Recurrent Overexpression of c-IAP2 in EBV-Associated Nasopharyngeal Carcinomas: Critical Role in Resistance to Toll-like Receptor 3-Mediated Apoptosis

Abstract

The oncogenic process leading to nasopharyngeal carcinoma (NPC) requires the combination of genetic and epigenetic alterations, latent infection by the Epstein-Barr virus and local inflammation. A transcriptome analysis of NPC xenografts identified the gene encoding the cellular inhibitor of apoptosis protein 2 (c-IAP2) among the top five most intensely expressed. Consistently, the very high levels of the c-IAP2 protein were detected in 11 of 13 NPC biopsies. RMT 5265, a structural analog of second mitochondria-derived activator of caspase (SMAC), induced the rapid degradation of c-IAP2 in nasopharyngeal epithelial cells, whether malignant or not, but blocked clonal cell growth in NPC cells only. In short-term experiments, RMT 5265 induced apoptosis in a fraction of NPC cells, and this apoptosis was dramatically enhanced when RMT 5265 was combined with Toll-like receptor 3 (TLR3) stimulation. By contrast, the cooperative effect with tumor necrosis factor alpha was only marginal. The apoptosis induced by the combination of RMT 5265 and TLR3 stimulation was mediated by caspase-8 and associated with a decrease in the cellular content of the long isoform of FLICE-like inhibitory protein. Similar caspase-8 activation was obtained when siRNA knockdown of c-IAP2 was combined with TLR3 stimulation. In conclusion, c-IAP2 has a specific protective function in NPC cells challenged by TLR3 agonists. This protective function is probably important to make NPC cells tolerant to their own production of small viral RNAs, which are potential agonists of TLR3. Our data will help to design a rational use of IAP inhibitors in NPC patients.

dsRNA induces apoptosis through an atypical death complex associating TLR3 to caspase-8

“…TLR3 also signals inflammation in normal bronchial and nasopharyngeal epithelial cells that, however, appear insensitive to poly(I:C)-induced apoptosis, even in the presence of Smac mimetic (our unpublished data and Friboulet et al 7 ). Thus, cIAPmediated RIP1 ubiquitination alone is unlikely to explain resistance to TLR3-triggered apoptosis in normal epithelial cells.…”

“…In addition, we and others have recently demonstrated that TLR3 can trigger caspase-8-dependent apoptosis in human cancers. [5][6][7] Related to this proapoptotic function, we also found that TLR3 expressed by breast cancer cells is a biomarker for the therapeutic efficacy of dsRNA. 8 Unlike classical death receptors (DRs), TLR3 lacks a death domain (DD) and thus the molecular basis of dsRNA-triggered apoptosis through caspase-8 remains an open question although recent reports indicate that TRIF and RIP1 are required for Poly(I:C) to engage the apoptotic machinery.…”

“…12,13 Conversely, cellular inhibitor of apoptosis protein (cIAP)1 and cIAP2 have been described as negative regulators of TLR3-induced apoptosis. 7,9,10 These E3 ubiquitin ligases were recently shown to inhibit the spontaneous formation of a molecular complex termed 'Ripoptosome', containing caspase-8, caspase-10, FADD, cFLIP, and RIP1 that associates with TRIF after Poly(I:C) stimulation. 10,14 While formation of this complex clearly involves TLR3 signaling, evidence of the molecular assembly of this machinery to TLR3 is lacking.…”

cIAP1 and TAK1 protect cells from TNF-induced necrosis by preventing RIP1/RIP3-dependent reactive oxygen species production

“…18 IAP family members protect cells from apoptosis by inhibiting caspases and by regulating RIP1 ubiquitination status. 12,13,16,30,31 In addition, IAPs have been implicated in several RIP1-dependent apoptotic triggers (such as stimulation of TNFR1, Fas, or toll-like receptor 3 (TLR3)) [14][15][16]18,[32][33][34] that can also induce necrotic cell death under certain conditions. We found that the IAP antagonist BV6 greatly sensitized L929 cells to TNF-induced necrotic cell death, but not to necrosis induced by poly(I:C) þ IFNb, anti-Fas þ zVADfmk, or H 2 O 2 .…”

Dual effects of Helicobacter pylori vacuolating cytotoxin on human eosinophil apoptosis in early and late periods of stimulation

“…siRNA against c-IAP2 (Birc3) was purchased from Gibco-Invitrogen (Stealth Select RNAi TM siRNA, HSS100562, Carlsbad, CA, USA) [50]. Transfection was performed according to the procedure described previously [27,51].…”