e€ects from other NF-kB inducible genes. systems. This was elegantly tested in the chicken spleen cells using temperature sensitive mutants of v-Rel to Future trends control the induction of exogenous ch-IAP1 (You et al., 1997). IAPs may not be the only anti-apoptotic Much work still needs to be done to establish the role genes induced by NF-kB. It has recently been of IAPs in cancer. However, new and exciting data are demonstrated that at least three other anti-apoptosis coming forth supporting just such a role for the IAPs; genes are induced by NF-kBorNF-kB-activating we believe it is likely that IAPs other than survivin and agents: they are A20 (Song et al., 1996), MnSOD HIAP1 will likely be demonstrated to have a role in (Wong and Goeddel, 1988; Wong et al., 1989; Jones et cancer. The IAPs may not be the initiating event in al., 1997; Mattson et al., 1997; Keller et al., 1998) and cancer as no obvious chromosomal translocations are an alternate splice product of an immediate early gene, associated with their genomic loci (Table 3) but they IEX-1L (Wu et al., 1998). It is also important to note are likely to play a role in disease progression and that NF-kB can induce survival factors in lymphoid resistance to therapy, and therefore represent new cells such as IL-6, and IL-2-receptors. Some or all of therapeutic targets for cancer treatment. these gene products likely determine the cells fate depending on the cell type, the gene program that is activated and the various environmental stimuli. The IAPs may be the predominant anti-apoptotic factor Acknowledgements induced by NF-kB but this remains to be clearly proven. Of special interest here is the fact that HIAP1 This article is dedicated to the original discoverer of the and HIAP2 are able to activate NF- B(Chuet al., IAPs; Lois Miller. Our thanks go to Drs D Altieri, J Reed, k J Schulz, M Weller, E Brown, A Ho€man, D Spengler, B 1997; Wang et al., 1998) and XIAP may also activate Tsang, M Hatano and K Matsumoto as well as members of NF-kB under some circumstances (Tanner, personal the MacKenzie, Korneluk and Apoptogen labs for sharing communication) and thus may lead to a positive information prior to publication. AEM and RGK are feedback loop whereby the HIAPs through their MRC of Canada Scientists; RGK is a Howard Hughes interaction with TRAF-1, TRAF-2 or RIP2/RICK/ Medical Institute International Research Scholar and AEM CARDIAK can potentiate the activation of NF-kB holds a Burroughs Wellcome Fund Clinical Translation which in turn leads to increased HIAP1 gene Award.

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