The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer

Seminars in Cancer Biology 14 (2004) 231–243

The inhibitor of apoptosis protein family (IAPs): an emerging therapeutic target in cancer Boaz Nachmias a , Yaqoub Ashhab a , Dina Ben-Yehuda a,∗ a

Department of Hematology, Hadassah University Hospital, Ein-Karem, P.O.B. 12000, Jerusalem 91120, Israel

Abstract Apoptosis is a crucial biological process that prevents uncontrolled cell proliferation and eliminates harmful cells. Resistance to apoptotic stimuli is a hallmark feature of various cancers. One of the mechanisms through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of inhibitor of apoptosis proteins (IAPs). IAPs are a group of structurally related proteins that were initially identified in baculoviruses. Mammalian IAPs block apoptosis either by binding and inhibiting caspases or through caspase-independent mechanisms. This family of proteins has become increasingly prominent in the field of cancer biology. To date, overexpression of several IAPs has been detected in various cancers. This paper reviews the recent advances in the research of IAPs. The differential expression and the biological significance of each IAP in various cancer types will be discussed. Finally, we review the most recent advances in the research efforts aimed at using IAPs as potential targets for cancer therapy. © 2004 Elsevier Ltd. All rights reserved. Keywords: Apoptosis; Cancer; IAP; Caspase; Molecular targets

1. Introduction Most of the currently available anti-cancer therapeutic strategies rely on the eradication of tumor cells. At first glance, approaches such as chemotherapy, radiotherapy, immunotherapy, or even oncogene targeted therapy may seem distinct. However, practically, they share the same biological mechanism in eliminating the malignant cells by programmed cell death (apoptosis). It is now increasingly accepted that part of the efficacy of anti-cancer drugs is due to their ability to activate apoptosis [1]. Unfortunately, the resistance of tumor cells to drug-induced apoptosis is emerging as a major category of cancer treatment failure. Therefore, amongst cancer biologists, there is increasing interest in understanding the regulatory mechanisms of apoptosis [2]. The current efforts in this field are focused on uncovering the cellular factors that determine the fate of the cell through their ability to control the balance between life and death. Understanding the biological role of these factors will enable the design of more efficient and selective drugs Abbreviations: IAP, inhibitor of apoptosis protein; BIR, baculovirus inhibitory repeat; RING, really interesting new gene; XIAP, X-linked IAP; TNF, tumor necrosis factor; TRIAL, TNF related apoptosis inducing ligand; JNK, Jun kinase 1; Smac, second mitochondria derived activator of caspase; XAF-1, XIAP associated factor 1 ∗ Corresponding author. Tel.: +972-2-6776744; fax: +972-2-6423067. E-mail address: [email protected] (D. Ben-Yehuda). 1044-579X/$ – see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2004.04.002

in order to overcome resistance to apoptosis. Apoptosis is an active mechanism leading to cell death, which controls the development and homeostasis of multicellular organisms. Tight regulation is required to ensure a delicate balance of life and death. Indeed, loss of apoptotic regulation results in a wide variety of diseases. Excess apoptosis might result in neurodegenerative disorders [3] and immunodeficiency [4]. On the other hand, cellular defects that halt apoptosis are frequently involved in cancer development and progression [5], as well as in autoimmune disorders [4]. A group of cysteine proteases, termed caspases, form the core activation cascade of this form of cell death with upstream, or initiator caspases (8–10), and downstream, or effector caspases (3, 6 and 7). Two major pathways of apoptosis initiation have been described so far: the intrinsic and extrinsic pathways [6]. The intrinsic pathway is activated in response to intra-cellular stress, such as DNA damage, hypoxia and growth factor deprivation. In this pathway, the caspase cascade is triggered by increasing the permeability of the mitochondrial membrane and the release of cytochrome c. These mitochondrial changes result in the formation of apoptosomes, which consist of procaspase 9, Apaf-1 and cytochrome c in the presence of dATP. The formation of this complex results in the activation of caspase 9 and in turn caspase 3, thereby leading to apoptosis. The extrinsic pathway is initiated by the death receptors (Fas/CD95, TNF receptor, TRAIL receptor). A complex of proteins then activate the initiator caspases

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Fig. 1. IAP family members. The primary sequence feature is indicated for the different IAP members. In addition, the molecular interaction either with caspases and other cellular targets are shown.

8 and 10 that in turn activate the effector caspases 3 and 7. These two pathways are not distinct, and the activation of one usually involves the other. In the last decade, a complex network of pro- and anti-apoptotic proteins that governs the tight regulation along these pathways was revealed. Some of these proteins, such as p53, act to regulate the expression of death receptors [7], while others act at the level of initiator caspases such the as Bcl-2 family members [8]. A third group of factors, which consists of structurally related proteins known as the inhibitor of apoptosis (IAP) family of proteins, possesses two unique features. First, they are the only cellular factors that can act both on initiator and effector caspases [9]. Second, the biological effect of these proteins can be converted from anti-apoptotic to pro-apoptotic [10,11].

2. IAP family Following their initial discovery in the baculoviral genome, IAPs were identified in species ranging from yeast, nematodes, flies and man. To date, eight human IAPs have been identified: c-IAP1, c-IAP2, NAIP, Survivin, XIAP, Bruce, ILP-2, and Livin [12] (Fig. 1). IAP family members are defined by one or more repeats of a highly conserved 70 amino acids domain termed the baculovirus IAP repeat (BIR), located at the amino-terminus. With the exception of NIAP and Survivin, human IAPs also contain a conserved sequence termed RING finger at the carboxy-terminus. As their name implies, IAP family proteins are able to inhibit apoptosis induced by a variety of stimuli (Fig. 2). This is mainly mediated by direct binding and inhibition of certain

caspases [9]. Nonetheless, our growing knowledge of IAPs proteins reveals a much more diverse range of functions that include, aside from regulation of the apoptotic thrust by caspase-dependent and independent manner, involvement in protein degradation [13]. In addition, Survivin has been shown to play a role in mitosis, mainly in microtubule organization [14], while c-IAP1 and c-IAP2 are an integral part of the type-2 TNF-receptor complex [15].

3. Mechanisms of IAPs-mediated inhibition of apoptosis 3.1. Caspase inhibition IAPs can block apoptosis through their ability to inhibit specific caspases. XIAP, c-IAP1, c-IAP2 and Survivin directly bind and inhibit caspases 3, 7 and 9 (Fig. 1). Among the human IAPs, XIAP is the best characterized. It has three BIR domains, which are not functionally equivalent. BIR 3 inhibits caspase 9 by binding to an area that is exposed after caspase 9 undergoes cleavage, while the region encompassing BIR 1 and BIR 2 was shown to act on caspases 3 and 7 [16]. Recently, crystallographic resolution studies of XIAP revealed that conserved amino acids in the linker region between BIR 1 and BIR 2 are the most critical for inhibiting caspase 3 and 7, through its ability to sterically hinder the substrate access [17]. Surprisingly, the BIR2 domain itself has almost no direct contact with caspases 3 or 7. Yet, the linker region alone is not sufficient, as the BIR domain is required to either align or stabilize the caspase-IAP structure [18,19]. IAPs with only one BIR domain such as Livin are also able to inhibit caspase 3, 7 and 9.

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Fig. 2. Targets and regulators of IAP. IAPs can block caspase 9, which is activated after the release of cytochrome c, induced by apoptotic stimuli such as radiation, stress, or chemotherapy. In addition, IAPs can block the activities of caspases 3 and 7, which are activated by death receptors through caspase 8. IAPs are regulated by proteolytic cleavage mediated by effector caspases. In addition, the IAPs are negatively regulated by cellular factors Smac, Omi, and XAF-1.

3.2. Signal transduction pathways Recently, several IAP family members have been shown to regulate apoptosis in a caspase-independent manner through the mitogen-activated protein (MAP) Jun kinase 1 (JNK1) signal transduction pathway [20]. JNK1 was first identified based on its ability to phosphorylate the c-JUN oncoprotein, thereby increasing its transcriptional activity. JNK has also been reported to activate other transcription factors such as ATF-2, Elk-1, p53 and c-myc. The role of JNK activation in cell death has been the subject of much debate, with suggestions of no role, pro-apoptotic or anti-apoptotic activities. To further complicate the picture, JNK activation regulates and apparently is regulated by NF␬B. Following certain stimuli such as inflammatory cytokines, this cross-talk determines life, death and inflammatory responses of the cells [21]. In order to simplify matters we will concentrate only on the involvement of IAP family members in JNK signaling pathways. NAIP, as well as XIAP and Livin, are able to activate JNK1, while c-IAP1, c-IAP2 and Survivin were unable to do so. XIAP was found to interact with the bone morphogenic protein (BMP) receptor through its RING domain, while the BIR domain directly binds to TAB1, a co-factor of TAK1, a MAP3 kinase that is downstream of BMP [22]. These direct interactions raise the possibility that XIAP bridges between key players in this pathway. NAIP and Livin are also able

to selectively activate JNK1, most probably by interaction with TAB1 and TAK1. Furthermore, the ability of XIAP, NAIP, or Livin to suppress apoptosis induced by certain stimuli, as TNF-␣ and ICE, but not other stimuli, is reduced by expression of TAK1 or JNK1 inhibitory mutants [20]. XIAP was also shown to induce NF␬B activation, which contributes to the pro-survival effect. Remarkably, this was inhibited in the presence of catalytically inactive TAK1 [23]. It becomes apparent that IAPs are involved in the signal transduction of JNK signaling pathways, especially in the context of inflammatory stimulation, and that their effect is pro-survival. Yet, conflicting results raise doubt whether IAPs activate or inhibit JNK activation. These data reveal a caspase-independent mechanism of apoptosis inhibition, the relevance of which is dependent on the cellular context, e.g. the cell type and the death stimuli. Further research on the interaction of IAPs in JNK signaling pathways is needed to clarify some of these controversies. 3.3. E-3 Ligase activity Degradation of proteins in the proteasome is a highly specific and coordinated cellular process. It is essential for cell cycle regulation, activation of transcriptional factors and elimination of misfolded proteins. Recently, growing evidence has proven the significance of the proteasome in apoptosis as well [24]. The targeted protein is labeled with

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covalent modification of 8 kDa ubiquitin molecules. The process is initiated by ubiquitin-activating enzyme (E1), while ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) actually attach the ubquitin. Repeated cycles of ubiquitinylation result in a multiubiquitin tree. Labeled proteins are recognized by the proteasome and are degraded. Interestingly, RING finger proteins might function as an E3 ubiquitin ligase. In response to apoptotic stimuli, XIAP and c-IAP1 undergo RING-domain-dependent autoubiquitylation, which in turn labels them for proteasomal degradation. [25]. The autoubiquitylation process is a mechanism through which certain IAPs can negatively regulate their own activity. By doing so, they act to lower the apoptotic barrier, thus allowing the cell to undergo apoptosis. Nonetheless, the E3 ligase activity of IAPs has been shown to promote degradation of other substrates as well. For example, XIAP can target active caspase 3 to proteasomal degradation [13]. In contrast to autoubiquitylation, the ubiquitinylation of caspase 3 can be considered as a mechanism to protect the cell from apoptosis by lowering the active caspases’ effect. These two mechanisms seem to work in a counteracting fashion to keep a fine balance and to decide by a certain, yet unclear regulatory mechanism, whether IAPs should enhance the degradation of themselves or their targets. Second mitochondria derived activator of caspases (Smac) is released from the mitochondria with cytochrome c upon apoptotic stimuli, and is able to promote caspase activation by inhibition of IAP. Recently, Smac was identified as a substrate for the E3-ligase activity of XIAP [26]. This study reveals another possible mechanism by which IAPs can reduce the activity of caspases in the cell through their E3-ligase activity.

4. IAPs regulation IAPs are positively and negatively regulated by several mechanisms. Expression levels of IAPs are tightly regulated at the level of gene transcription. Although they are structurally and functionally similar proteins, IAPs have a differential pattern of gene expression. This phenomenon suggests that the different members of this multigene family are unique rather than redundant. For instance, we found different expression patterns for Livin, XIAP, and Survivin during the course of lymphocyte activation. As expected, the expression of Survivin is cell-cycle dependent, whereas the amount of Livin and XIAP transcripts were inversely correlated with cell division (unpublished results). NF␬B was shown to control the transcription of several apoptosis regulating genes, including some IAPs such as XIAP, c-IAP1, and c-IAP2. A unique feature of XIAP mRNA is that it has an unusual long 5 untranslated region, which contains a special sequence termed internal ribosomal entry site (IRES). IRES sequences have been discovered in several other life and death regulators in the cell, such as VEGF, PDGF, c-myc, c-JUN, and Apaf-1. During apopto-

sis, translation of cap-dependent proteins is inhibited due to caspase 3-mediated cleavage of cap-dependent translation proteins such as eIF4G I and II [27]. It is believed that IRES sequences allow translation under these abnormal cellular circumstances [28]. The ability of XIAP as well as other oncogenes to overcome stress conditions might be one of the advantages of tumor cells which enables them to resist chemotherapy or radiotherapy [29]. The BIR domain, in addition to its functional role, has a regulatory role as the binding domain of IAPs inhibitory proteins, such as Smac/DIABLO, Omi/Htra2, and XIAP associated factor 1 (XAF-1). Smac and XAF-1 have been shown to directly bind XIAP and reduce its ability to inhibit caspases [30–34]. Interestingly, very low levels of XAF-1 were detected in various cancer cell lines tested, in comparison to almost all normal adult and fetal tissues. This might provide the cancer cell with an advantage, as XIAP activity is not inhibited. Smac is released from the mitochondria along with cytochrome c, while XAF-1 is a nuclear protein. Remarkably, overexpression of XAF-1 was able to neutralize the anti-apoptotic effect of XIAP by sequestrating it in the nucleus [30]. Smac interferes with XIAP ability to bind caspases by binding to the BIR3 of XIAP. A four amino acids motif at the N-terminus of Smac mediates this binding. This motif is highly conserved, and is present in Smac homologous proteins in Drosophila: Hid, Grim and Reaper [34]. Remarkably, a recent study showed that the interaction between Survivin and Smac is crucial for the ability of Survivin to inhibit taxol-induced apoptosis. This might be achieved by not allowing Smac to inhibit other IAP members, such as XIAP [35]. Another fascinating example of a bi-directional effect is found in the intimate association of IAPs with caspases. IAPs inhibit caspases, yet this interaction comprises an intrinsic regulatory mechanism, as the caspases can cleave the IAPs. So far XIAP, cIAP-1, and most recently Livin have been shown to undergo specific and functional cleavage by caspases. In the case of XIAP, cleavage results in two sub-units: one that encompasses BIR 1 and 2, and the second, BIR3-RING. The N-terminal BIR 1-2 fragment shows a reduced ability to inhibit caspases 3 and 7, while the BIR3-RING fragment retains its ability to inhibit caspase 9 [36]. The cleavage of cIAP-1 occurs immediately after the BIR3 domain and produces a pro-apoptotic C-terminal fragment, which the RING domain is preceded by a spacer sequence of amino acids [10]. The pro-apoptotic activity of c-IAP1 fragment, which does not contain BIR, is not surprising since RING domains of other baculoviral and mammalian IAPs are able to induce apoptosis when expressed without their BIR domains [37]. We recently described a novel regulatory mechanism by which Livin is specifically cleaved by effector caspases at Asp52 to produce a large C-terminal sub-unit containing both the BIR and RING domains [11]. In contrast to XIAP and c-IAP1, our results regarding Livin, showed the first example of an IAP cleavage product that acts as a pro-apoptotic

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factor although it contains a BIR domain. A possible explanation for this unique behavior is that an additional, as yet undetermined, motif in the first 52 amino acids of Livin can modulate the anti-apoptotic effect of the BIR domain. The absence of this motif might enhance the E3-ubiquitin ligase activity of the RING domain that in turn targets other anti-apoptotic proteins to proteasome-mediated degradation. Interestingly, effector caspases 3, 6 and 7 and not upstream initiator caspases 8 and 9 are responsible for the specific proteolytic cleavage of the different IAP proteins. This is despite the fact that these anti-apoptotic factors are able to interact with both types of caspases. This might enable the cell to form a gradient of inhibition along the apoptotic cascade. At the upstream level, IAPs inhibit caspase 9, which cannot cleave any IAP. Yet, once the cells are committed to apoptosis and downstream caspases are active, they can overcome IAP inhibition by a specific cleavage. Phosphorylation has also been shown to be involved in the regulation of certain IAPs. Most recently, Akt with a pro-survival effect, was shown to interact with and phosphorylate XIAP. Phosphorylation of XIAP decreased its ubiquitination, which resulted in greater stability of XIAP [38]. Nonetheless, the relevance of this pathway is still unclear. Akt activity in endometrial cancer cells was found in correlation only with high levels of c-IAP1 and not with XIAP and c-IAP2 [39].

5. Differential expression of IAPs in malignancies Tumors develop due to rapid proliferation, decreased death, or the combination of both factors. The natural mechanisms which evolved to ensure that randomly mutated cells will not develop into a malignant tumor include: contact inhibition, nutritional deprivation, and immune surveillance. The elimination of the abnormal cells is virtually always accomplished by the induction of apoptosis. In most cancer patients, these mechanisms are fully functional, yet the tumor cells become resistant to their effect. Despite some controversy, recently accumulated evidence has indicated that anti-cancer therapies exert their cytotoxic effects mainly by activating apoptosis in tumor cells. Many studies demonstrated the role of different apoptosis regulators in rendering tumors cells resistant to apoptosis in vitro and in vivo [40]. Obviously, resistant tumors pose a serious problem in the treatment of cancer patients by these agents. In this regard, upregulation of IAP family members would certainly be advantageous for tumors. As data regarding different tumors accumulate, a widespread expression of IAPs, especially Survivin, has been revealed. Despite this almost universal expression, a careful analysis to discriminate between studies with and without clinical correlation should be made. Such a thorough analysis will help in estimating the relative contribution of each IAP in a given tumor, which will rationally enable more successfully tailored treatment.

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Of all IAPs, Survivin involvement in tumors has been most extensively studied, and has been found to be an important feature. Fetal and embryonic tissues show high expression of Survivin, while it is undetectable in normal, fully differentiated tissues. This is in marked contrast to high levels of expression observed in a broad range of malignancies. High level of Survivin which correlated with clinical status have been reported in colorectal cancer [41,42], esophageal cancer [43–46] and soft tissue sarcoma [47,48]. Survivin expression has also been associated with poor prognostic implications in several central nervous system tumors including glioma [49,50]. Although the prognostic relevance is still not clear, high levels of Survivin were also detected in carcinoma of the stomach [51], pancreas [52], liver [53], uterus [54] and in pheochromocytoma [55]. In breast cancer, Survivin expression did not correlate with tumor stage, histological stage, or nodal status. Remarkably however, nuclear Survivin was associated with a better outcome in relapse-free and overall survival [56]. Similar results were reported regarding osteosarcoma. While cytoplasmic Survivin showed no correlation with patient outcome, nuclear Survivin was correlated with prolonged survival [57]. This pattern of sub-localization and its relevance in tumors warrant further study. In ovarian cancer, Survivin expression was associated with tumor progression and poor prognosis and resistance to treatment [58–61]. Another study reported correlation with poor prognostic parameters, but not with overall survival [62]. Downregulation of XIAP has been shown to play a role in sensitizing ovarian cancer cells to cisplatin which is crucial for the treatment of ovarian cancer, in a p53-dependent manner [63,64]. Several studies have demonstrated a prominent role for Survivin in carcinoma of the bladder. Higher expression has been demonstrated as compared with normal tissue, and was associated with an increased rate of recurrence [65,66], but not with tumor grade, or pathological stage [66]. In yet another study, comparing mRNA levels of Survivin in bladder carcinoma, correlation was found with a more advance pathological stage and grade and disease recurrence, but not with survival [67]. Remarkably, detection of Survivin in urine has been associated with a higher risk of bladder cancer [68,69]. Survivin was also detected in TCC of the upper urinary tract, as compared to normal urothelium, but with no pathological or clinical correlation [70]. A recent study reported that nuclear staining of Survivin in TCC correlated with a longer disease-free period, although the difference is not statistically significant [71]. This coincides with the data regarding breast cancer and osteosarcoma mentioned earlier. We have previously identified two splicing variants of Livin, termed Livin ␣ and ␤. The two proteins are highly similar, except for 18 amino acids located between the BIR and the RING domains, which are present in the ␣ but not the ␤ isoform. Despite the high similarity, we showed different anti-apoptotic properties of the two isoforms [72]. A study examining both Livin and Survivin expression by RT-PCR,

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found a correlation between Livin ␣ expression and a higher risk of relapse. No such correlation was found with Survivin expression [73]. High levels of XIAP were also found in a high percentage of bladder carcinoma samples, but were not in correlation with tumor stage or grade. However, XIAP was shown to convey resistance to apoptosis in the TCC cell lines tested [74]. Survivin, XIAP and c-IAP have all been shown to be upregulated in non small-cell lung cancer (NSCLC) cells, yet the clinical relevance is still not clear [75]. Higher rate of Survivin expression were correlated with a low apoptosis rate and decreased survival [76–79]. Another study, which focused on early stage NSCLC, demonstrated a high expression level in most tumors, suggesting an earlier role in tumorigenesis. However, no correlation was found with survival [80]. One study reported preferential high levels of c-IAP2 mRNA in patients with NSCLC adenocarcinoma versus squamous cell carcinoma [78]. Also, higher levels of cIAP-2 were reported in NSCLC versus SCLC [81]. The involvement of XIAP in NSCLC is more controversial. A recent study showed that XIAP inhibits the apoptosome (mithochondrial) activation of the apoptotic cascade in human NSCLC cell lines. Treatment with XIAP antagonist Smac, relieved this inhibition. Remarkably, in a mouse model, treatment with Smac sensitized cells to apoptosis, and in combination with chemotherapy reduced tumor growth [82]. Furthermore, antisense treatment against XIAP sensitized tumor cells to chemotherapy both in vitro and in vivo [83]. However, in marked contrast, Ferreira at al. [84], reported that high XIAP expression did not correlate with the apoptosis rate, but with a lower proliferation rate and a longer survival. Thus, in NSCLC XIAP might serve as a prognostic factor, with higher expression levels implying a paradoxical better prognosis. However, whether XIAP actually has a negative effect on proliferation is still to be determined. High levels of Survivin, cIAP-1, cIAP-2, NAIP and XIAP were reported in prostate cancer cell lines [85] and prostate cancer samples, as compared to normal tissue. However, IAP expression did not correlate with Gleason grade or prostate-specific antigen levels [86,87]. Other studies have reported a correlation between Survivin expression and a higher Gleason sum and a more aggressive carcinoma [88]. Survivin expression has been correlated with poor prognosis in several hematological malignancies including: diffuse large B cell lymphoma [89], mantle cell lymphoma [90], acute lymphocytic leukemia [91,92] and chronic myeloid leukemia [93]. Survivin was found at a high rate in high-grade non-Hodgkin’s lymphoma, but not in low-grade lymphomas [94]. A role for Survivin has also been suggested in thyroid lymphoma [95], myelodysplastic syndrome [96] and chronic lymphocytic leukemia [97]. However, the clinical significance is yet to be determined. Several studies have reported involvement of both Survivin and XIAP in acute myelogenous leukemia (AML), although some controversies exist. One group reported a strong cor-

relation between XIAP expression and a shorter survival [98]. Survivin has also been demonstrated to be an unfavorable prognostic factor [99]. On the other hand, a recent study that analyzed the expression of Survivin and XIAP in primary AML blasts revealed that while expression was detected in all samples, no correlation has been found with cytogenetics, remission, or overall survival of the patients. Interestingly, caspase inhibition in AML cells did not inhibit apoptosis induced by various chemotherapeutic agents [100]. This data suggest a caspase independent pathway, which might explain the lack of clinical correlation. High levels of XIAP have also been detected in primary cells of Hodgkin’s disease (HD). Furthermore, in vitro studies demonstrated the ability of XIAP to inhibit mitochondrial-mediated apoptosis. Antagonizing XIAP using Smac made HD derived B cells less resistant to Staurosporine. These results suggest a role for XIAP in HD, yet the clinical relevance should still be proven [101]. c-IAP2 was shown to be involved in the pathogenesis of a malignant lymphoma known as mucosa-associated lymphoid tissue (MALT). In this distinctive sub-type of B-cell non-Hodgkin’s lymphoma, the recurrent chromosomal translocation t(11;18)(q21;q21) results in the expression of a chimeric transcript fusing c-IAP2 on chromosome 11 to a gene known as MLT on chromosome 18 [102]. The truncation of the c-IAP2 gene is distal to its three copies of BIR domain and fusion with the carboxy-terminal region of MLT may act as an oncogenic lesion that might promote MALT lymphomas. Melanoma is the most aggressive form of skin cancer and is highly resistant to the various anti-cancer modalities. Several defects in the apoptotic cascade were suggested to play role in the drug resistance of this malignancy [103–105]. We have shown that Livin has a differential expression pattern in primary cultures derived from melanoma patients, while ubiquitous expression of Survivin and XIAP was observed. Remarkably, we found a correlation between Livin expression level and the resistance of the cells to chemotherapy both in vitro, and in melanoma patients receiving chemotherapy. High levels of expression were also correlated with a lower survival rate [11]. Recently, a few reports detected over-expression of Survivin in malignant and invasive melanoma whereas no levels were detected in normal human melanocytes [105–107]. In a follow-up study on a group of melanoma patients, Survivin expression significantly correlated with the progression of the disease and a lower survival rate [108]. Interestingly, etoposide-resistant melanoma cells showed decreased caspase activation but this was not correlated with Survivin expression [109].

6. Possible roles of IAPs in cancer In view of the almost universal and notable levels of IAPs in tumors, several questions arise. First, what is the relative contribution of each IAP in a specific tumor? meaning,

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is there a tumor-specific IAP, while other expressed family members play no role in the same tumor? In this regard, it is crucial to separate the expression studies, even in combination with an in vitro effect, from studies showing a clear correlation with clinical parameters. Second, what is the role of IAP in tumorgenesis? It might be beneficial to differentiate involvement of IAPs in tumor initiation, progression, metastasis and drug resistance. Third, in light of the combined IAPs expression in the same tumor, can we identify a common regulatory pathway? Finally, what is the potential of IAPs as targets for new anti-cancer drugs? These questions will be discussed briefly. 6.1. What is the relative contribution of each IAP in a specific tumor? As already mentioned, several studies have demonstrated the expression of multiple IAPs in various tumors and cancer cell lines [98] although they have not always shown a clinical correlation [86]. Still, the exact role of each IAP and their interplay is unclear. A recent study demonstrated a high level of caspases 3 and 8 activities in cancer cell lines without apoptotic stimuli. Concomitantly, high levels of Survivin and XIAP were detected, as compared to normal counterpart cells. Inhibition of IAPs in these cells was sufficient to induce apoptosis. Remarkably, blocking both XIAP and Survivin augmented the pro-apoptotic effect, suggesting a synergistic action [82]. These results are in agreement with previous studies regarding Survivin [110,111]. On the other hand, in NSCLC Survivin and XIAP expression is associated with an opposing prognostic implication. Expression of Survivin was correlated with a low apoptosis rate and poorer prognosis [76], while XIAP expression did not correlate with the apoptosis rate, but remarkably, with a lower proliferation rate and longer survival [84]. Recently, a possible interplay between IAP family members has been suggested. Survivin was shown to bind Smac, and by doing so relieves its inhibition of XIAP and allows the latter to function [35]. Further studies should provide a better understanding of the distinct functions of each IAP in the same tumor. 6.2. What is the role of IAP in tumorigenesis? Recently, studies performed on skin tumors in transgenic mice expressing high levels of Survivin implied that Survivin participates in tumor progression rather than initiation. In these mice the papillomas did not regress and some progressed into squamous cell carcinomas. In contrast, although normal mice developed more papillomas, most of them regressed and none transformed into carcinomas [112]. On the other hand, several studies demonstrated high levels of IAPs in early stages and even pre-malignant lesions, indicating an early role in these tumors. For example, XIAP, cIAP-1, cIAP-2 and Survivin have been detected in prostatic intraepithelial neoplasia lesions (carcinoma in situ)

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[86]. Survivin has been detected in pre-malignant lesions at similar levels observed in overt malignancy [105]. In colon cancer, several reports suggest an early role for Survivin in tumorigenesis. Intestinal epithelial cells undergo rapid proliferation at the base of the intestinal crypts, followed by differentiation, migration to the surface, and finally apoptosis. One of the earliest events in the development of colon cancer is upregulation of cyclooxygenase (COX)-2, which in turn increases cAMP production, which promotes growth and is anti-apoptotic. Remarkably, as cells migrate to the surface of the villi, a lowered level of cAMP is seen [113]. Recently, c-IAP and Livin expression were shown to be positively regulated by cAMP in colon epithelial cells [114]. Furthermore, Survivin was preferentially expressed in the lower crypts, and showed an increase in expression in the transition from adenoma to carcinoma [42]. These findings coincide with the ability of adenomatous polyposis coli (APC) to down regulate Survivin expression in colon cancer cell lines [115]. One possible mechanism by which IAPs can promote tumorigenesis is by keeping mutated cells alive. Active caspase 3 cleaves inhibitor of caspase 3-activated DNase (ICAD) and therefore allows breakdown of DNA. As IAPs inhibit cell death, cells that suffered DNA breaks might be rescued, raising the possibility of malignant transformation [40]. Indeed, several studies have demonstrated upregulation of certain IAPs in response to chemotherapy [116] and radiation [29]. This upregulation have been shown to mediate cell resistance to apoptosis. Furthermore, it is not unlikely that these processes could predispose to the development of secondary malignancies. This suggests possible dual benefits from targeting IAPs in combination with these treatments, that is, better control of the primary tumor and less chance of developing secondary malignancies. 6.3. Can we identify a common regulatory pathway? As several IAPs are often detected in the same tumor, a possible common upregulatory pathway can be presumed. A recent study comparing IAPs expression in human leukemia HL-60 and its multidrug resistant variant (HL60R) showed overexpression of c-IAP2, XIAP, NAIP and Survivin in the HL60R variant. Interestingly, only the HL60R variant expressed the p65-activated form of NF␬B, necessary to form the NF␬B heterodimer and to increase transcription [117]. The pro-survival effect of NF␬B activation has been linked to the upregulation of several IAPs, including c-IAP2 and XIAP [118–121]. c-H-Ras was also suggested as a possible candidate, and was shown to upregulate Survivin [122]. Several studies have demonstrated that Survivin expression is suppressed by p53 [123,124]. Thus, loss of p53 might result in upregulation of Survivin expression. It would be interesting to study the relevance of p53 regulation on other IAP family members. Another mentioned pathway is by ␤-catenin signaling, as a high ␤-catenin activity results in upregulation of Survivin in colon cancer [115]. Finally, a decline in proteasomal activity that results in an inability to

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efficiently degrade IAPs, has also been shown as a possible mechanism [125]. 6.4. What is the potential of IAPs as targets for new anti-cancer drugs? Conventional cancer chemotherapies are cytotoxic chemicals that kill rapidly growing malignant cells. These drugs are indiscriminate toxins that lack cell selectivity. Therefore, they cause harmful effects to many types of healthy cells and consequently lead to potentially devastating and rarely even irreversible side effects to the treated patients. There has been an increasing interest in the development of anti-cancer targeted therapy based on rational drug design. The starting point for developing more selective and less harmful anti-cancer drugs is to single out targets associated with the signal transduction network within cells that are critical for proliferation, cell death, and angiogenesis. In this context, IAPs seem to fit ideally as a specific molecular target for cancer treatment. These cellular factors are differentially overexpressed in many cases of malignant cells and not in their healthy counterparts. They are vital for keeping the tumor cells alive and making them resistant to high doses of chemotherapy. The third and the most unique advantage of IAPs as anti-cancer targets is that they act at the effector level of the apoptosis pathways. Therefore, several apoptotic defects that occur frequently in many tumor types can be bypassed.

Theoretically, inhibition of an IAP expression can be achieved at various biological levels (Fig. 3). At the nucleic acid level, gene expression can be reduced or even blocked by means of antisense nucleic acids, ribozyme, or small interfering RNA (siRNA). In addition to blocking gene expression by acting on the RNA levels, the protein itself can be either directly inhibited or its function can be modulated so as to induce apoptosis. Finally, the protein can be a useful target for immune-mediated tumor destruction. 6.4.1. Targeting IAP at the nucleic acid level In this approach, short DNA or RNA molecules that are complementary to the mRNA of the IAP of interest are introduced into malignant cells. In addition to inhibiting the translation into protein, they induce degradation of the specific mRNA molecules. Survivin was the subject of several studies to inhibit its expression. In one study, the apoptotic threshold of JR8 human melanoma cell line was significantly lowered by the addition of active ribozyme that targeted Survivin mRNA prior to topotecan treatment [126]. In another study, which employed an antisense oligonucleotide specific for Survivin, several lymphoma cell lines were rendered more sensitive to apoptotic signals. In addition, the development of tumors as well as the growth of established tumors in a lymphoma xenograft model were inhibited by these antisense molecules [127]. In three in vitro studies on cell lines derived from non-small lung cancer,

Fig. 3. Targeting IAPs. IAPs can be downregulated at nucleic acid level by antisense or small interfering RNA (siRNA). At the protein levels, IAPs can be blocked by inhibitory antibodies or small synthetic molecules.

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acute myeloid leukemia, and bladder cancer, administration of antisense against XIAP mRNA caused a significant reduction of chemoresistance to several conventional drugs [74,83,128]. Small interference RNA (RNAi) technology was used to reduce Livin gene expression. Silencing of Livin was correlated with caspase 3 activation and a strongly increased apoptotic rate in response to different pro-apoptotic stimuli. Interestingly, the effects were specific for Livin-expressing tumor cells [129].

nation of taxol followed by a CDC2 inhibitor has been employed. Remarkably, this protocol resulted in a release from the mitotic block, induced by taxol, and massive apoptosis in vivo [110,135]. Recently, new studies implicated Survivin in the anti-apoptotic effect of PDGF following vascular injury [136]. This might suggest that targeting Survivin might also inhibit the angiogenesis that is crucial for tumor development.

6.4.2. Targeting IAP by blocking protein function Blocking protein function is usually achieved by designing molecules that can specifically bind and inhibit the biological activity of that protein. Different approaches can be used to achieve this goal. One possible method is to use monoclonal antibodies against the IAP, which is highly expressed by tumor cells. In this model, antibodies would be expected to induce apoptosis, by interfering with the antiapoptoic effects of the IAP. Alternatively, mutant proteins or short polypeptides that are derived from endogenous proteins, which are negative regulators of IAPs, such as Smac, XAF1, or Omi can be used as semi-natural inhibitors for IAPs. Several groups did actually attempted the latter approach and they were able to show that short peptides derived from Smac can enhance the pro-apoptotic effect of various chemotherapeutic agents [130,131]. Remarkably, these peptides were also active in sensitizing cells to apoptosis though they suffered from defects in apoptosis signaling such as loss of caspase 8 expression, impaired Apaf1 expression, Bcl-2 overexpression. Furthermore, the Smac peptides were able to enhance the anti-tumor activity of apoptotic stimuli in an intracranial malignant glioma as well as non-small cell lung cancer xenograft models in vivo [82,131]. Similar interest in deriving Livin inhibitory peptides leads to the generation of Smac-based peptides which have significant affinity for Livin [132]. Currently, there is increasing interest in a novel approach that is based on using combinatorial chemical libraries to screen for small pharmacological molecules, which can act as IAP antagonists. A library of 89,856 polyphenylurea compounds was screened for molecules that can reverse XIAP-mediated inhibition of caspase 3. Interestingly, some of the selected compounds were able to directly induce apoptosis of many types of tumor cells lines. Additionally, they were shown to sensitize cancer cell to chemotherapy [133]. By searching a library of approximately 160,000 compounds, a completely different group of small molecules were found to overcome the inhibitory effect of XIAP on caspase 3. Nevertheless, these compounds were not tested for their ability to directly trigger apoptosis [134].

6.4.4. IAP as targets for immune-mediated tumor destruction Immune-mediated tumor destruction is emerging as an interesting modality to cure cancer patients. Indeed, several immunotherapeutic strategies have shown that immune manipulation can induce the regression of established tumors. However, the mechanisms that regulate the immune system’s attack on malignant cells are unclear. A significant effort in this field is aimed at the elucidation of tumor associated antigens. Several recent reports showed that various IAP are among the tumor antigens that serve as potential targets for immune-mediated tumor destruction. Anti-Survivin antibodies were detected in the sera of patients suffering from melanoma, gastrointestinal, and lung tumors [137,138]. Livin antibodies were also detected in several patients that suffered from gastrointestinal tumors [139]. Most recently, several epitopes which are HLA-A restricted, and derived from Survivin, as well as Livin, were revealed as specific targets for cytotoxic T-lymphocyte responses in melanoma, breast cancer, and chronic lymphocytic leukemia [140,141]. By in vitro analysis, these lymphocytes were found to be cytotoxic against HLA-matched tumor cells [142]. The identification of the IAP as tumor-associated antigens can be used to develop effective anti-cancer vaccinations against these antigens. These immunizations could activate both humoral and cellular-mediated immunity to eradicate the malignant cells.

6.4.3. Targeting IAP by modulating their functions Another approach utilizes the crucial Thr34 phosphorylation of Survivin. Microtubule poisons, such as taxol, cause mitotic arrest which in turn upregulates CDC2. CDC2 phosphorylates Survivin, increasing its stability. Thus, a combi-

7. Conclusion and future perspectives IAPs are a group of structurally related anti-apoptotic proteins. In addition to other biological functions, they are the only endogenous caspase inhibitors with the ability to act on various points of the apoptotic cascade. Recently, an emerging role of these proteins in tumor development and progression has been proven. The IAPs are differentially overexpressed in many cases of malignant tissues and not in their healthy counterparts. They are crucial in keeping the tumor cells alive and causing resistance to high doses of chemotherapy. Therefore, understanding the biological role of these factors will enable the design of more efficient and selective drugs that can overcome apoptosis resistance in several cancer types. As shown above several lines of evidence suggest that attenuation or modulation of IAP proteins can be considered as attractive targets for new therapeutic in-

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terventions in various cancers. These novel drugs can be used either alone or in combination with safer doses of conventional anti-cancer therapies in order to enhance their efficacy. The last few years have witnessed significant advances in our understanding of the role of IAPs in apoptosis and cancer. However, we still need to know a great deal about their precise involvement in different tumor types. The use of these cellular factors as targets for cancer therapy is still in its infancy. Much effort is needed to focus on the identification of new compounds that can act as selective inhibitors of the various IAPs. The biological and pharmacological evaluation of these novel drugs necessitate the establishment of new in vivo experimental models that mimic the authentic biological and microenvironmental characteristics of the tumor.

Acknowledgements The authors would like to thank Prof. Deborah Rund, Dr. Reki Perlman and Dr. Gillian Dank for their helpful comments and suggestions. A special thank for Mr. Mohammad Abdeen for the help in graphical design.

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