Survivin, Versatile Modulation of Cell Division and Apoptosis in Cancer

Survivin, versatile modulation of cell division and apoptosis in cancer

Dario C Altieri*,1

1Department of Cancer Biology and the Cancer Center, LRB-428, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA

Survivin is a member of the inhibitor of apoptosis (IAP) spliced survivin transcripts (Figure 1). In addition to gene family that has attracted attention from several wild-type survivin (142 amino acids), two survivin viewpoints of basic and translational research. Its cell isoforms are generated by insertion of an alternative cycle-regulated expression at mitosis and association with exon 2 (survivin-2B, 165 amino acids) or removal of the mitotic apparatus have been of interest to cell exon 3 (survivin-DEx-3, 137 amino acids) (Mahotka biologists studying faithful segregation of sister chroma- et al., 1999). In survivin-DEx-3, the splicing event tids and timely separation of daughter cells. Investigators introduces a frame shift that generates a unique – interested in mechanisms of apoptosis have found survivin COOH terminus of potential functional significance an evolving challenge:while survivin inhibits apoptosis in (Mahotka et al., 2002) (Figure 1). vitro and in vivo, this pathway may be more selective as A unique property of survivin is a sharp cell-cycle- compared to cytoprotection mediated by other IAPs. dependent expression at mitosis. This is largely, but not Finally, basic and translational researchers in cancer exclusively, controlled at the level of gene transcription biology have converged on survivin as a pivotal cancer and involves canonical CDE/CHR boxes (Badie et al., gene, not simply for its sharp expression in tumors and not 2000) in the survivin promoter (Kobayashi et al., 1999; in normal tissues, but also for the potential exploitation of Li and Altieri, 1999b), acting as potential G1-repressor this pathway in cancer diagnosis and therapy. The elements (Figure 2). Among the post-translational objective of the present contribution is to line up current modifications that affect survivin levels, interest has evidence and emerging concepts on the multifaceted recently focused on the control of protein stability. 1 functions of survivin in cell death and cell division, and Survivin is a relatively short-lived protein ðt2 ¼ 30 minÞ, how this pathway is being pursued for novel cancer and polyubiquitylation followed by proteasomal de- therapeutic strategies. struction has been shown to contribute to cell cycle Oncogene (2003) 22, 8581–8589. doi:10.1038/sj.onc.1207113 periodicity by keeping survivin levels low at interphase (Zhao et al., 2000). In addition, mitotic phosphorylation Keywords: survivin; apoptosis; cell cycle; cancer; check- of survivin on Thr34 by p34cdc2-cyclin B1 has been point associated with increased survivin stability at metaphase (O’Connor et al., 2002). There are also examples of modulation of survivin expression independently of cell cycle progression. For Survivin structure–function CD34 þ bone-marrow-derived stem cells, stimulation with hematopoietic cytokines resulted in survivin At 16.5 kDa, survivin is the smallest mammalian expression in the absence of cyclin D and with member of the inhibitor of apoptosis (IAP) gene family hypophosphorylated Rb (Fukuda et al., 2002), and (Salvesen and Duckett, 2002). Structurally, it contains a vascular remodeling by angiopoietin-1 (Ang-1) upregu- single Baculovirus IAP Repeat (BIR), an B70-amino- lated survivin in nonproliferating endothelial cells acid zinc-finger fold that is the hallmark of all IAPs and (Papapetropoulos et al., 2000; Harfouche et al., 2002). an extended –COOH terminus a-helical coiled-coil, but The search for signaling intermediates that control no other identifiable domain (Ambrosini et al., 1997) survivin expression has generated interesting findings typically found in IAPs. Based on X-ray crystallography (Figure 2). One common denominator of this pathway is of the human (Chantalat et al., 2000; Verdecia et al., the requirement for PI3-kinase/Akt activation, a general 2000) or mouse (Muchmore et al., 2000) protein, antiapoptotic signal (Datta et al., 1999) that has been survivin is a stable homodimer in solution, with the – implicated in the upregulation of survivin induced by COOH terminus a-helices protruding from the core GM-CSF, CSF and Ang-1 (Papapetropoulos et al., dimer. A single-copy survivin gene located on chromo- 2000; Carter et al., 2001; Fukuda et al., 2002). In some 17q25 (human; Li et al., 1998) or 11E2 (mouse; Li addition, for endothelial cells stimulated with nonmito- and Altieri, 1999a) gives rise to three alternatively genic IL-11 (Mahboubi et al., 2001), or pleural effusion lymphoma cells sustained by autocrine VEGF, IL-6 or IL-10 (Aoki et al., 2003), increased survivin expression *Correspondence: DC Altieri; E-mail: [email protected] This work was supported by NIH Grants HL54131, CA78810 and was shown to be dependent on STAT3 activation. This CA90917 is potentially important for the known role of STAT3 in Survivin in cell division and apoptosis DC Altieri 8582 Role of survivin in cell division

Upon expression at mitosis, survivin localizes to various components of the mitotic apparatus, including centrosomes, microtubules of the metaphase and anaphase spindle, and the remnants of the mitotic apparatus – midbodies (Li et al., 1998, 1999; Skoufias et al., 2000; Jiang et al., 2001; Fortugno et al., 2002). A direct association between survivin and polymerized tubulin has been demonstrated in vitro (Li et al., 1998), potentially involving the –COOH terminus a-helices (Figure 2). A subcellular pool of survivin has also been localized to kinetochores of metaphase chromosomes (Skoufias et al., 2000; Wheatley et al., 2001), and may B Figure 1 Structure–function of survivin proteins generated by comprise up to 20% of total cellular survivin alternative splicing. Organization of survivin and its alternatively (Fortugno et al., 2002). On the other hand, survivin is spliced variants involving insertion of an alternative exon (survivin largely excluded from the nucleus (Fortugno et al., 2002) 2B) or skipping of exon 3 (survivin-DEx3). Discrete regions through a CRM1-dependent mechanism that may implicated in dimerization, microtubule binding, nuclear localiza- involve the survivin –COOH terminus (Rodriguez tion/export and subcellular targeting to mitochondria are indi- cated. Residues involved in the Zn2 þ coordination sphere (Cys84) et al., 2002). and increased protein stability following phosphorylation by The complex localization of survivin to the mitotic p34cdc2 (Thr34) are shown apparatus reflects its important function at cell division (Table 1). This first surfaced in targeting experiments using antisense or dominant-negative mutants, which resulted in a dual phenotype of apoptosis (see below) and aberrant mitotic progression, with supernumerary centrosomes, multipolar mitotic spindles, failed cytokinesis and multinucleation (Li et al., 1999). Parallel experiments targeting survivin-like molecules in yeast (Uren et al., 1999) or C. elegans (Fraser et al., 1999) also revealed lethal cell division defects with failure to assemble a cleavage furrow and inability to complete cytokinesis. The phenotype of survivin knockout mice is consistent with an essential role of this pathway at mitosis (Uren et al., 2000). Beginning at embryonic day (E) 2.5, homozygous deletion of the survivin gene resulted in catastrophic defects of microtubule assembly, Figure 2 Differential regulation of survivin expression in cell with absence of mitotic spindles, complete failure of cell cycle-dependent and -independent pathways. Transcriptional and division and multinucleation with 100% lethality by E post-transcriptional mechanisms controlling survivin gene expres- 3.5–4.5 (Uren et al., 2000). An identical phenotype was sion involve CDE/CHR G1 repressor elements in the survivin independently confirmed on three different mouse promoter. Non-cell-cycle-dependent mechanisms influencing survivin expression involve response to hematopoietic and vascular genetic backgrounds (Conway et al., 2002). Therefore, remodeling cytokines, STAT3-dependent signaling and PI3 kinase one model that has been put forward is that survivin activity functions in an essential and evolutionary conserved step in late-stage cell division, that is, cytokinesis, potentially involving cleavage furrow formation (Uren cytoprotection (Shen et al., 2001), which may have et al., 2000; Adams et al., 2001). In support of this, direct consequences for oncogenic transformation survivin was shown to form a complex with molecules (Turkson and Jove, 2000) (Figure 2). thought to regulate cytokinesis, including Aurora B

Table 1 Survivin functions Mitotic regulator Cell death inhibitor

Localization to the mitotic apparatus Inhibition of extrinsic/intrinsic apoptotic pathways Catastrophic defects of mitosis in survivin À/À embryos Resistance to apoptosis in survivin transgenic mice Association with Aurora B Increased sensitivity to apoptosis of survivin +/À mice Failed cytokinesis and polyploidy induced by antisense Spontaneous apoptosis induced by antisense, DN mutants, or DN mutants siRNA or ribozyme Depletion of microtubules and defects in Association with caspases spindle assembly and spindle checkpoint after antibody microinjection Association with Smac/DIABLO

Oncogene Survivin in cell division and apoptosis DC Altieri 8583 kinase and INCENP on kinetochores and the anaphase by microtubule survivin in the control of spindle central spindle (Speliotes et al., 2000; Wheatley et al., formation upon entry into metaphase (Giodini et al., 2001), and to enhance the activity of Aurora B kinase in 2002; Tran et al., 2002). A continuum regulation of the both mammalian cells (Chen et al., 2003) and Xenopus mitotic spindle checkpoint by survivin is also in line with laevii (Bolton et al., 2002). However, recent data antibody microinjection data, where inhibition of demonstrate that this model is probably unsatisfactory. survivin abrogated cell cycle arrest in response to A broader role of survivin in cell division became spindle poisons (Kallio et al., 2001). This model is also apparent in antibody microinjection studies, which satisfactory if one considers the biology of Aurora revealed a composite phenotype of prolonged meta- kinases, which, like survivin, are found frequently phase arrest, occasionally followed by apoptosis, for- overexpressed in human cancers, and may participate mation of multipolar mitotic spindles, and defects in in oncogenic transformation (Ota et al., 2002) (Table 1). chromosome attachments (Kallio et al., 2001; Giodini Altogether, the combined overexpression of both et al., 2002). By immunoﬂuorescence, cells microinjected survivin and Aurora in cancer may obliterate the with an antibody to survivin exhibited ﬂattened and surveillance mechanism of the spindle assembly check- abortive mitotic spindles severely depleted of micro- point, allowing cells with spindle defects, aberrant tubules (Giodini et al., 2002). Conversely, retroviral or chromosome congression or misaligned/unattached ki- adenoviral expression of survivin restored spindle netochores to proceed through cell division, thus stability and dynamics against microtubule poisons in enhancing tumor-associated aneuploidy. tumor cells (Giodini et al., 2002) as well as endothelial cells (Tran et al., 2002). Clearly, these data point to a much different model in which survivin participates in Role of survivin in apoptosis inhibition earlier phases of mitosis than cytokinesis, and is required for the assembly of a bipolar mitotic apparatus The question as to whether survivin had also a role in by controlling microtubule stability. apoptosis inhibition, similar to other mammalian IAPs Is there a framework that reconciles these two sets of (Salvesen and Duckett, 2002), or whether its function observations? One possibility is that the two pools of was limited to cell division, as with IAPs in yeast and C. survivin localized to centrosomes/microtubules and elegans (Fraser et al., 1999; Uren et al., 1999), has kinetochores (Fortugno et al., 2002) actually mediate frequently been asked. This was dubbed as a ‘con- distinct functions at various phases of cell division: troversy’ that at times reached emotional tones (Adams regulation of microtubule stability and spindle forma- et al., 2001; Silke and Vaux, 2001). However, the tion telephase congression and control of cleavage published record on this topic has been uniformly furrow formation of completion telophase. This is consistent, and not a single peer-reviewed article has consistent with the observation that microtubule- and appeared that experimentally refutes a role of survivin in kinetochore-associated survivin are differentially regu- apoptosis inhibition. Three lines of experimental evi- lated during the cell cycle, exhibit nonoverlapping dence underscore this conclusion (Table 1). patterns of phosphorylation, and are recognized by First, overexpression of survivin has been associated monoclonal antibodies in a mutually exclusive fashion with inhibition of cell death initiated via the extrinsic or (Fortugno et al., 2002). Alternatively, the differently intrinsic apoptotic pathways (Ambrosini et al., 1997; spliced survivin isoforms could mediate separate func- Tamm et al., 1998; Kobayashi et al., 1999; Mahotka tions at cell division (Mahotka et al., 1999). This et al., 1999; Islam et al., 2000; Kasof and Gomes, 2000; remains a possibility that awaits formal testing when Suzuki et al., 2000; Hoffman et al., 2001; Mirza et al., antibodies capable of discriminating between the var- 2002; Zaffaroni et al., 2002). Second, transgenic expres- ious endogenous survivin isoforms become available. sion of survivin resulted in apoptosis inhibition in vivo However, wild-type survivin is clearly the overwhelming (Grossman et al., 2001a), and livers isolated from form in all cell types tested so far (Mahotka et al., 1999), heterozygous survivin þ /À animals exhibited exaggerated and, at least in overexpression experiments, survivin-2B apoptosis in response to suboptimal ligation of Fas does not associate with the mitotic apparatus, and (Conway et al., 2002). Third, molecular antagonists of survivin-DEx-3 has actually been localized to the nucleus survivin including antisense, ribozymes, siRNA se- potentially through a bipartite nuclear localization quences or dominant-negative mutants resulted in signal in the new ÀCOOH terminus (Mahotka et al., caspase-dependent cell death, enhancement of apoptotic 2002; Rodriguez et al., 2002). stimuli and anticancer activity, in vivo (Li et al., 1999; Perhaps a third and more inclusive possibility is that Chen et al., 2000; Kasof and Gomes, 2000; Olie the various survivin pools provide a continuum in the et al., 2000; Jiang et al., 2001; Kanwar et al., 2001; dynamic regulation of the mitotic spindle checkpoint, Shankar et al., 2001; Xia et al., 2002; Yamamoto et al., from kinetochore dynamics through spindle assembly. 2002; Zhou et al., 2002; Choi et al., 2003; Pennati et al., This is consistent with recent data identifying a role for 2003; Williams et al., 2003). The apoptosis inhibitory Aurora B in chromosome congression and microtubule function of survivin is evolutionary conserved, and a arrangement at earlier stages of mitosis than cytokinesis survivin-like molecule in Drosophila, Deterin, functions (Shannon and Salmon, 2002). In this context, a role of interchangeably with survivin to block apoptosis (Jones the Aurora/survivin complex in kinetochore dynamics et al., 2000) in mammalian or insect cells (Jiang et al., (Bolton et al., 2002; Chen et al., 2003) may be integrated 2001).

Oncogene Survivin in cell division and apoptosis DC Altieri 8584 Although the notion that survivin inhibits apoptosis is a survivin-DEx3-like molecule in the herpes simplex well established (Table 1), elucidation of the mechan- virus (HSV) genome localized to mitochondria and ism(s) by which this occurs has not been straightfor- inhibited apoptosis by associating with Bcl-2 and by ward. As with other IAPs (Salvesen and Duckett, 2002), suppressing caspase-3 activity through its BIR (Wang a physical interaction between survivin and initiator or et al., 2002). Whether survivin-DEx3 behaves similar to effector caspases has been reported by several groups its viral counterpart remains to be seen, but its potential (Tamm et al., 1998; Kobayashi et al., 1999; Kasof and import to mitochondria would make it ideally suited to Gomes, 2000), and a physical proximity between act, alone or by dimerizing with wild-type survivin, as survivin and effector caspase-3 has been seen in vivo as an upstream regulator of mitochondrial-dependent well (Li et al., 1999). However, with the exception of two apoptosis. published studies (Conway et al., 2000; Shin et al., 2001), this did not seem to translate in meaningful inhibition of caspase activity, in vitro (Banks et al., 2000; Role of survivin in cancer biology Kasof and Gomes, 2000; Verdecia et al., 2000). In addition, the crystal structure of survivin does not A sharp differential expression in cancer versus normal suggest the presence of a ‘hook and sinker’ region that tissues is one of the most intriguing features of survivin, mediates caspase binding in other IAPs (Verdecia et al., and is unlike any other IAPs (Salvesen and Duckett, 2000), and the residues in the linker upstream BIR2 that 2002). Survivin is strongly and broadly expressed in in other IAPs (i.e. XIAP) dock to caspase-3 (Shi, 2002) embryonic and fetal organs (Adida et al., 1998b; are not present in survivin. One can take the view that Kobayashi et al., 1999), but becomes undetectable in this does not automatically rule out that survivin may most terminally differentiated normal tissues (Ambrosi- still function as an inhibitor of effector caspases similar ni et al., 1997), perhaps with the exception of to other IAPs (Salvesen and Duckett, 2002), and thymocytes (Ambrosini et al., 1997), CD34 þ stem cells arguments to explain the lack of activity in cell-free (Carter et al., 2001; Fukuda and Pelus, 2001) and basal systems may include folding requirements of the colonic epithelial cells (Gianani et al., 2001; Zhang et al., recombinant protein (Shin et al., 2001), or the need for 2001). The strict developmental pattern of survivin additional protein partners, in vivo. On the other hand, expression is evolutionary conserved, and similar data evidence is accumulating that survivin may play a more were observed for survivin-like molecules in Drosophila selective role than other IAPs in antagonizing mito- (Jones et al., 2000) and X. laevii (Murphy et al., 2002). chondrial-dependent apoptosis. Overexpression of sur- By contrast, dramatic overexpression of survivin has vivin is more efficient at blocking mitochondrial- but not been demonstrated in tumors of lung (Monzo et al., death-receptor-induced apoptosis (Grossman et al., 1999), breast (Tanaka et al., 2000), colon (Kawasaki 2001a), a complex between survivin and the upstream et al., 1998), stomach (Lu et al., 1998), esophagus (Kato mitochondrial initiator caspase-9 has been demon- et al., 2001; Grabowski et al., 2003), pancreas (Satoh strated in vivo (O’Connor et al., 2000a), and survivin et al., 2001), liver (Ikeguchi et al., 2002), uterus (Saitoh has been shown to associate with Smac/DIABLO (Du et al., 1999), ovaries (Yoshida et al., 2001), Hodgkin’s et al., 2000), a mitochondrial-released apoptogenic disease (Garcia et al., 2003), non-Hodgkin’s lymphoma protein that relieves the inhibitory effect of IAPs on (Adida et al., 2000a; Kuttler et al., 2002), leukemias caspase activation (Shi, 2002). Moreover, cell death (Adida et al., 2000b; Kamihira et al., 2001), myelodys- induced by molecular antagonists of survivin or by plastic syndrome with refractory anemia (Badran et al., heterozygous reduction of survivin levels (Conway et al., 2003), neuroblastoma (Adida et al., 1998a; Islam et al., 2002) has the characteristics of mitochondrial-depen- 2000), pheochromocytoma (Koch et al., 2002), soft dent apoptosis with cytochrome c release (Mesri et al., tissue sarcomas (Wurl et al., 2002), brain tumors 2001b), caspase-9 activation (O’Connor et al., 2000a), (Chakravarti et al., 2002; Kajiwara et al., 2003) and and involvement of the apoptosome components melanoma (Grossman et al., 1999; Gradilone et al., (O’Connor et al., 2002), caspase-9 and Apaf-1 (Wang, 2003). Genome-wide searches independently confirmed 2001). Collectively, these data suggest that survivin, the differential expression of survivin in tumors versus differently from other IAPs that inhibit initiator or normal tissues (Velculescu et al., 1999). The two effector caspases through their independent BIRs (Shi, alternatively spliced forms of survivin are also differen- 2002), may selectively target the multimolecular process tially expressed in tumors (Hirohashi et al., 2002). of caspase-9 activation. This pathway may well be Several retrospective studies used protein and/or centered on the survivin–Smac/DIABLO interaction nucleic acid detection strategies (immunohistochemistry, (Du et al., 2000), which was recently characterized in RT–PCR, in situ hybridization, DNA array profiling, vitro and in vivo, and proposed as a critical modulator of etc.) to map the presence of survivin in various tumor caspase-9 processing during taxol-induced apoptosis series, and to determine its impact on disease parameters (Song et al., 2003). Additional data also implicate and clinical outcome. The results proved that survivin is survivin, or at least its splice variant, survivin-DEx3, in a reliable marker of aggressive and unfavorable disease, mitochondrial-dependent apoptosis. It was recently signaling abbreviated overall survival (Kawasaki et al., reported that survivin-DEx3 contains a mitochondrial 1998; Monzo et al., 1999; Adida et al., 2000a; Sarela localization signal and a BH2 domain in the new – et al., 2000; Tanaka et al., 2000; Kappler et al., 2001; COOH terminus (Wang et al., 2002) (Figure 1), and that Kato et al., 2001; Chakravarti et al., 2002; Ikeguchi and

Oncogene Survivin in cell division and apoptosis DC Altieri 8585 Kaibara, 2002; Wurl et al., 2002), increased rates of Table 2 Deregulated survivin gene expression in cancer recurrences (Swana et al., 1999), resistance to therapy Survivin promoter activity in cancer cell lines but not in normal cells (Tran et al., 2002; Zaffaroni et al., 2002; Hausladen Cancer-specific gene expression by the survivin promoter, in vivo et al., 2003) and reduced apoptotic index, in vivo Amplification of the survivin locus on 17q25 (neuroblastoma) (Kawasaki et al., 1998; Tanaka et al., 2000; Satoh Demethylation of survivin exon 1 (ovarian cancer) et al., 2001). Array-based gene profiling studies are Transcriptional repression by wild-type p53 Transcriptional activation by Wnt/TCF (colorectal cancer) consistent with this view, identifying survivin as a ‘risk- Correlates with abbreviated survival, associated’ gene for unfavorable outcome in breast accelerated rates of recurrences and resistance to therapy cancer (van ‘t Veer et al., 2002), large cell non- Hodgkin’s lymphoma (Kuttler et al., 2002) and colorectal cancer (van de Wetering et al., 2002; Williams et al., but prominently expressed in tumor cell lines (Li and 2003). Altieri, 1999a), and cells transfected with a reporter gene How does survivin bring about so many unfavorable under the control of a survivin promoter exhibited consequences in tumors? Recent experiments of stepwise cancer-specific activity, in vitro and in vivo (Bao et al., skin carcinogenesis in survivin transgenic mice began to 2002). Secondly, molecular lesions associated with address this question (Allen et al., 2003). Skin tumor cancer have been associated with deregulated survivin (papilloma) formation was actually found less fre- expression. These include amplification of the survivin quently in survivin transgenic mice than in control locus on 17q25 in neuroblastoma (Islam et al., 2000), littermates, whether or not on a wild-type or hetero- demethylation of survivin exon 1 in ovarian cancer but zygous p53 background (Allen et al., 2003). However, not in normal ovaries (Hattori et al., 2001), and, papillomas never regressed in survivin transgenic mice importantly, loss of p53. Three groups have indepen- and occasionally evolved into squamous carcinoma, dently reported that survivin is one of the genes that is whereas papillomas in control mice frequently regressed transcriptionally repressed by wild-type p53 (Hoffman and never transitioned to squamous cell carcinoma et al., 2001; Mirza et al., 2002; Zhou et al., 2002). (Allen et al., 2003). These data suggest that survivin may Although it is still unclear whether this involves a participate in tumor progression rather than in the bipartite p53-responsive element in the survivin promo- initial step of oncogenic transformation, and that its ter (Hoffman et al., 2001), or changes in chromatin consequences may not become apparent until additional structure (Mirza et al., 2002), transcriptional repression mutations are accumulated. This is consistent with the of survivin contributed, at least in part, to p53- general paradigm that deregulated apoptosis inhibition dependent apoptosis (Mirza et al., 2002; Zhou et al., contributes to malignancy by favoring the accumulation 2002), whereas survivin expression antagonized p53- of additional ‘cancer traits’ (Hanahan and Weinberg, induced cell death (Hoffman et al., 2001). Another 2000), codifying more aggressive disease behaviors molecular abnormality of human cancer that was found (Evan and Vousden, 2001). The paradoxical ‘protective’ to drive survivin expression is deregulated Wnt/TCF effect of survivin on the onset of skin tumors (Allen signaling (Zhang et al., 2001). This may be particularly et al., 2003) mirrors similar data obtained with other relevant for the expression and role of survivin in apoptosis inhibitors, and may be explained as to the colorectal cancer (Williams et al., 2003), and is need for an initial wave of apoptosis of neighboring consistent with an embryonic-like pattern of survivin keratinocytes to allow physical expansion of the gene expression, potentially exploited during transfor- mutated cloned. mation (Zhang et al., 2001) (Table 2). The molecular basis for the overexpression of survivin in cancer has been intensely investigated (Table 2). Survivin gene transcription being linked to Translational exploitation of survivin for cancer mitotic progression, it was originally thought that therapeutics therapy the overexpression seen in cancer simply reflected a higher number of proliferating cells, and that the The differential expression in cancer and what appears proportion of cycling, potentially survivin-positive cells to be a requirement to preserve tumor cell viability in normal tissues was below the limit of detection identify survivin as a novel therapeutic target in cancer (Silke and Vaux, 2001). However, fresh experimental (Altieri, 2003). Three independent strategies have evidence discounted this view. First, comparable ex- generated promising results, and passed proof of pression of survivin is found in tumors regardless of principle in preclinical testing (Figure 3). The first their mitotic indices (Grossman et al., 1999), and, approach relied on the generation of an antigen-specific second, survivin reactivity is typically observed in the immune response against survivin-bearing tumor cells vast majority of tumor cells, in vivo, far exceeding the for potential cancer vaccination strategies (Andersen number of cycling cells detected by S-phase-specific and Thor, 2002). Survivin being selectively expressed in markers. This has prompted an alternative possibility tumors, it may not be surprising that host T cells that the survivin gene may be globally deregulated in recognize it as a non-self-protein and mount a vigorous cancer, driving overexpression of the protein at all cell cytolytic and antibody response to survivin peptides, cycle phases, not just mitosis. Several experimental data in vitro and in vivo (Rohayem et al., 2000; Schmitz are consistent with this view (Table 2). Firstly, survivin et al., 2000; Andersen et al., 2001; Yagihashi et al., promoter activity is largely silent in normal cell types 2001; Hirohashi et al., 2002; Schmidt et al., 2003).

Oncogene Survivin in cell division and apoptosis DC Altieri 8586 sis, and anticancer activity, in vivo (O’Connor et al., 2002; Wall et al., 2003). The reverse combination (Purv.A followed by taxol) was without effect, and Cdk inhibition after a mitotic block with depolymerized microtubules (mediated by vinca alkaloids) was also ineffective (O’Connor et al., 2002). This provided some mechanistic insights into the role of survivin in cytoprotection, and suggested that survivin functions in a relatively narrow time window at metaphase that requires elevated p34cdc2 activity and polymerized spindle microtubules. These data may also explain some conflicting results as to the actual role of p34cdc2 in apoptosis. A premature arrest of the cell cycle before the Figure 3 Current approaches for survivin targeting in cancer critical junction when the antiapoptotic function of therapy. Generation of antigen-specific cytolytic T cells against survivin peptides is being considered for cancer vaccination survivin is required would be expected to have strategies. Molecular antagonists of survivin including antisense, little effects on cell death, and, accordingly, early siRNA, ribozymes and dominant-negative mutants induce sponta- suppression of p34cdc2 activity by p21Waf1 has been neous apoptosis, enhance cell death stimuli and inhibit tumor associated with reduced apoptosis induced by taxol (Yu growth, in vivo. Inhibition of survivin phosphorylation on Thr34 by et al., 1998). sequential administration of cyclin-dependent kinase inhibitors flavopiridol or Purv.A after mitotic arrest with stabilized micro- It is difficult to predict which of these three modalities tubules (i.e. taxane) results in anticancer activity in vitro and in vivo. is likely to first see clinical testing in patients. New A similar response has been observed by adenoviral overexpression mechanistic insights have been obtained into the of a phosphorylation-deficient survivin Thr34Ala mutant (pAd- dominant-negative mode of action of the survivin T34A) Thr34Ala mutant (O’Connor et al., 2000a). In protein turnover experiments, survivin Thr34Ala exhibited a Indeed, HLA class I-restricted cytolytic T cells against 4–5-fold accelerated clearance (O’Connor et al., 2002), survivin peptides have been demonstrated in patients suggesting that dimerization of such unstable mutants with breast cancer, leukemia and melanoma, in vivo with endogenous survivin may favor destruction of the (Andersen et al., 2001). The antibody response to heterocomplex and lower endogenous survivin levels survivin peptides may also offer concrete diagnostic below a critical antiapoptotic threshold. In addition, a opportunities, and detection of antibodies to survivin in replication-deficient adenovirus encoding survivin combination with other cancer genes may provide a Thr34Ala has been recently generated, and shown to useful screening tool for patient follow-up strategies possess antitumor efficacy when locally administered by (Zhang et al., 2003). intratumor injection (Mesri et al., 2001b), an approach As anticipated above, a second approach involved the that has already been validated in clinical trials for other use of ‘molecular antagonists’, including antisense, anticancer gene therapy strategies, that is, p53 (Khuri ribozymes, siRNA and dominant-negative survivin et al., 2000). On the other hand, progress has been made mutants (Figure 3). In these studies, interference with at improving the antitumor efficacy of antisense survivin expression/function resulted in suppression of oligonucleotides, and various active sequences have tumor growth in various cancer models, alone or in been independently reported to suppress survivin combination with other anticancer strategies, and expression and induce tumor cell apoptosis in vitro appearance of tumor cell apoptosis, in vivo (Kanwar and in vivo (Li et al., 1999; Olie et al., 2000; Jiang et al., et al., 2001; Grossman et al., 2001b; Yamamoto et al., 2001; Kanwar et al., 2001; Xia et al., 2002). Lastly, the 2002; Williams et al., 2003). antitumor activity of the sequential combination of A third strategy focused on pharmacologic inhibition taxanes followed by Cdk inhibition has already been of mitotic phosphorylation of survivin on Thr34 validated in recent clinical trials in solid tumors (Figure 3). Mutation of Thr34 in survivin abolishes a (Schwartz et al., 2002). Consistent with the in vitro data phosphorylation site for the mitotic kinase p34cdc2 (O’Connor et al., 2002), these studies may pave the way (O’Connor et al., 2000a), and results in a dominant- for exploitation of a more selective class of Cdk negative phenotype with induction of apoptosis and inhibitors, which may exhibit improved specificity anticancer activity, in vivo (Grossman et al., 2001b). To and reduced toxicity in sequential combination with reproduce this effect with a more flexible pharmacologic taxanes. approach, antagonists of cyclin-dependent kinases There is, therefore, clear experimental evidence that (Cdk), flavopiridol (Sausville, 2002), or the more interfering with survivin results in anticancer activity p34cdc2-specific inhibitor, Purv.A (Gray et al., 1998), potentially suitable for clinical testing. But what are the were tested in tumor cells arrested at mitosis with taxol, actual cellular targets of survivin antagonists? The most which induces hyperphosphorylation of survivin on intuitive hypothesis is that inhibition of survivin results Thr34 (Zaffaroni et al., 2002). With this protocol, in apoptosis of the proliferating tumor cell compart- sequential administration of the Cdk inhibitors resulted ment, a model supported by the kinetics of apoptosis in in escape from the mitotic block imposed by taxol, cell cycle-synchronized cultures (O’Connor et al., massive activation of mitochondrial-dependent apopto- 2000a). However, there is evidence that survivin may

Oncogene Survivin in cell division and apoptosis DC Altieri 8587 provide a broader cytoprotective role for the tumor pathway has emerged as a multifaceted and essential microenvironment as a whole. This stemmed from the cellular infrastructure controlling chromosome congres- observation that survivin becomes strongly expressed in sion, spindle microtubule function and also the up- endothelial cells during the proliferative (Tran et al., stream initiation of mitochondrial-dependent apoptosis. 1999; O’Connor et al., 2000b) as well as remodeling The original premise that this pathway was dramatically (Papapetropoulos et al., 2000; Harfouche et al., 2002) exploited in many human cancers (Ambrosini et al., phases of angiogenesis, and mediates apoptosis resis- 1997) was intensely scrutinized, and has now been tance in these cells (O’Connor et al., 2000b; Tran et al., unanimously validated. The blueprints as to how the 2002). Conversely, antisense ablation of survivin during survivin pathway may couple to mitotic transition and angiogenesis removed the cytoprotective effect of the control of apoptosis have recently come into better VEGF, caused endothelial cell apoptosis and prompted focus. However, how survivin is wired into the deep rapid involution of three-dimensional capillaries, in vitro cellular circuitry controlling checkpoint function for cell (Mesri et al., 2001a). Therefore, it is possible that proliferation and cell death in normal and transformed molecular targeting of survivin may provide a dual cells remains to be elucidated. An answer to these advantage for anticancer strategies in vivo, by promoting questions is expected to provide greater opportunities tumor cell apoptosis and collapse of tumor-associated for the translational applicability of the survivin path- angiogenesis. way in cancer diagnosis and therapy.

Concluding remarks Note added in proof

Over the past few years, compelling experimental A role of survivin in the regulation of mitochondrial- evidence has accumulated to identify survivin as a dependent apoptosis has been recently further charac- unique member of the IAP gene family, positioned at the terized: Marusawa H, Matzusawa S, Welsh K, Zou H, crossroad between cell death and mitotic progression. Armstrong R, Tamm I and Reed JC. (2003) EMBO J., Through the contribution of several groups, the survivin 22, 2729–2740.

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