Oncogene (2011) 30, 575–587 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Caspase-3 cleaves XIAP in a positive feedback loop to sensitize melanoma cells to TRAIL-induced apoptosis MHo¨rnle1, N Peters1, B Thayaparasingham1,HVo¨rsmann1, H Kashkar2 and D Kulms1 1Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Baden-Wu¨rttemberg, Germany and 2Institute for Medical Microbiology, Immunology and Hygiene, Center of Molecular Medicine Cologne, University of Cologne, Cologne, Germany Successful treatment of melanoma is still challenging, 57 kDa protein which appears to be ubiquitously because metastasis remain chemoresistant and radio- expressed in adult and fetal tissues (Hunter et al., resistant. Accordingly, combinational treatments invol- 2007). XIAP consists of three BIR domains (baculoviral ving death ligands are mandatory. In a recent study from IAP repeat 1–3), a RING-finger domain conferring our lab, the majority out of 18 melanoma cell lines ubiquitin (Ub) protein ligase (E3) activity and a recently remained resistant against treatment with the death ligand discovered evolutionarily conserved Ub-acceptor (UBA) TRAIL (tumor necrosis factor related apoptosis inducing domain (Gyrd-Hansen et al., 2008; Srinivasula and ligand). Resistance was shown to be mainly due to Ashwell, 2008). XIAP was identified as the only cellular incomplete processing of caspase-3 into catalytically protein being able to bind caspase-3, -7 and -9 directly inactive p21 by binding of the anti-apoptotic protein and to inhibit both the initiation and the execution X-linked inhibitor of apoptosis protein (XIAP). Co- phase of apoptosis (Deveraux et al., 1999). Given its role irradiation with sub-lethal ultraviolet (UV) B caused in apoptosis and its frequently elevated expression in depletion of XIAP resulting in synergistic sensitization of malignant cells, XIAP has been considered as a all but two melanoma cell lines to TRAIL. We show here promising therapeutic target in mammalian cancer. the XIAP depletion to essentially require initial caspase- Consequently, research efforts have lately been focused mediated cleavage, which promotes proteasomal degrada- on the development of drugs targeting XIAP (IAP tion of XIAP. Utilizing specific caspase inhibitors and inhibitors), which are currently being evaluated in small interfering RNA-mediated knockdown, we further clinical trials (Schimmer et al., 2006). identified caspase-3 to be responsible for performing the Pro-apoptotic second mitochondria-derived activator initial cleavage of XIAP after UVB treatment. Additional of caspase/direct inhibitor of apoptosis-binding protein evidence suggests an accelerated mitochondrial outer with low pI (Smac/DIABLO), being released from membrane permeabilization in response to co-treatment mitochondria during the onset of intrinsic apoptosis, is with TRAIL and UVB, which directs the release of XIAP known to functionally inhibit XIAP by distracting the antagonizing factors including Smac. Distraction of XIAP protein from caspases (Du et al., 2000; Sun et al., 2008). consequently liberates caspase-3 to autocatalytically Full autocatalytic processing of released caspases con- process into active p17. Activated caspase-3 cleaves XIAP sequently executes the apoptotic process, while the fate and further enhances its activation in a positive regulatory of XIAP is still discussed. Smac/DIABLO-dependent feedback loop. The molecular mechanism discovered here and -independent mechanisms have been described to appears to have broader implications, because cleavage of mediate ubiquitination and proteasomal degradation of XIAP was also shown to accompany cisplatin-induced XIAP thereby enhancing apoptosis (Fu et al., 2003; sensitization of melanoma cells to TRAIL. Yang and Du, 2004; Sohn et al., 2006; Liu et al., 2007; Oncogene (2011) 30, 575–587; doi:10.1038/onc.2010.434; Galban and Duckett, 2009). Vice versa, there are published online 20 September 2010 numerous reports implying E3 activity of XIAP to cause ubiquitination and immediate proteasomal degradation Keywords: caspase-3; XIAP; melanoma; apoptosis of pro-apoptotic XIAP counterparts, including cytosolic Smac/DIABLO (MacFarlane et al., 2002; Morizane et al., 2005; Rehm et al., 2006), caspase-9 (Morizane Introduction et al., 2005), caspase-7 (Creagh et al.,2004)andcaspase-3 (Suzuki et al.,2001;Chenet al., 2003), thereby directing The most intensively studied inhibitor of apoptosis them to proteasomal degradation. Taken together, the protein (IAP) family member, XIAP (X-linked IAP), is a cellular decision between pro- and anti-apoptotic re- sponses underlies the regulation of caspase activity mainly provided by a multilayered cross-talk between Correspondence: Dr D Kulms, Institute of Cell Biology and XIAP and caspases and intracellular regulator circuits Immunology, University of Stuttgart, Allmandring 31, Stuttgart, including mitochondria and proteasomes. Baden-Wu¨rttemberg 70569, Germany. E-mail: [email protected] Malignant melanoma represents one of the most Received 15 April 2010; revised 4 August 2010; accepted 16 August 2010; aggressive malignancies with a highly chemoresistant published online 20 September 2010 and radioresistant metastatic stage (Flaherty, 2006), XIAP cleavage by caspase-3 sensitizes melanoma to TRAIL MHo¨rnle et al 576 offering a poor prognosis for affected patients active p17 fragment (Figure 1b). To inquire whether (Lasithiotakis et al., 2006). Therefore, therapeutic XIAP depletion during apoptosis induction is dependent strategies combining chemotherapeutic drugs and tumor on proteasomal degradation, cells were treated with the selective death ligands are under investigation to proteasome inhibitor MG132 before TRAIL þ UVB improve cancer treatment (Ganten et al., 2006; Zhang stimulation. Strikingly, proteasomal inhibition regained et al., 2006; Koschny et al., 2007). An effective offence only a truncated 29 kDa form of XIAP, whereas no to malignant melanoma with the tumor selective ligand accumulation of the full-length protein could be TRAIL (tumor necrosis-related apoptosis inducing detected. This suggests proteolytic processing of XIAP ligand; Walczak et al., 1999) is curtailed by the fact to be responsible for generation of the 29 kDa XIAP that many melanoma cell lines are a priori resistant fragment, which is subsequently committed to protea- against TRAIL-induced apoptosis (Zeise et al., 2004). somal degradation (Figure 1c). This assumption was On investigating 18 melanoma cell lines, we could supported by the observation that apoptosis induc- recently show that TRAIL susceptibility was completely tion—as documented by poly (ADP-ribose) polymerase independent of the tumor progression stage, while only cleavage—clearly coincided with decrease in wt-XIAP, the minority of cell lines responded to TRAIL at all but with accumulation of the c-terminal cleavage (Thayaparasingham et al., 2009). Co-exposure to a non- product D1-241-XIAP in cells ectopically expressing apoptotic UV (ultraviolet) B dose was shown to highly both variants and treated with the proteasome inhibitor synergistically sensitize all melanoma cell lines to MG132 (Figure 1d). Of note, in primary melanocytes, TRAIL-induced apoptosis, providing an excellent tool XIAP depletion did not occur when using identical to study the mechanism underlying TRAIL resistance. TRAIL þ UVB doses and even remained absent when TRAIL insensitivity was based on incomplete caspase-3 treating these primary cells with a high dose of 500 ng/ processing into the catalytic inactive p21 fragment only, ml TRAIL and 400 J/m2 UVB (Figure 1c), proposing a while UVB-induced sensitization coincided with XIAP tumorselective mechanism. depletion, completion of caspase-3 activation yielding Based on biochemical cell-free analyses, caspases have the catalytically active p17, and consequently apoptosis been previously shown to be capable of cleaving XIAP induction (Thayaparasingham et al., 2009). Investigat- and the data provided identified a specific caspase- ing TRAIL resistance of melanoma cells and their cleavage motive in XIAP (Deveraux et al., 1999). synergistic sensitization by sub-lethal UVB, we revealed Accordingly, the contribution of caspases in XIAP a novel mechanism involving caspase-3-induced clea- processing was exemplarily investigated using the vage of XIAP as a prerequisite for its proteasomal melanoma cell line WM-115 (vertical growth phase). degradation. Our data elucidate the nodal point causing Application of pan caspase inhibitors (Q-Val-Asp(non- TRAIL resistance in melanoma and may consequently omethylated)-OPhMe)inhibitor (QVD); z-Val-Ala-Asp help to open new doors for tumorselective killing. (OMe)-FMK (zVAD)) completely prevented depletion of XIAP upon TRAIL þ UVB treatment and also circumvented stabilization of truncated XIAP upon Results concomitant proteasome inhibition (Figure 2a). Using specific inhibitors for caspase-6 (z-Val-Glu-Ile-Asp(OMe)- Caspase-mediated proteolytic cleavage of XIAP promotes FMK, (zVEID)), -8 (z-Ile-Glu-Thr-Asp(OMe)-FMK its proteasomal degradation and sensitization of (zIETD)), -3 (z-Asp-Glu-Val-Asp(OMe)-FMK, (zDEVD)) melanoma cells to TRAIL and -9 (z-Leu-Glu-His-Asp(OMe)-FMK, (zLEHD)), ana- Resistance of melanoma cells to TRAIL-induced lysis of XIAP depletion revealed that any of the tested apoptosis has been shown to be independent of the caspases could potentially be involved in XIAP processing, tumor progression stage