The transcription factor SP3 drives TNF-α expression in response to Smac mimetics.

The controlled production and downstream signaling of the inflammatory cytokine tumor necrosis factor-α (TNF-α) are important for immunity and its anticancer effects. Although chronic stimulation with TNF-α is detrimental to the health of the host in several autoimmune and inflammatory disorders, TNF-α-contrary to what its name implies-leads to cancer formation by promoting cell proliferation and survival. Smac mimetic compounds (SMCs), small-molecule antagonists of inhibitor of apoptosis proteins (IAPs), switch the TNF-α signal from promoting survival to promoting death in cancer cells. Using a genome-wide siRNA screen to identify factors required for SMC-to-TNF-α-mediated cancer cell death, we identified the transcription factor SP3 as a critical molecule in both basal and SMC-induced production of TNF-α by engaging the nuclear factor κB (NF-κB) transcriptional pathway. Moreover, the promotion of TNF-α expression by SP3 activity confers differential sensitivity of cancer versus normal cells to SMC treatment. The key role of SP3 in TNF-α production and signaling will help us further understand TNF-α biology and provide insight into mechanisms relevant to cancer and inflammatory disease.

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-ß), 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.

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 mimetic compounds potentiate interleukin-1beta-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-1ß. Here, we investigated the potential impact of IL-1ß 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-1ß treatment, which required IL-1ß-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-1ß-mediated cell death. Importantly, SMC-resistant cells that produced TNFα in response to IL-1ß 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-1ß 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.

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.

Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK.

Recent studies suggest that nuclear factor kappaB-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-kappaB 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.

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.

The role of XAF1 in cancer.

The inhibitors of apoptosis (IAP) proteins have emerged as important cancer targets. The cellular control of IAP expression is regulated by survival signaling pathways and by a variety of known intrinsic antagonists. Among these antagonists, the X-linked IAP-associated factor (XAF)1 is unique in its control of IAP function and in its ability to sensitize cancer cells to apoptosis. Studies have demonstrated that XAF1 is strongly pro-apoptotic, is inducible by IFN and is a tumor suppressor. Thus, this antagonist may have significant value in the treatment of cancer.

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-beta-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.

Isolation and characterization of LCHN: a novel factor induced by transient global ischemia in the adult rat hippocampus.

Using mRNA differential display to identify cerebral ischemia-responsive mRNAs, we isolated and cloned a cDNA derived from a novel gene, that has been designated LCHN. Antisense mRNA in situ hybridization and immunoblotting confirmed LCHN expression to be induced in the rat hippocampus following transient forebrain ischemia. The deduced amino acid sequence of the novel LCHN cDNA contains an open reading frame of 455 amino acids, encoding a protein with a predicted molecular mass of approximately 51 kDa. Although LCHN is highly conserved between rat, mouse, and human, the deduced amino acid sequence of LCHN does not possess significant homology to other known genes. LCHN immunoreactivity is detected within the somatodendritic compartment of neurons, is also present on dendritic growth cones, but is not detected on astrocytes. The induction of LCHN in the hippocampus following ischemic injury may have functional consequences, as the ectopic over-expression of LCHN generated neurons with longer and more branched axons and dendrites. Taken together, these data suggest that LCHN could play a role in neuritogenesis, as well as in neuronal recovery and/or restructuring in the hippocampus following transient cerebral ischemia.

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

BACKGROUND: XIAP-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-beta. METHODS: We 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-beta. Stable XAF1 glioblastoma knock-down cell lines were established to characterize the direct implication of XAF1 in IFN-beta-mediated sensitization to TRAIL-induced cell death. RESULTS: We found a strong variability in xaf1 promoter methylation profile and responsiveness to IFN-beta 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-beta-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-beta 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-beta-mediated reactivation of endogenous XAF1 plays a critical role in TRAIL-induced cell death since XAF1 knock-down cell lines completely lost their IFN-beta-mediated TRAIL sensitivity. CONCLUSION: Together, these results suggest that promoter demethylation is not the sole factor determining xaf1 gene induction under IFN-beta treatment. Furthermore, our study provides evidence that XAF1 is a crucial interferon-stimulated gene (ISG) mediator of IFN-induced sensitization to TRAIL in cancer.

Involvement of caspase-2 and caspase-9 in endoplasmic reticulum stress-induced apoptosis: a role for the IAPs.

Dysregulation of apoptosis is involved in a wide spectrum of disease ranging from proliferative to degenerative disorders. An emerging area of study in apoptosis is the critical contribution of the endoplasmic reticulum (ER) in both mitochondrial and ER specific apoptosis pathways. Here we show that brefeldin A and tunicamycin-mediated ER stress lead to caspase-dependent apoptosis involving caspase-2. Confocal microscopy and subcellular fractionation indicate that caspase-2 is localized to the ER, and following ER stress, the processing of caspase-2 and -9 is an early event preceding the activation of caspase-3 and -7 and the cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP). Inhibition and silencing of either caspase-2 or caspase-9 suppress ER stress-induced apoptosis, as demonstrated by annexin V binding. Similarly, transduction with an adenovirus encoding either Inhibitors of Apoptosis (IAP) protein HIAP1/c-IAP2 or HIAP2/c-IAP1 also suppresses ER stress-induced apoptosis. However, among HIAP1, HIAP2 and XIAP, only HIAP2 binds and inhibits caspase-2. Our results thus indicate a novel mechanism by which HIAP2 can regulate ER-initiated apoptosis by modulating the activity of caspase-2.

Abnormalities of cell structures in tumors: apoptosis in tumors.

A conceptual shift has occurred in recent years from considering cancer as simply a disease of deregulated cell proliferation to a view that incorporates the aberrant control of apoptosis into the equation. Apoptosis is an organized, genetically programmed cell death process by which multicellular organisms specifically destroy, dismantle and dispose of cells. In cancer cells, this tightly controlled process is suppressed by genetic lesions, allowing cancer cells to survive beyond their normal life span even in hostile environments that are prone to hypoxia and lack many trophic factor supports. In the last two decades, cancer researchers have made great strides in our understanding of the underlying molecular mechanism of apoptosis in chemoresistance generation and tumorigenesis. This tremendous increase in our knowledge of apoptosis in tumors has greatly impacted our perspective on carcinogenesis. Key regulators of apoptosis such as members of the Inhibitors of Apoptosis family and Bcl-2 family have been shown to play a pivotal role in allowing most cancer cells to escape apoptosis. The identification of specific targets involved in the suppression of apoptosis in cancer cells has facilitated the design and development of therapeutic strategies based on rational molecular approaches that aim to modulate apoptotic pathways. Many promising apoptosis-dependent strategies have been translated into clinical trials in the continued assessment of regimens that can effectively eradicate cancers.

Lateral hypothalamic signaling mechanisms underlying feeding stimulation: differential contributions of Src family tyrosine kinases to feeding triggered either by NMDA injection or by food deprivation.

In rats, feeding can be triggered experimentally using many approaches. Included among these are (1) food deprivation and (2) acute microinjection of the neurotransmitter l-glutamate (Glu) or its receptor agonist NMDA into the lateral hypothalamic area (LHA). Under both paradigms, the NMDA receptor (NMDA-R) within the LHA appears critically involved in transferring signals encoded by Glu to stimulate feeding. However, the intracellular mechanisms underlying this signal transfer are unknown. Because protein-tyrosine kinases (PTKs) participate in NMDA-R signaling mechanisms, we determined PTK involvement in LHA mechanisms underlying both types of feeding stimulation through food intake and biochemical measurements. LHA injections of PTK inhibitors significantly suppressed feeding elicited by LHA NMDA injection (up to 69%) but only mildly suppressed deprivation feeding (24%), suggesting that PTKs may be less critical for signals underlying this feeding behavior. Conversely, food deprivation but not NMDA injection produced marked increases in apparent activity for Src PTKs and in the expression of Pyk2, an Src-activating PTK. When considered together, the behavioral and biochemical results demonstrate that, although it is easier to suppress NMDA-elicited feeding by PTK inhibitors, food deprivation readily drives PTK activity in vivo. The latter result may reflect greater PTK recruitment by neurotransmitter receptors, distinct from the NMDA-R, that are activated during deprivation-elicited but not NMDA-elicited feeding. These results also demonstrate how the use of only one feeding stimulation paradigm may fail to reveal the true contributions of signaling molecules to pathways underlying feeding behavior in vivo.

Translational induction of the inhibitor of apoptosis protein HIAP2 during endoplasmic reticulum stress attenuates cell death and is mediated via an inducible internal ribosome entry site element.

Prolonged endoplasmic reticulum (ER) stress leads to activation of caspases and cell death. The inhibitor of apoptosis (IAP) proteins are intrinsic inhibitors of apoptosis by virtue of inhibiting distinct caspases and are, therefore, critical regulators of cell death. Here we demonstrate that the expression of one member of the IAP family, HIAP2, is induced in response to ER stress and attenuates ER stress-induced cell death. The induction of HIAP2 is executed at the level of protein synthesis and is mediated by an inducible internal ribosome entry site (IRES) element. The triggering of ER stress results in caspase-mediated proteolytic processing of eukaryotic initiation factor p97/DAP5/NAT1, producing a fragment that specifically activates HIAP2 IRES. These data suggest an existence of a novel mechanism that regulates apoptotic response in ER stress.

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor.

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N-methyl-d-aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four-vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src-family kinases (SFKs) and Pyk2 were increased in post-synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post-ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia-induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia-induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia-induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src-family kinases.