ORIGINAL ARTICLE MnSOD protects colorectal cancer cells from TRAIL-induced apoptosis by inhibition of Smac/DIABLO release A Mohr 1 , C Bu¨ neker 1,2 , RP Gough 1 and RM Zwacka 1 1 National Centre for Biomedical Engineering Science, Molecular Therapeutics Group, National University of Ireland Galway, Galway, Ireland and 2 Division of Gene Therapy, University of Ulm, Ulm, Germany The mitochondrial enzyme manganese superoxide dismu- tase (MnSOD) has been shown to have two faces with regard to its role in tumor development. On the one side, it is well documented that overexpression of MnSOD slows down cancer cell growth, whereas on the other side MnSOD also has a metastasis-promoting activity. We set out to examine the role of MnSOD in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, thought to be a first-line tumor surveillance mechanism and failure to undergo apoptosis might contribute to metastasis formation. We show that over- expression of MnSOD at moderate levels is able to protect cells from TRAIL-induced apoptosis. While caspase-8 activation and Bid cleavage were not affected by MnSOD, we detected a marked decrease in caspase-3 activation pointing to a mitochondrial resistance mechan- ism. Indeed, we found that MnSOD-overexpressing cells showed reduced cytochrome c and no Smac/DIABLO release into the cytosol. The resulting lack of X-linked inhibitor of apoptosis (XIAP) inhibition by cytosolic Smac/DIABLO most likely caused the TRAIL resistance as RNAi against XIAP-rescued caspase-3 activity and TRAIL sensitivity. Our results show that reactive oxygen species are involved in TRAIL-induced Smac/DIABLO release and in TRAIL-triggered apoptosis. Hence, high levels of MnSOD, which decompose and neutralize these reactive oxygen species, might contribute to metastasis formation by allowing disseminated tumor cells to escape from TRAIL-mediated tumor surveillance. As part of TRAIL regimens, adjuvant treatment with XIAP inhibi- tors in the form of Smac/DIABLO mimetics or MnSOD inhibitors might be able to break TRAIL resistance of malignant tumor cells. Oncogene (2008) 27, 763–774; doi:10.1038/sj.onc.1210673; published online 23 July 2007 Keywords: apoptosis; MnSOD; TRAIL; XIAP; Smac/ DIABLO; colon cancer Introduction The tumor-specific apoptosis-inducing activity of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) suggests it is a promising tumoricidal agent (Ashkenazi et al., 1999; Duiker et al., 2006). However, only about 50% of all cancer cells tested so far respond to TRAIL, leaving the other half, including some malignant tumors with high metastasizing poten- tial, resistant to the apoptosis-inducing effects of TRAIL (Vigneswaran et al., 2005; Zhang and Fang, 2005). Moreover, endogenously produced TRAIL has been described as a tumour-surveillance mechanism (Cretney et al., 2002; Takeda et al., 2002; Wajant, 2006) and factors that impair this function by blocking apoptosis in malignant cells can contribute to the escape of metastasizing tumor cells from this TRAIL-mediated control. Hence, a molecular understanding of the resistance mechanisms in tumors is the first step in the identification of strategies that can circumvent these hurdles for TRAIL cancer therapy and possibly more importantly may aid in the development of approaches that promote the inherent antimetastasizing potential of endogenously produced TRAIL. Various TRAIL-resistance mechanisms have been identified stretching from low TRAIL-receptor (TRAIL-R1 and TRAIL-R2), increased levels of the Fas-associated death domain (FADD)-like interleukin- 1b-converting enzyme-inhibitory protein (Thome et al., 1997) or the antiapoptotic caspase-8L splice variant (Mohr et al., 2005) to loss of the proapoptotic Bcl-2 members such as Bax as well as overexpression of the antiapoptotic members Bcl-2 and/or Bcl-x L . In addition, nuclear factor-k B (NF-kB), mitogen-activated protein kinases and decreased Smac/DIABLO release from mitochondria have been implicated in TRAIL resistance in certain types of cancer cells (Zhang and Fang, 2005). We were particularly interested in putative resistance mechanisms that are prominently present in metastasiz- ing tumors and that could explain the marked TRAIL- resistance in malignant tumor cells as compared to cells from early-stage disease. A gene product that has been reported to be particularly highly expressed in metas- tases as compared to early-stage growth, where it is frequently found at low levels, is manganese superoxide dismutase (MnSOD) (Oberley, 2005). MnSOD is a mito- chondrial matrix protein that catalyses the dismutation Received 12 February 2007; revised 31 May 2007; accepted 11 June 2007; published online 23 July 2007 Correspondence: Dr RM Zwacka or A Mohr, National Centre for Biomedical Engineering Science, Molecular Therapeutics Group, National University of Ireland Galway, University Road, Galway, Ireland. E-mails: ralf.zwacka@nuigalway.ie or andrea.mohr@nuigalway.ie Oncogene (2008) 27, 763–774 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc