Inhibitor of Apoptosis (IAP) proteins as therapeutic targets for radiosensitization of human cancers

Cancer Treatment Reviews 38 (2012) 760–766

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New Drugs

Inhibitor of Apoptosis (IAP) proteins as therapeutic targets for radiosensitization of human cancers Simone Fulda ⇑ Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, Komturstr. 3a, 60528 Frankfurt, Germany

a r t i c l e

i n f o

Article history: Received 17 December 2011 Received in revised form 17 January 2012 Accepted 23 January 2012

Keywords: IAP proteins Smac Apoptosis Radiotherapy

s u m m a r y Radiotherapy initiates a variety of signaling events in cancer cells that eventually lead to cell death in case the DNA damage cannot be repaired. However, the signal transduction pathways that mediate cell death in response to radiation-inflicted DNA damage are frequently disturbed in human cancers, contributing to radioresistance. For example, aberrant activation of antiapoptotic programs such as high expression of Inhibitor of Apoptosis (IAP) proteins has been shown to interfere with the efficacy of radiotherapy. Since IAP proteins have been linked to radioresistance in several malignancies, therapeutic targeting of IAP proteins may open new perspectives to overcome radioresistance. Therefore, molecular targeting of IAP proteins may provide novel opportunities to reactivate cell death pathways that mediate radiation-induced cytotoxicity. A number of strategies have been developed in recent years to antagonize IAP proteins for the treatment of cancers. Some of these approaches have already been translated into a clinical application. While IAP protein-targeting agents are currently being evaluated in early clinical trials alone or in combination with conventional chemotherapy, they have not yet been tested in combination with radiation therapy. Therefore, it is a timely subject to discuss the opportunities of antagonizing IAP proteins for radiosensitization. Preclinical studies demonstrating the potential of this concept in relevant in vitro and in vivo models underscore that this combination approach warrants further clinical investigation. Thus, combination protocols using IAP antagonists together with radiotherapy may pave the avenue to more effective radiation-based treatment options for cancer patients. Ó 2012 Elsevier Ltd. All rights reserved.

Introduction The anticancer activity of most cytotoxic therapies which are currently used in the clinical management of cancer patients including radiotherapy is based on their ability to activate cell death programs such as apoptosis in cancer cells. Apoptosis or programmed cell death is an intrinsic program that is in place in every cell of the human body, including cancer cells.1 A characteristic feature of human cancers is the inability to mount a proper apoptotic response during tumor progression or upon treatment with cytotoxic therapies.2 Therefore, evasion of apoptosis constitutes a critical cause of primary or acquired treatment resistance that frequently occurs in various human cancers. This also applies to the resistance of cancers to radiotherapy, one of the main pillars of cancer therapy. Therefore, a better understanding of the molecular events that are responsible for the defects encountered in the apoptosis signaling network may offer novel perspectives to tackle treatment resistance, including radioresistance of human cancers. This review focuses on apoptosis resistance caused by aberrant

⇑ Tel.: +49 69 67866557; fax: +49 69 6786659157. E-mail address: [email protected] 0305-7372/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ctrv.2012.01.005

expression and/or function of Inhibitor of Apoptosis (IAP) proteins, a family of antiapoptotic proteins, and discusses the opportunities of how the targeting of IAP proteins can be translated into the design of new radiation-based treatment protocols.

Signaling pathways in radiation-induced cell death Radiation-induced signaling events can be initiated in distinct cellular compartments, for example the nucleus, the cytosol or the plasma membrane.3 In response to DNA damage that is sensed in the nucleus, the tumor suppressor and checkpoint protein p53 accumulates and becomes activated, thereby initiating cell cycle arrest, DNA repair and, in case of severe DNA damage, the induction of cell death.4 Furthermore, radiation can stimulate the production of reactive oxygen species (ROS) that can cause perturbation of the mitochondrial pathway of apoptosis.5 In addition, ionizing radiation can damage the plasma membrane, leading to the generation of ROS which may cause lipid oxidative damage and production of bioactive molecules.6 In addition, ROS production in response to irradiation has been associated with glutathione depletion and mitochondrial perturbation.7 Radiation-induced DNA damage can also lead to the activation of stress-activated protein kinases, e.g.

S. Fulda / Cancer Treatment Reviews 38 (2012) 760–766

c-Jun NH2-terminal kinase (JNK).8 All these cellular events in response to irradiation can eventually lead to the activation of a common effector phase of apoptosis, which is executed by a family of proteases called caspases.9 Caspase activation can result in amplification of the death signal via the protease cascade, since caspases can activate each other via proteolytic cleavage.10 In addition, feedback loops linking caspase activation to mitochondrial dysfunction contribute to amplification of cell death induction upon irradiation.11 Cell death by apoptosis can in principle proceed via two major routes, i.e. the extrinsic (death receptor) pathway and the intrinsic (mitochondrial) pathway.12 In the extrinsic pathway, transmembrane death receptors of the tumor necrosis factor (TNF) receptor superfamily can trigger caspase activation and cell death upon binding of their cognate ligands.13 The mitochondrial pathway of apoptosis is initiated by a variety of intracellular stimuli, including ROS and bioactive lipids, resulting in the release of proteins from the mitochondrial intermembrane space into the cytosol, for example cytochrome c and Smac.14 Once in the cytosol, cytochrome c promotes caspase-3 activation, while Smac binds to and neutralizes IAP proteins, thereby releasing the breaks on caspase activation. Inhibitor of Apoptosis (IAP) proteins are a family of endogenous antiapoptotic proteins.15 All IAP proteins contain a baculoviral IAP repeat (BIR) domain which is the structural constituent for their classification as IAP proteins.15 In addition to one to three of these BIR domains, IAP proteins can also contain other structural domains including the Really Interesting New Gene (RING) domain and the caspase-activating and recruitment domain (CARD).15 The RING domain is an E3 ubiquitin ligase that is responsible for ubiquitination and proteasomal degradation of multiple substrates, including Smac and IAP proteins.16 Among the eight human IAP proteins, XIAP has been reported to exert the strongest antiapoptotic function,17 which has been linked to its ability to bind to caspase-3,-7 and -9. In addition, ubiquitination and proteasomal degradation of proapoptotic factors, for example Smac and activated caspases, via the E3 ligase activity of XIAP can contribute to its antiapoptotic properties.16 Also, XIAP has been implicated in the regulation of additional signaling pathways, including NF-jB activation.18,19cIAP1 and cIAP2 are involved in both canonical and non-canonical NF-jB signaling.20 In the canonical NF-jB pathway, cIAP1 and cIAP2 are important mediators of non-degradative ubiquitination of the kinase RIP1, which is important for receptor-mediated NF-jB activation.20 Furthermore, cIAP1 and cIAP2 restrain non-canonical NF-jB activation in unstimulated cells by triggering the constitutive degradation of NIK via the proteasome.20 NIK is a key kinase that is involved in the initiation of the non-canonical NF-jB cascade.

Targeting IAP proteins for radiosensitization On theoretical grounds, targeting IAP proteins may present a particularly promising approach to overcome radioresistance, since some IAP proteins such as XIAP and cIAP1 are among the proteins that can be regulated at the level of translation under cellular stress conditions.21 Accordingly, the messenger RNA (mRNA) molecules of XIAP and cIAP1 are translated via an internal ribosome entry site (IRES).22,23 This IRES site allows continued translation of the protein even under cellular stress conditions when CAPdependent translation is usually blocked, for example following irradiation, endoplasmic reticulum (ER) stress, UV exposure or anoxia, and ensures that XIAP and cIAP1 protein expression levels are maintained.21,24,25 Recently, MDM2 has been reported to be involved in the regulation of XIAP translation upon irradiation.25 Irradiation-triggered DNA damage induced dephosphorylation of MDM2 and its cytoplasmic localization, thereby promoting IRES-

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dependent translation of XIAP.25 Interestingly, MDM2 was shown to physically interact with the IRES of the 50 -UTR region of XIAP, and to stimulate XIAP IRES activity.25 This ability to keep up IAP protein expression levels contributed to the ability of cancer cells, e.g. MDM2-overexpressing cells, to evade the induction of apoptosis following irradiation and thus conferred radioresistance.25 Therefore, interfering with IAP protein expression or function may prove to be particularly suitable to restore sensitivity to radiation-induced apoptosis in cancer cells. Against this background, a number of different strategies have been developed in recent years to antagonize aberrant IAP protein expression and/or functions in human cancers in order to overcome radioresistance and to increase radiosensitivity. In a first approach, genetic intervention studies tested the functional relevance of IAP proteins as targets for radiosensitization. Based on the evidence that XIAP exerts the most pronounced antiapoptotic functions among the eight human IAP proteins,17 antisense oligonucleotides were designed to downregulate XIAP expression levels.26 Furthermore, small molecule antagonists were developed that mimick the N-terminal part of Smac, which is the critical domain for the binding of Smac to XIAP as well as to other IAP proteins.27,28 Structural studies identifying the binding pockets and interaction sites of XIAP and its endogenous antagonist Smac have greatly facilitated these structure-guided drug development efforts.29

Genetic interventions to target IAP proteins To test the therapeutic potential of neutralizing IAP proteins to increase radiosensitivity of human cancers, a number of different genetic strategies have been employed (Table 1). For example, overexpression of Smac, the endogenous inhibitor of IAP proteins, has been reported to present a potent mean to increase radiation-induced cell death in a variety of human cancers, including neuroblastoma, glioblastoma, pancreatic and breast carcinoma.30,31 Both the full-length and the mature forms of Smac were reported to significantly potentiate radiation-induced apoptosis and to reduce clonogenic survival.30,31 Full-length Smac, which contains a mitochondrial translocation sequence, resides in the mitochondrial intermembrane space and is released from the mitochondria into the cytosol upon the induction of apoptosis. The mature version of Smac lacks the mitochondrial translocation sequence and is therefore constitutively expressed in the cytosol. Overexpression of either form of Smac increased c-irradiation-induced activation of the caspase cascade, promoted mitochondrial outer membrane permeabilization and the release of cytochrome c from the mitochondria into the cytosol, leading to increased caspase-3 activation and caspase-dependent apoptosis.30 Experiments showing that a broad-range caspase inhibitor or a relatively selective caspase-2 inhibitor blocked mitochondrial outer membrane permeabilization upon c-irradiation of Smac-overexpressing cells suggested that Smac facilitates caspase activation upstream of mitochondria.30 Similarly, overexpression of full-length Smac or the mature form of Smac was demonstrated in another study to promote irradiation-induced apoptosis in breast cancer cells.31 Ectopic expression of Smac caused increased interaction of Smac with IAP proteins following irradiation, resulting in enhanced caspase-3 activation and apoptosis.31 By comparison, overexpression of Smac did not affect the initial DNA damage and/or cellular stress response, as no differences in cH2AX or RAD51 foci formation, NFjB activation, p53 accumulation or cell cycle arrest in response to c-irradiation were observed in Smac-overexpressing cells compared to vector control cells.30 In addition to overexpression of Smac, knockdown of XIAP by RNA interference technology resulted in a significant increase in

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Table 1 Overview of genetic strategies to target IAP proteins for radiosensitization. Strategy

Cancer type

Results

Reference

Smac overexpression

Neuroblastoma, glioblastoma & pancreatic cancer

30

Smac overexpression XIAP knockdown

Breast cancer Pancreatic cancer

XIAP knockdown XIAP knockdown

Laryngeal cancer Lung cancer

XIAP knockdown XIAP knockdown Mutant survivin

Chondrosarcoma Colorectal cancer Lung & ovarian cancer

Radiosensitization in vitro, increased caspase activation & mitochondrial outer membrane permeabilization, caspase-dependence, no effect on DNA damage response Radiosensitization in vitro Radiosensitization in vitro, increased caspase activation & mitochondrial outer membrane permeabilization, caspase-dependence Radiosensitization in vitro Radiosensitization in vitro, better radiosensitization in TP53 mutant than wildtype cells Radiosensitization in vitro Radiosensitization in vitro Radiosensitization in vitro, reduced interaction of survivin/Smac

c-irradiation-induced apoptosis of pancreatic carcinoma cells.32 Also, silencing of XIAP cooperated with irradiation to suppress clonogenic growth, demonstrating that XIAP antagonism affects longterm survival in response to c-irradiation.32 The XIAP knockdownmediated radiosensitization was associated with increased activation of caspases and drop of mitochondrial membrane potential.32 Also, knockdown of XIAP initiated a mitochondrial amplification loop via the increase in caspase activity, since caspase inhibition also interfered with the concerted action of XIAP silencing and irradiation to cause mitochondrial outer membrane permeabilization.32 In addition, silencing of XIAP by RNA interference resulted in increased radiosensitivity in laryngeal and colorectal carcinoma cells as well as in chondrosarcoma cells.33–35 In a pair of p53 wildtype and mutant non-small cell lung cancer cell lines, RNA interference-mediated downregulation of XIAP significantly increased radiation-induced apoptosis in both wildtype p53 as well as in mutated p53 cell lines.36,37 Interestingly, cells with mutated p53 proved to be even more susceptible to radiation-induced caspase-3 activation and apoptosis compared to wildtype p53.36,37 While the underlying molecular mechanisms for this observation remain to be determined, these findings indicate that XIAP knockdown may provide a possibility to radiosensitize also cancer cells with mutated p53.

Pharmacological interventions to target IAP proteins In addition to genetic strategies to interfere with aberrant expression of IAP proteins in order to increase radiosensitivity of cancers, a number of pharmacological approaches have been developed to neutralize IAP proteins (Table 2). These efforts

31 32 34 36,37 33 35 63

focused on the development of small molecule compounds that mimick the N-terminal stretch of the endogenous Smac protein. Small molecule IAP inhibitors were reported to sensitize pancreatic carcinoma cells for irradiation-induced apoptosis.32 Similarly, IAP inhibitors were shown to cooperate with c-irradiation to reduce cell viability, to induce apoptosis and to suppress clonogenic survival of glioblastoma cells.38 In addition to established cell lines, IAP inhibitors also enhanced irradiation-induced cell death in primary cultured glioblastoma cells that were generated from patients’ derived tumor samples.38 Of particular interest is the fact that IAP inhibitors were shown to potentiate the efficacy of radiotherapy in glioblastoma-initiating cancer stem cells,38 which have been described to confer radioresistance.39 In contrast to malignant cells, no increased cytotoxicity against non-malignant cells of the central nervous system such as neurons and glial cells was observed following irradiation in the presence of IAP inhibitors.38 In addition, IAP inhibitors did not enhance irradiation-induced apoptosis in non-malignant fibroblasts.32 These results point to some tumor selectivity and indicate that IAP inhibitors may radiosensitize cancer cells while sparing normal non-transformed cells. Furthermore, the bivalent small molecule Smac mimetic BV6 was reported to prime glioblastoma cells to c-irradiation-mediated apoptosis.40 Calculation of combination index demonstrated that BV6 cooperated with irradiation in a highly synergistic manner to trigger apoptosis.40 Interestingly, NF-jB was identified as a critical mediator of Smac mimetic-mediated radiosensitization.40 Accordingly, the Smac mimetic stimulated increased DNA binding of NF-jB subunits.40 The proapoptotic function of NF-jB in this model of apoptosis was underscored by experiments showing that

Table 2 Overview of pharmacological strategies to target IAP proteins for radiosensitization. Compound

Cancer type

IAP inh. IDN

Pancreatic cancer

IAP inh. IDN

Smac mimetic BV6 Smac Smac Smac Smac

mimetic mimetic mimetic mimetic

BV6 LBW242 SM-164 SM-164

Smac mimetic SH-130 Smac mimetic JP-1201 XIAP antagonists 1396-11 and 1396-12 XIAP antisense Embelin

Results

Reference

Radiosensitization in vitro, increased caspase activation & mitochondrial outer membrane permeabilization, caspase-dependence, no sensitization of fibroblasts Glioblastoma Radiosensitization in vitro including primary glioblastoma cultures & glioblastoma stem cells, increased caspase activation & mitochondrial outer membrane permeabilization, caspasedependence, no sensitization of neurons or glial cells Glioblastoma Radiosensitization in vitro (synergism) including primary glioblastoma cultures & glioblastoma stem cells, caspase-dependence, NF-jB-dependent apoptosis Lung cancer Radiosensitization in vitro Glioblastoma Radiosensitization in vitro and in vivo Breast cancer Radiosensitization in vitro, caspase-dependence Head and neck squamous Radiosensitization in vitro and in vivo, caspase-dependence, sensitization involves NF-jB activation cell carcinoma and TNFa secretion Prostate cancer Radiosensitization in vitro and in vivo, no systemic toxicity, inhibition of NF-jB Colorectal cancer Radiosensitization in vitro, reduced DNA repair Pancreatic cancer Radiosensitization in vitro

32

Lung cancer Prostate cancer

48 51

Radiosensitization in vitro and in vivo, no systemic toxicity Radiosensitization in vitro and in vivo, minimal systemic toxicity

38

40 41 42 43 44 45 46 47

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genetic inhibition of NF-jB via ectopic expression of the dominantnegative superrepressor IjBa significantly reduced Smac mimeticand radiation-induced caspase activation, loss of mitochondrial membrane potential, cytochrome c release and apoptosis.40 Similarly, overexpression of a kinase dead mutant of IKK2, used as an alternative approach to block NF-jB activation, suppressed Smac mimetic-triggered radiosensitization.40 Experiments using primary cultured glioblastoma cells derived from tumor specimens and glioblastoma-initiated cancer stem cells underlined the clinical relevance of the Smac mimetic-mediated radiosensitization, as the Smac mimetic was similarly effective to prime these patients’ derived tumor cells for radiation-induced apoptosis.40 Besides glioblastoma, the Smac mimetic BV6 significantly enhanced radiation-induced apoptosis in non-small cell lung cancer lines.41 Moreover, the small molecule IAP inhibitor LBW242 was reported to potentiate the efficacy of a combination therapy with irradiation plus temozolomide, which represents the standard-ofcare therapy for malignant glioma.42 LBW242 both increased the radiotherapy-induced cytotoxicity and reduced the survival in clonogenic assays.42 Importantly, the combination treatment with LBW242 and irradiation showed higher cytotoxic activity than either treatment alone against the population of glioblastoma-initiating stem cells in neurosphere assays.42 Also, LBW242 acted in concert with radiation and temozolomide to suppress tumor growth in a orthotopic glioblastoma xenograft model.42 These results indicate that Smac mimetics can increase the efficacy of standard-of-care therapy for glioblastoma, which may have important implications for the design of experimental protocols. The bivalent Smac mimetic compound SM-164 at nanomolar concentrations was reported to prime breast carcinoma cells to radiotherapy-induced cell death.43 This radiosensitization by SM164 was associated with cIAP1 downregulation, reduced binding of XIAP to active caspase-9 and increased activation of caspases-9, -8 and -3.43 Inhibition of caspase activation by a pharmacological inhibitor or by RNA interference-mediated knockdown also inhibited the SM-164-mediated radiosensitization, pointing to caspasedependent apoptosis.43 In addition to breast carcinoma, the Smac mimetic compound SM-164 sensitized head and neck squamous cell carcinoma for irradiation-induced apoptosis.44 Investigations into the underlying molecular mechanisms showed that caspase activation was required for the SM-164-conferred radiosensitization, since inhibition of caspases by a pharmacological pan-caspase inhibitor or by short interfering RNA knockdowns blocked the Smac mimetic-conferred radiosensitization.44 Furthermore, the radiosensitization effect involved Smac mimetic-stimulated NF-jB activation, transcriptional activation of TNFa and secretion of TNFa, which in turn contributed to the induction of cell death.44 Of note, SM-164 also enhanced suppression of tumor growth following radiation of tumor xenografts in a mouse model with minimal toxicity.44 The monovalent Smac mimetic compound SH-130 was reported to increase radiation-induced caspase activation and apoptosis in prostate cancer.45 Pull-down and immunoprecipitation assays revealed that SH-130 bound to XIAP and cIAP1 and interrupted the binding of XIAP and cIAP1 to Smac. Also, SH-130 was shown to reduce radiation-stimulated activation of NF-jB.45 In vivo, SH-130 cooperated with radiation therapy to suppress tumor growth with over 80% of complete regression of tumors in a prostate cancer xenograft mouse model.45 Based on the evidence that the resistance to chemoradiation correlates with expression levels of IAP proteins in human cancers, the ability of the Smac mimetic JP-1201 was evaluated in radioresistant colorectal cancer cells.46 The addition of JP-1201 was found to potentiate the effect of ionizing irradiation to decrease the survival of colorectal cancer cells. The addition of JP-1201 also reduced tumor load in a xenograft mouse model compared to the treatment group that received irradiation alone.46

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Moreover, compounds 1396-11 and 1396-12, two XIAP small molecule antagonists, showed synergistic inhibition of pancreatic carcinoma growth when combined with radiation and reduced colony formation in soft agar.47 In addition to small molecule IAP inhibitors, antisense oligonucleotides against XIAP were developed to neutralize XIAP expression. XIAP antisense oligonucleotides significantly increased apoptosis following irradiation in lung carcinoma cells.48 Of note, treatment with XIAP antisense oligonucleotides caused tumor growth delay in a xenograft lung cancer mouse model following radiotherapy compared to the group receiving radiotherapy alone, while no weight loss or toxicity was observed upon the administration of XIAP antisense.48 Furthermore, the natural compound embelin has been tested as a radiosensitization agent. Embelin is an active ingredient of traditional herbal medicine and has previously been shown to inhibit XIAP.49,50 The combination of embelin together with radiation proved to be superior to either treatment alone to suppress prostate cancer cell proliferation, to cause cell cycle arrest at G2/M phase and to trigger caspase-independent apoptosis.51 Also, embelin cooperated with radiation to suppress tumor growth in a xenograft model of prostate cancer.51 This was accompanied by increased tumor growth delay and prolonged time to tumor progression in the combination treatment group with minimal systemic toxicity.51 Immunohistochemical analysis of tumor tissues showed that embelin in combination with radiation cooperated to inhibit cell proliferation, to trigger apoptosis and to reduce microvessel density when compared to single treatment.51 This indicates that the combination of embelin and radiation exerts a cooperative effect on both tumor suppression and angiogenesis. Molecular mechanisms The following common picture has emerged so far from these preclinical studies into the underlying molecular mechanisms that mediate the cooperative induction of apoptosis by IAP antagonists and radiotherapy (Fig. 1). Neutralizing IAP proteins in conjunction with irradiation results in increased activation of the caspase cascade as well as enhanced mitochondrial outer membrane permeabilization involving increased loss of mitochondrial membrane potential and cytochrome c release from the mitochondrial intermembrane space into the cytosol. This culminates in increased activation of the effector caspase-3 and caspase-dependent apoptosis, which is profoundly inhibited by broad-range caspase inhibitors such as zVAD.fmk. This activation of the extrinsic and intrinsic apoptosis signaling pathways also involves amplification loops, for example between caspase activation and mitochondrial outer membrane permeabilization. Accordingly, the release of cytochrome c into the cytosol promotes caspase-3 activation, which in turn acts back to mitochondria to trigger further mitochondrial perturbations.

Fig. 1. Scheme of radiosensitization by inhibition of IAP proteins. Inhibition of IAP proteins by IAP inhibitors or XIAP antisense cooperates with radiotherapy to increase activation of the caspase cascade, loss of mitochondrial membrane potential and release of cytochrome c and Smac from mitochondria into the cytosol. This culminates in increased activation of the effector caspase-3 and caspase-dependent apoptosis.

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Table 3 Overview of IAP inhibitors in phase I/II clinical trials.

a

Compound

Mechanism

Regimen

Cancer

Reference

GDC-0917 LCL-161 AT-406 AEG40826/HGS1029 TL32711 AEG35156 AEG35156

Monovalent IAP antagonist Monovalent IAP antagonist Monovalent IAP antagonist Bivalent IAP antagonist Bivalent IAP antagonist XIAP antisense XIAP antisense

Monotherapy Monotherapy Monotherapy Monotherapy Monotherapy Combination therapya Combination therapya

Solid tumors Solid tumors Solid tumors, lymphoma Solid tumors Solid tumors, lymphoma Leukemia Leukemia

58 59 60 62 61 56,64 57

Plus Cytarabine, idarubicin.

However, there are still several open questions concerning the signaling pathways that mediate the cooperative cytotoxicity of radiation and IAP antagonists. For example, the role of NF-jB and TNFa in the IAP antagonist-mediated sensitization to radiation therapy-induced apoptosis is not yet clear and may depend on the cellular context. Accordingly, Yang et al. reported that cotreatment with irradiation plus the bivalent Smac mimetic SM164 stimulated NF-jB activation and increased mRNA expression and secretion of TNFa, which contributed to caspase-8 activation and apoptosis.44 These findings are consistent with a Smac mimetic-initiated, NF-jB-mediated autocrine TNFa loop that promotes caspase activation and apoptosis. Similarly, treatment of glioblastoma cells with the bivalent Smac mimetic BV6 was reported to trigger NF-jB activation that was critically required for the Smac mimetic-mediated radiosensitization, since overexpression of the dominant-negative superrepressor IjBa also completely abolished the Smac mimetic-mediated increased apoptosis following irradiation.40 However in the latter study, an autocrine TNFa loop was not involved in mediating the synergism of Smac mimetic and radiotherapy, since addition of the TNFa-neutralizing antibody Enbrel failed to block apoptosis induction.40 Furthermore, the monovalent IAP inhibitor SH-130 was shown to inhibit rather than stimulate TNFa- and radiation-induced NF-jB activation in prostate cancer cells.45 In response to genotoxic damage, cIAP1 has recently been reported to be part of a cytoplasmic complex containing ATM, TRAF6 and cIAP1 that links nuclear DNA damage to NF-jB activation.52 Since activation of NF-jB may contribute to radioresistance,53 Smac mimetics may enhance radiosensitivity by interfering with radiation-induced NF-jB activation via degradation of cIAP proteins. Therefore, additional studies are required to explore the contribution of NF-jB and its downstream target genes in the Smac mimetic-conferred radiosensitization. Furthermore, it is not yet clear whether neutralization of IAP proteins has an impact on the DNA damage response and repair mechanisms. For example, it has been reported that overexpression of Smac sensitized for c-irradiation-induced apoptosis without altering the DNA damage/DNA repair response, as Smac did not affect c-irradiation-induced cH2AX and RAD51 foci formation.30 In contrast, the Smac mimetic JP-1201 has been shown to reduce the ability of colon carcinoma cells to repair doublestranded DNA breaks upon ionizing radiation.46 Therefore, further studies are required to investigate whether or not IAP proteins and/or Smac-mimicking compounds are involved in the regulation of DNA damage or its repair following irradiation. In addition, cIAP proteins have recently been shown to regulate the formation of a RIP1/caspase-8/FADD-containing complex that drives caspase-8 activation in genotoxic drug-dependent, receptor-independent pathways.54,55 Whether or not cIAP proteins may exert similar functions in the control of RIP1/caspase-8/FADD complex formation in response to irradiation will be an interesting question to address in future studies.

Clinical development of IAP protein-targeting agents The concept to target IAP proteins for therapeutic purposes has been transferred into a clinical context in recent years. Both antisense oligonucleotides as well as small molecule inhibitors of IAP proteins have been evaluated in phase I/II clinical trials either as monotherapy or in combination with conventional chemotherapeutics (Table 3). In general, XIAP antisense oligonucleotides and IAP antagonists were well tolerated in these early clinical trials,56–61 with the exception of some dose-limiting toxicities (i.e. severe fatigue, elevated pancreatic enzymes) that were observed for HGS1029.62 Efficient target inhibition of IAP proteins was demonstrated by downregulation of XIAP or cIAP1 expression in tumor or surrogate tissues.56–62 Of note, biomarkers such as cleaved cytokeratin-18 and caspase-3/-7 in the circulation indicated that cell death was induced upon systemic administration of these compounds.59,61,62 Together, these results from initial clinical trials support the further clinical evaluation of IAP protein-targeting agents to activate cell death pathways in cancer cells. However, no clinical trials have yet been launched to combine IAP antagonists or XIAP antisense oligonucleotides together with radiotherapy. Conclusions IAP proteins are aberrantly expressed in various human cancers and represent promising molecular targets for therapeutic intervention in order to restore the ability of cancer cells to undergo cell death in response to radiotherapy. IAP protein-targeting agents have shown synergistic antitumor activity in combination with irradiation in a wide variety of preclinical models. Therefore, the concept to antagonize IAP proteins in order to increase radiosensitivity is considered as a promising strategy to enhance the efficacy of radiotherapy. Of note, IAP antagonists were proved to be able to increase irradiation-induced cytotoxicity also in cancer-initiating stem cells that have been accused to confer radioresistance. However, little is yet known about the molecular markers which could be used to select the patient population that most likely will benefit from this molecular targeted approach. Also, it is not yet clear which IAP protein(s) is/are the most important regulator(s) of radioresistance in human cancers. Therefore, it remains to be determined whether broad-range IAP antagonists that neutralize several IAP proteins, i.e. XIAP, cIAP1 and cIAP2, or more selective IAP antagonists will be better suitable for radiation therapy protocols with no or minimal side effects. Taken together, incorporation of IAP antagonists into radiation protocols represents a particularly interesting approach to enhance radiosensitivity of human cancers that warrants further investigation. Conflict of interest None to declare.

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