Herman H. Cheung

Activation of noncanonical NF-κB requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2, TRAF3 and the kinase NIK

Recent studies suggest that nuclear factor κB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-κB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.

Degradation of survivin by the X-linked inhibitor of apoptosis (XIAP)-XAF1 complex.

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a putative tumor suppressor in which expression is significantly reduced in human cancer cell lines and primary tumors. The proapoptotic effects of XAF1 have been attributed to both caspase-dependent and -independent means. In particular, XAF1 reverses the anti-caspase activity of XIAP, a physiological inhibitor of apoptosis. We further investigated the function of XAF1 by examining its relationship with other IAPs. Immunoprecipitation studies indicate that XAF1 binds to XIAP, cIAP1, cIAP2, Livin, TsIAP, and NAIP but not Survivin, an IAP that prevents mitotic catastrophe and in which antiapoptotic activity is exerted through direct XIAP interaction and stabilization. We found that overexpressed XAF1 down-regulates the protein expression of Survivin. Under these conditions, Survivin expression was restored in the presence of the proteasome inhibitor MG132 or a XIAP RING mutant that is defective in ubiquitin-protein isopeptide ligase (E3) activity, suggesting that XAF1 interaction activates E3 activity of XIAP and targets Survivin by direct ubiquitination. In addition, RNA interference targeting endogenous XIAP protected Survivin degradation by XAF1. Furthermore, interferon-β-mediated XAF1 induction promoted formation of an endogenous XIAP-XAF1-Survivin complex. This complex facilitated Survivin degradation, which was prevented in XAF1-/- stable clones. Altogether, our study demonstrates that XAF1 mediates Survivin down-regulation through a complex containing XIAP, supporting dual roles for XAF1 in apoptosis and mitotic catastrophe.

Modulation of immune signalling by inhibitors of apoptosis

The inhibitor of apoptosis (IAP) genes are critical regulators of multiple pathways that control cell death, proliferation, and differentiation. Several members of the IAP family regulate innate and adaptive immunity through modulation of signal transduction pathways, cytokine production, and cell survival. The regulation of immunity by the IAPs is primarily mediated through the ubiquitin ligase function of cellular IAP (cIAP)1, cIAP2, and X-linked IAP (XIAP), the targets of which impact nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signalling pathways. In addition, neuronal apoptosis inhibitory protein (NAIP), cIAP1, and cIAP2 modulate innate immune responses through control of the inflammasome complex. This review examines the role of mammalian IAPs in regulating immunity and describes the implications of a new class of pan-IAP antagonists for the treatment of immune disorders.

Smac mimetics and innate immune stimuli synergize to promote tumor death

Smac mimetic compounds (SMC), a class of drugs that sensitize cells to apoptosis by counteracting the activity of inhibitor of apoptosis (IAP) proteins, have proven safe in phase 1 clinical trials in cancer patients. However, because SMCs act by enabling transduction of pro-apoptotic signals, SMC monotherapy may be efficacious only in the subset of patients whose tumors produce large quantities of death-inducing proteins such as inflammatory cytokines. Therefore, we reasoned that SMCs would synergize with agents that stimulate a potent yet safe “cytokine storm.” Here we show that oncolytic viruses and adjuvants such as poly(I:C) and CpG induce bystander death of cancer cells treated with SMCs that is mediated by interferon beta (IFN-β), tumor necrosis factor alpha (TNF-α) and/or TNF-related apoptosis-inducing ligand (TRAIL). This combinatorial treatment resulted in tumor regression and extended survival in two mouse models of cancer. As these and other adjuvants have been proven safe in clinical trials, it may be worthwhile to explore their clinical efficacy in combination with SMCs.

Down-regulation of c-FLIP Enhances Death of Cancer Cells by Smac Mimetic Compound

Smac mimetic compounds (SMC) are novel small molecules being developed for cancer therapy. The mechanism of SMC-induced sensitivity in cancer cells depends on autocrine release of tumor necrosis factor alpha (TNFalpha); however, potential mechanisms of resistance remain unknown. Here, we investigated the molecular profile and cytotoxic responsiveness of a diverse panel of 51 cancer cell lines to combinations of a dimeric SMC (AEG40730), death ligand TNFalpha, and tumor necrosis factor-related apoptosis-inducing ligand. Synergy was seen in combination with death receptor agonists in some cells, although single-agent activity was limited to a fewsensitive lines. Unexpectedly, the majority of cell lines resistant to combinations of SMC-AEG40730 and death ligands expressed caspase-8, FADD, RIP1, and ligand receptors necessary for apoptosis execution. Furthermore, TNFalpha-mediated ubiquitination of RIP1 was repressed by SMC-AEG40730 treatment, leading to the formation of the proapoptosis complex II. However, in resistant cancer cells, SMC-AEG40730 repressed TNFalpha-mediated c-jun-NH(2)-kinase activation and the levels of caspase-8 inhibitor c-FLIP were persistently elevated, in contrast to SMC-responsive cancer cells. Importantly, the silencing of c-FLIP restored SMC sensitivity in previously resistant cancer cells by allowing ligand-mediated activation of caspase-8 and caspase-3 to proceed. Together, these results provide mechanistic insight into the action of SMCs, demonstrating that the deciphering of the relevant molecular signature in cancer cells leads to the prediction of cancer cell responsiveness to SMC treatment. Furthermore, a majority of resistant cancer cells were sensitized to SMC-AEG40730 and TNFalpha by down-regulating c-FLIP, suggesting novel approaches in the use of SMCs and c-FLIP antagonists in treating cancer.

The RING Domain of cIAP1 Mediates the Degradation of RING-bearing Inhibitor of Apoptosis Proteins by Distinct Pathways

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR-mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.

Silencing of the XAF1 gene by promoter hypermethylation in cancer cells and reactivation to TRAIL-sensitization by IFN-β

BackgroundXIAP-associated factor 1 (XAF1) is a putative tumor suppressor that exerts its proapoptotic effects through both caspase-dependent and – independent means. Loss of XAF1 expression through promoter methylation has been implicated in the process of tumorigenesis in a variety of cancers. In this report, we investigated the role of basal xaf1 promoter methylation in xaf1 expression and assessed the responsiveness of cancer cell lines to XAF1 induction by IFN-β.MethodsWe used the conventional bisulfite DNA modification and sequencing method to determine the methylation status in the CpG sites of xaf1 promoter in glioblastoma (SF539, SF295), neuroblastoma (SK-N-AS) and cervical carcinoma (HeLa) cells. We analysed the status and incidence of basal xaf1 promoter methylation in xaf1 expression in non-treated cells as well as under a short or long exposure to IFN-β. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-β-mediated sensitization to TRAIL-induced cell death.ResultsWe found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-β across the four cancer cell lines studied. At the basal level, aberrant promoter methylation was linked to xaf1 gene silencing. After a short exposure, the IFN-β-mediated reactivation of xaf1 gene expression was related to the degree of basal promoter methylation. However, in spite of continued promoter hypermethylation, we find that IFN-β induced a transient xaf1 expression, that in turn, was followed by promoter demethylation upon a prolonged exposure. Importantly, we demonstrated for the first time that IFN-β-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-β-mediated TRAIL sensitivity.ConclusionTogether, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-β treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer.

X-Linked Inhibitor of Apoptosis Antagonism: Strategies in Cancer Treatment

X-linked inhibitor of apoptosis (XIAP), first identified in 1996 ([1][1]), is a member of the IAP family of endogenous caspase inhibitors that blocks the execution of apoptosis. The inhibitory function of XIAP can be reversed by its antagonists, whereas its abundance can be regulated in a proteasome

SMG1 and NIK regulate apoptosis induced by Smac mimetic compounds

Smac mimetic compounds (SMCs) are experimental small molecules that induce tumour necrosis factor alpha (TNFα)-dependent cancer cell death by targeting the inhibitor of apoptosis proteins. However, many cancer cell lines are resistant to SMC-mediated apoptosis despite the presence of TNFα. To add insight into the mechanism of SMC-resistance, we used functional siRNA-based kinomic and focused chemical screens and identified suppressor of morphogenesis in genitalia-1 (SMG1) and NF-κB-inducing kinase (NIK) as novel protective factors. Both SMG1 and NIK prevent SMC-mediated apoptosis likely by maintaining FLICE inhibitory protein (c-FLIP) levels to suppress caspase-8 activation. In SMC-resistant cells, the accumulation of NIK upon SMC treatment enhanced the activity of both the classical and alternative nuclear factor-κB pathways, and increased c-FLIP mRNA levels. In parallel, persistent SMG1 expression in SMC-resistant cells repressed SMC-mediated TNFα-induced JNK activation and c-FLIP levels were sustained. Importantly, SMC-resistance is overcome by depleting NIK and SMG1, which appear to facilitate the downregulation of c-FLIP in response to SMC and TNFα treatment, leading to caspase-8-dependent apoptosis. Collectively, these data show that SMG1 and NIK function as critical repressors of SMC-mediated apoptosis by potentially converging on the regulation of c-FLIP metabolism.

Smac Mimetic Compounds Potentiate Interleukin-1β-mediated Cell Death

Smac mimetic compounds (SMCs) potentiate TNFα-mediated cancer cell death by targeting the inhibitor of apoptosis (IAP) proteins. In addition to TNFα, the tumor microenvironment is exposed to a number of pro-inflammatory cytokines, including IL-1β. Here, we investigated the potential impact of IL-1β on SMC-mediated death of cancer cells. Synergy was seen in a subset of a diverse panel of 21 cancer cell lines to the combination of SMC and IL-1β treatment, which required IL-1β-induced activation of the NF-κB pathway. Elevated NF-κB activity resulted in the production of TNFα, which led to apoptosis dependent on caspase-8 and RIP1. In addition, concurrent silencing of cIAP1, cIAP2, and X-linked IAP by siRNA was most effective for triggering IL-1β-mediated cell death. Importantly, SMC-resistant cells that produced TNFα in response to IL-1β treatment were converted to an SMC-sensitive phenotype by c-FLIP knockdown. Reciprocally, ectopic expression of c-FLIP blocked cell death caused by combined SMC and IL-1β treatment in sensitive cancer cells. Together, our study indicates that a positive feed-forward loop by pro-inflammatory cytokines can be exploited by SMCs to induce apoptosis in cancer cells.