Abstract. Toll-like receptors (TLRs) as well as the receptors for tumor necrosis factor (TNF-R) and interleukin-1 (IL-1R) play an important role in innate immunity by regulating the activity of distinct transcription factors such as nuclear factor-kB (NF-kB). TLR, IL-1R and TNF-R signaling to NF-kB converge on a common IkB kinase complex that phosphorylates the NF-kB inhibitory protein IkBa. However, upstream signaling components are in large part receptor-specific. Nevertheless, the principles of signaling are similar, involving the recruitment of specific adaptor proteins and the activation of kinase cascades in which protein-protein interactions are controlled by poly-ubiquitination. In this review, we will discuss our current knowledge of NF-kB signaling in response to TLR-4, TNF-R and IL-1R stimulation, with a special focus on the similarities and dissimilarities among these pathways.Keywords. Toll-like receptor 4, interleukin-1, tumor necrosis factor, NF-kB, signal transduction. NF-kB, does it still need to be introduced?Nuclear factor kB (NF-kB) is the generic name of a family of transcription factors that regulate the expression of a large number of genes involved in immune and inflammatory responses, as well as in cell survival, cell proliferation and cell differentiation. NFkB transcription factors are activated in response to various stimuli, including cytokines, infectious agents, injury and other stressful conditions requiring rapid reprogramming of gene expression. Inappropriate activation of the NF-kB signaling pathway is implicated in the pathogenesis of chronic inflammation and autoimmunity, certain hereditary disorders and various cancers. In mammals, the NF-kB family consists of five proteins sharing a highly conserved Rel homology domain: c-Rel, RelB, p65 (= RelA), p105 (= NF-kB1) and p100 (=NF-kB2). The first three contain Cterminal transactivation domains, while the others share a long C-terminal domain with multiple copies of ankyrin repeats, which inhibit their activation.

The IkB kinases (IKKs) IKK-a and IKK-b, and the IKK-related kinases TBK1 and IKK-e, have essential roles in innate immunity through signal-induced activation of NF-kB, IRF3 and IRF7, respectively. Although the signaling events within these pathways have been extensively studied, the mechanisms of IKK and IKK-related complex assembly and activation remain poorly defined. Recent data provide insight into the requirement for scaffold proteins in complex assembly; NF-kB essential modulator coordinates some IKK complexes, whereas TANK, NF-kB-activating kinase-associated protein 1 (NAP1) or similar to NAP1 TBK1 adaptor (SINTBAD) assemble TBK1 and IKK-e complexes. The different scaffold proteins undergo similar post-translational modifications, including phosphorylation and non-degradative polyubiquitylation. Moreover, increasing evidence indicates that distinct scaffold proteins assemble IKK, and potentially TBK1 and IKK-e subcomplexes, in a stimulus-specific manner, which might be a mechanism to achieve specificity. Review GlossaryCaspase-recruitment domain (CARD): the CARD is found in some initiator caspases, but also in some adaptor proteins, and mediates protein-protein interactions. Classical and alternative NF-kB-activating pathways: the classical pathway is triggered by various stimuli, including proinflammatory cytokines and TLR ligands, and leads to the activation of the IKK complex that includes IKK-a and IKK-b and also the scaffold protein NEMO. This complex targets the inhibitory IkBa protein for phosphorylation, which is followed by its degradation through the proteasome pathway. NF-kB heterodimers (typically composed of p50 and p65) subsequently move into the nucleus to drive the expression of proinflammatory molecules and chemokines. The alternative pathway is triggered by stimuli such as lymphotoxin-b and requires the kinase NIK in addition to an IKK-a homodimer. NEMO is dispensable for this pathway to be activated. The targeted inhibitory molecule is p100 instead of IkBa, and the NFkB heterodimers are typically composed of p52 and RelB. The target genes of this pathway are required for adaptive immunity. Conventional myeloid and plasmacytoid dendritic cells: dendritic cells (DCs) take up antigens, are activated and migrate to lymphoid tissues in order to present the antigenic peptides on the MHC molecules. They can be broadly divided into plasmacytoid DCs (pDCs) and conventional myeloid DCs, based on the expression of a variety of cell surface markers and their responses to pathogen molecules. pDCs are defined as a subset of cells, the appearance under the microscope of which is similar to that of plasmablasts. These cells are the main producers of type I IFNs in response to viral infections. CpG DNAs: CpG DNAs are DNA oligodeoxynucleotide sequences that include a cytosine-guanosine sequence and some flanking nucleotides. The CpG DNAs induce innate immunity through binding to the TLR9 receptor. Cytosolic NF-kB and IRF activating pathways: these pathways include the RIG-I family (comprising MDA5 and RI...

Type I interferon gene induction relies on IKK-related kinase TBK1 and IKK⑀-mediated phosphorylations of IRF3/7 through the Toll-like receptor-dependent signaling pathways. The scaffold proteins that assemble these kinase complexes are poorly characterized. We show here that TANK/I-TRAF is required for the TBK1-and IKK⑀-mediated IRF3/7 phosphorylations through some Toll-like receptor-dependent pathways and is part of a TRAF3-containing complex. Moreover, TANK is dispensable for the early phase of doublestranded RNA-mediated IRF3 phosphorylation. Interestingly, TANK is heavily phosphorylated by TBK1-IKK⑀ upon lipopolysaccharide stimulation and is also subject to lipopolysaccharide-and TBK1-IKK⑀-mediated Lys 63 -linked polyubiquitination, a mechanism that does not require TBK1-IKK⑀ kinase activity. Thus, we have identified TANK as a scaffold protein that assembles some but not all IRF3/7-phosphorylating TBK1-IKK⑀ complexes and demonstrated that these kinases possess two functions, namely the phosphorylation of both IRF3/7 and TANK as well as the recruitment of an E3 ligase for Lys 63 -linked polyubiquitination of their scaffold protein, TANK.

Osteopontin (OPN) is a secreted protein involved in most aspects of tumor progression and metastasis development. Elevated OPN expression has been reported in multiple types of cancer including glioblastoma (GBM), the highest grade and most aggressive brain tumor. GBMs contain a subpopulation of glioma-initiating cells (GICs) implicated in progression, therapeutic resistance and recurrence. We have previously demonstrated that OPN silencing inhibited GBM cell growth in vitro and in vivo. Moreover, activation of CD44 signaling upon OPN ligation has been recently implicated in the acquisition of a stem cell phenotype by GBM cells. The present study is aimed to explore OPN autocrine function using shRNA silencing strategy in GICs enriched from GBM cell lines and a human primary GBM grown in EGF and bFGF defined medium. The removal of these growth factors and addition of serum induced a significant loss of OPN expression in GICs. We showed that OPN-silenced GICs were unable to grow as spheres and this capacity was restored by exogenous OPN. Importantly, the expression of Sox2, Oct3/4 and Nanog, key stemness transcription factors, was significantly decreased in GICs upon OPN targeting. We identified Akt/ mTOR/p70S6K as the main signaling pathway triggered following OPN-mediated EGFR activation in GICs. Finally, in an orthotopic xenograft mouse model, the tumorigenic potential of U87-MG sphere cells was completely abrogated upon OPN silencing. Our demonstration of endogenous OPN major regulatory effects on GICs stemness phenotype and tumorigenicity implies a greater role than anticipated for OPN in GBM pathogenesis from initiation and progression to probable recurrence.

Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikke in Wnt-driven tumor development. We found that Ikke was activated in intestinal tumors forming upon loss of the tumor suppressor Apc. Genetic ablation of Ikke in b-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikke to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikke was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding proinflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikke-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikke phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikke to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. Cancer Res; 76(9);

Endosomes have important roles in intracellular signal transduction as a sorting platform. Signaling cascades from TLR engagement to IRF3-dependent gene transcription rely on endosomes, yet the proteins that specifically recruit IRF3-activating molecules to them are poorly defined. We show that adaptor protein containing a pleckstrin-homology domain, a phosphotyrosine-binding domain, and a leucine zipper motif (APPL)1, an early endosomal protein, is required for both TRIF- and retinoic acid–inducible gene 1–dependent signaling cascades to induce IRF3 activation. APPL1, but not early endosome Ag 1, deficiency impairs IRF3 target gene expression upon engagement of both TLR3 and TLR4 pathways, as well as in H1N1-infected macrophages. The IRF3-phosphorylating kinases TBK1 and IKKε are recruited to APPL1 endosomes in LPS-stimulated macrophages. Interestingly, APPL1 undergoes proteasome-mediated degradation through ERK1/2 to turn off signaling. APPL1 degradation is blocked when signaling through the endosome is inhibited by chloroquine or dynasore. Therefore, APPL1 endosomes are critical for IRF3-dependent gene expression in response to some viral and bacterial infections in macrophages. Those signaling pathways involve the signal-induced degradation of APPL1 to prevent aberrant IRF3-dependent gene expression linked to immune diseases.

<p>This file includes some material and methods with corresponding references, Supplementary figure legends as well as 14 supplementary figures</p>

<div>Abstract<p>Constitutive Wnt signaling promotes intestinal cell proliferation, but signals from the tumor microenvironment are also required to support cancer development. The role that signaling proteins play to establish a tumor microenvironment has not been extensively studied. Therefore, we assessed the role of the proinflammatory Ikk-related kinase Ikkϵ in Wnt-driven tumor development. We found that Ikkϵ was activated in intestinal tumors forming upon loss of the tumor suppressor <i>Apc</i>. Genetic ablation of Ikkϵ in β-catenin-driven models of intestinal cancer reduced tumor incidence and consequently extended survival. Mechanistically, we attributed the tumor-promoting effects of Ikkϵ to limited TNF-dependent apoptosis in transformed intestinal epithelial cells. In addition, Ikkϵ was also required for lipopolysaccharide (LPS) and IL17A-induced activation of Akt, Mek1/2, Erk1/2, and Msk1. Accordingly, genes encoding pro-inflammatory cytokines, chemokines, and anti-microbial peptides were downregulated in Ikkϵ-deficient tissues, subsequently affecting the recruitment of tumor-associated macrophages and IL17A synthesis. Further studies revealed that IL17A synergized with commensal bacteria to trigger Ikkϵ phosphorylation in transformed intestinal epithelial cells, establishing a positive feedback loop to support tumor development. Therefore, TNF, LPS, and IL17A-dependent signaling pathways converge on Ikkϵ to promote cell survival and to establish an inflammatory tumor microenvironment in the intestine upon constitutive Wnt activation. <i>Cancer Res; 76(9); 2587–99. ©2016 AACR</i>.</p></div>