NF-κB-inducing Kinase Research Articles

Mangiferin, a novel nuclear factor kappa B-inducing kinase inhibitor, suppresses metastasis and tumor growth in a mouse metastatic melanoma model

Advanced metastatic melanoma, one of the most aggressive malignancies, is currently without reliable therapy. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone and exerts many beneficial biological activities. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we evaluated the effect of mangiferin on metastasis and tumor growth in a mouse metastatic melanoma model. We found that mangiferin inhibited spontaneous metastasis and tumor growth. Furthermore, mangiferin suppressed the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated NF-κB-inducing kinase (NIK), inhibitor of kappa B kinase (IKK), and inhibitor of kappa B (IκB) and increases the expression of IκB protein in vivo. In addition, we found that mangiferin inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs) in vivo. Mangiferin treatment also increased the expression of cleaved caspase-3, cleaved Poly ADP ribose polymerase-1 (PARP-1), p53 upregulated modulator of apoptosis (PUMA), p53, and phosphorylated p53 proteins, and decreased the expression of Survivin and Bcl-associated X (Bcl-xL) proteins in vivo. These results indicate that mangiferin selectivity suppresses the NF-κB pathway via inhibition of NIK activation, thereby inhibiting metastasis and tumor growth. Importantly, the number of reported NIK selective inhibitors is limited. Taken together, our data suggest that mangiferin may be a potential therapeutic agent with a new mechanism of targeting NIK for the treatment of metastatic melanoma.

NIBP impacts on the expression of E-cadherin, CD44 and vimentin in colon cancer via the NF-κB pathway.

NIBP, a novel nuclear factor-κB (NF-κB)-inducing kinase (NIK) and IκB kinase β (IKKβ) binding protein, directly interacts with NIK and IKKβ, and acts as the 'bridge' of the NF‑κB classical and alternative signaling pathways. However, its influence on epithelial‑mesenchymal transition markers in colon cancer remains to be fully elucidated. The aim of the present study was to investigate the roles of NIBP impacting on the expression of E‑cadherin, CD44 and vimentin. In the present study, the associations between NIBP and E‑cadherin, CD44 and vimentin in clinical samples were analyzed by making pairwise comparisons between normal colon tissue, non‑metastatic colon cancer tissue and metastatic colon cancer tissue. In invitro experiments, after changing the expression of NIBP in cells, the protein expression levels of CD44, vimentin, E‑cadherin were analyzed by western blot analysis. The results revealed that the protein expression levels of NIBP, CD44 and vimentin were markedly increased, and E‑cadherin was markedly decreased, in metastaticcolon cancer tissue compared with normal colon tissue and non‑metastatic colon cancer tissue. Upregulation of NIBP expression decreased the levels of E‑cadherin, whereas the downregulation of NIBP increased E‑cadherin levels, while no significant differences were observed in the levels of CD44 and vimentin. In addition, cells that were treated with the NF‑κB inhibitor, pyrrolidine dithiocarbamate (PDTC), also tended to exhibit increased levels of CD44 and vimentin expression in the NIBP upregulated expression group (29‑NIBP group) compared with the mock group, whereas the expression levels of E‑cadherin, CD44 and vimentin were similar in the NIBP downregulated expression group (116‑NIBPmir group) and the HCT116 blank control group (116‑mock group) on treatment of the cells with tumor necrosis factor‑α. These findings indicated that NIBP, E‑cadherin, CD44 and vimentin are possibly associated with metastasis in colon cancer. When the NF‑κB pathway is not subjected to any interventions, NIBP may predominantly regulate the NF‑κB classical pathway, rather than the alternative pathway. When the classical pathway was completely inhibited, NIBP was able to activate the NF‑κB alternative pathway. NIBP is therefore necessary for the interaction between the NF‑κB classical and alternative pathways. In conclusion, NIBP impacts on the expression levels of E‑cadherin, CD44 and vimentin via the NF‑κB classical and alternative pathways. Therapeutic regimens for patients with colorectal cancer may comprise NIBP inhibitors in the future.

Mangiferin induces apoptosis in multiple myeloma cell lines by suppressing the activation of nuclear factor kappa B-inducing kinase

Mangiferin is a naturally occurring glucosyl xanthone, which induces apoptosis in various cancer cells. However, the molecular mechanism underlying mangiferin-induced apoptosis has not been clarified thus far. Therefore, we examined the molecular mechanism underlying mangiferin-induced apoptosis in multiple myeloma (MM) cell lines. We found that mangiferin decreased the viability of MM cell lines in a concentration-dependent manner. We also observed an increased number of apoptotic cells, caspase-3 activation, and a decrease in the mitochondrial membrane potential. In addition, mangiferin inhibited the nuclear translocation of nuclear factor kappa B (NF-κB) and expression of phosphorylated inhibitor kappa B (IκB) and increased the expression of IκB protein, whereas no changes were observed in the phosphorylation levels of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal protein kinase 1/2 (JNK1/2), and mammalian target of rapamycin (mTOR). The molecular mechanism responsible for mangiferin-induced inhibition of nuclear translocation of NF-κB was a decrease in the expression of phosphorylated NF-κB-inducing kinase (NIK). Moreover, mangiferin decreased the expression of X-linked inhibitor of apoptosis protein (XIAP), survivin, and Bcl-xL proteins. Knockdown of NIK expression showed results similar to those observed with mangiferin treatment. Our results suggest that mangiferin induces apoptosis through the inhibition of nuclear translocation of NF-κB by suppressing NIK activation in MM cell lines. Our results provide a new insight into the molecular mechanism of mangiferin-induced apoptosis. Importantly, since the number of reported NIK inhibitors is limited, mangiferin, which targets NIK, may be a potential anticancer agent for the treatment of MM.

NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

The control of NFκB in CNS neurons appears to differ from that in other cell types. Studies have reported induction of NFκB in neuronal cultures and immunostaining in vivo, but others have consistently detected little or no transcriptional activation by NFκB in brain neurons. To test if neurons lack some component of the signal transduction system for NFκB activation, we transfected cortical neurons with several members of this signaling system along with a luciferase-based NFκB-reporter plasmid; RelA was cotransfected in some conditions. No component of the NFκB pathway was permissive for endogenous NFκB activity, and none stimulated the activity of exogenous RelA. Surprisingly, however, the latter was inhibited by cotransfection of NFκB-inducing kinase (NIK). Fluorescence imaging of RelA indicated that co-expression of NIK sequestered RelA in the cytoplasm, similar to the effect of IκBα. NIK-knockout mice showed elevated expression of an NFκB-reporter construct in neurons in vivo. Cortical neurons cultured from NIK-knockout mice showed elevated expression of an NFκB-reporter transgene. Consistent with data from other cell types, a C-terminal fragment of NIK suppressed RelA activity in astrocytes as well as neurons. Therefore, the inhibitory ability of the NIK C-terminus was unbiased with regard to cell type. However, inhibition of NFκB by full-length NIK is a novel outcome that appears to be specific to CNS neurons. This has implications for unique aspects of transcription in the CNS, perhaps relevant to aspects of development, neuroplasticity, and neuroinflammation. Full-length NIK was found to inhibit (down arrow) transcriptional activation of NFκB in neurons, while it elevated (up arrow) activity in astrocytes. Deletion constructs corresponding to the N-terminus or C-terminus also inhibited NFκB in neurons, while only the C-terminus did so in astrocytes. One possible explanation is that the inhibition in neurons occurs via two different mechanisms, including the potential for a neuron-specific protein (e.g., one of the 14-3-3 class) to create a novel complex in neurons, whereas the C-terminus may interact directly with NFκB. [Structure of NIK is based on Liu J., Sudom A., Min X., Cao Z., Gao X., Ayres M., Lee F., Cao P., Johnstone S., Plotnikova O., Walker N., Chen G., and Wang Z. (2012) Structure of the nuclear factor κB-inducing kinase (NIK) kinase domain reveals a constitutively active conformation. J Biol Chem. 287, 27326-27334); N-terminal lobe is oriented at top].

TNF-α Modulation of Intestinal Tight Junction Permeability Is Mediated by NIK/IKK-α Axis Activation of the Canonical NF-κB Pathway

Tumor necrosis factor (TNF)-α, a key mediator of intestinal inflammation, causes an increase in intestinal epithelial tight junction (TJ) permeability by activating myosin light chain kinase (MLCK; official name MYLK3) gene. However, the precise signaling cascades that mediate the TNF-α-induced activation of MLCK gene and increase in TJ permeability remain unclear. Our aims were to delineate the upstream signaling mechanisms that regulate the TNF-α modulation of intestinal TJ barrier function with the use of invitro and invivo intestinal epithelial model systems. TNF-α caused a rapid activation of both canonical and noncanonical NF-κB pathway. NF-κB-inducing kinase (NIK) and mitogen-activated protein kinase kinase-1 (MEKK-1) were activated in response to TNF-α. NIK mediated the TNF-α activation of inhibitory κB kinase (IKK)-α, and MEKK1 mediated the activation of IKK complex, including IKK-β. NIK/IKK-α axis regulated the activation of both NF-κB p50/p65 and RelB/p52 pathways. Surprisingly, the siRNA induced knockdown of NIK, but not MEKK-1, prevented the TNF-α activation of both NF-κB p50/p65 and RelB/p52 and the increase in intestinal TJ permeability. Moreover, NIK/IKK-α/NF-κB p50/p65 axis mediated the TNF-α-induced MLCK gene activation and the subsequent MLCK increase in intestinal TJ permeability. In conclusion, our data show that NIK/IKK-α/regulates the activation of NF-κB p50/p65 and plays an integral role in the TNF-α-induced activation of MLCK gene and increase in intestinal TJ permeability.

The NIK of time for B cells.

NF-κB-inducing kinase (NIK) is a key mediator of the noncanonical NF-κB signaling pathway, which is critical for B-cell development and function. Although complete deletion of NIK in mice has been shown to result in defective B cells and impaired secondary lymphoid organogenesis, the consequences of deleting NIK exclusively in B cells have not been determined. In this issue of the European Journal of Immunology, Hahn et al. [Eur. J. Immunol. 2016. 46: 732-741] describe mice in which the NF-κB2 pathway mediator, NIK, is deleted at different points in B-cell lineage differentiation and activation. The results show that the survival of mature peripheral B cells, as well as appropriate kinetics of germinal center reactions, rely on noncanonical NF-κB signaling. These findings confirm and extend prior observations implicating a nonredundant role for NF-κB2 downstream of BAFF signaling via BAFF-R, and prompt assessment of the growing literature regarding the relative roles of BCR and BAFF signals in B-cell homeostasis, as well as the downstream pathways responsible.

Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway

A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.

Mono-(2-Ethylhexyl) Phthalate Promotes Pro-Labor Gene Expression in the Human Placenta.

Women exposed to phthalates during pregnancy are at increased risk for delivering preterm, but the mechanism behind this relationship is unknown. Placental corticotropin-releasing hormone (CRH) and cyclooxygenase-2 (COX-2) are key mediators of parturition and are regulated by the non-canonical NF-kB (RelB/p52) signaling pathway. In this study, we demonstrate that one of the major phthalate metabolites, mono-(2-ethylhexyl)-phthalate (MEHP), increased CRH and COX-2 mRNA and protein abundance in a dose-dependent manner in primary cultures of cytotrophoblast. This was coupled with an increase in nuclear import of RelB/p52 and its association with the CRH and COX-2 promoters. Silencing of NF-kB inducing kinase, a central signaling component of the non-canonical NF-kB pathway, blocked MEHP-induced upregulation of CRH and COX-2. These results suggest a potential mechanism mediated by RelB/p52 by which phthalates could prematurely induce pro-labor gene activity and lead to preterm birth.

API2-MALT1 oncoprotein promotes lymphomagenesis via unique program of substrate ubiquitination and proteolysis.

Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin's lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB (NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor (TNF) receptor associated factor (TRAF) 2 and receptor interacting protein 1 (RIP1), two proteins essential for TNF receptor-induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While the intrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for non-canonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase (NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to generate a stable, constitutively active fragment. Thus, API2-MALT1 harnesses multiple unique pathways to achieve deregulated NF-κB activation. Emerging data from our group and others have also detailed additional gain-of-function activities of API2-MALT1 that extend beyond NF-κB activation. Specifically, API2-MALT1 recruits and subverts multiple other signaling factors, including LIM domain and actin-binding protein 1 (LIMA1) and Smac/DIABLO. Like NIK, LIMA1 represents a unique substrate for API2-MALT1 protease activity, but unlike NIK, its cleavage sets in motion a major NF-κB-independent pathway for promoting oncogenesis. In this review, we highlight the most recent results characterizing these unique and diverse gain-of-function activities of API2-MALT1 and how they contribute to lymphomagenesis.

NF-κB-inducing kinase is essential for B-cell maintenance in mice.

NF-κB-inducing kinase (NIK) is a key mediator of the noncanonical NF-κB signaling pathway, which is critical for normal B-cell development and function. It is well established that the complete deletion of NIK in mice results in defective B cells and impaired secondary lymphoid organogenesis. To address the role of NIK deficiency specifically in B cells, we generated a new mouse strain for the conditional deletion of this kinase. Deletion of NIK during B-cell development results in a drastic reduction of mature B cells from the transitional 2 stage on, while B-1 B cells are less affected. Moreover, deletion of NIK in the germinal centers decreases the numbers of germinal center B cells and impairs the ability of NIK-deficient B cells to develop into class-switched cells in vivo. This new mouse strain will be helpful for studying the role of NIK in different cell types of the body.

The NFκB-inducing kinase is essential for the developmental programming of skin-resident and IL-17-producing γδ T cells.

γδ T cells contribute to first line immune defense, particularly through their ability for rapid production of proinflammatory cytokines. The cytokine profile of γδ T cells is hard-wired already during thymic development. Yet, the molecular pathways underlying this phenomenon are incompletely understood. Here we show that signaling via the NFκB-inducing kinase (NIK) is essential for the formation of a fully functional γδ T cell compartment. In the absence of NIK, development of Vγ5(+) dendritic epidermal T cells (DETCs) was halted in the embryonic thymus, and impaired NIK function caused a selective loss of IL-17 expression by γδ T cells. Using a novel conditional mutant of NIK, we could show in vivo that NIK signaling in thymic epithelial cells is essential for the thymic hardwiring of γδ T cell cytokine production.

Enhanced NF-Kb Non-Canonical Signaling Impairs Hematopoietic Stem Cell Self-Renewal

We previously demonstrated that the NF-kB non-canonical signaling way positively and intrinsically regulates hematopoietic stem/progenitor cell (HSPC) self-renewal and maintains stromal/osteoblastic niches (Stem Cells 2012 30:709-18). These results lead us to think that persistent activation of NF-kB non-canonical signaling would have favorable effects on the HSPC pool size and self-renewal capacity. NF-kB-inducing kinase (NIK) plays a critical role in non-canonical NF-kB signaling by directly phosphorylating IKKk. It is constitutively degraded by TRAF3 in unstimulated cells to prevent unwanted NF-kB activation. To investigate the enhanced NF-kB non-canonical signaling specifically in hematopoietic cells, we crossed Vav-Cre mice with a mouse strain in which a mutated form of NIK lacking the TRAF3-binding domain is expressed under the control of the ROSA26 promoter after Cre-mediated deletion of the LoxP-flanked STOP cassette (NIKΔT3Cre mice). In contrast to what we expected in these preliminary studies, the NIKΔT3Cre mice rapidly developed anemia, pancytopenia, with a reduced HSPC pool and marrow cellularity and postnatal lethality, mimicking many of the findings in humans with bone marrow failure syndrome, and different from recently published mice with deficiency in A20, which also activates NF-kB signaling. Furthermore, the NIK activated HSPCs have profoundly impaired engraftment and self-renewal activity after transplantation into wild-type recipients. Further analysis showed that the mutant cells are proliferate faster and predispose to apoptosis than wild type cells. These observations suggest that finely controlled NF-kB activity is crucial for HSC maintenance. Currently, we are focusing on the analysis of the underlying molecular mechanisms. DisclosuresNo relevant conflicts of interest to declare.

Mir-302c mediates influenza A virus-induced IFNβ expression by targeting NF-κB inducing kinase

Little is known about the role of microRNA during influenza A virus (IAV) infection. We observed that NIK 3′UTR luciferase activity was elevated during IAV infection. Further studies demonstrated that miR-302c reduced NIK expression, resulting in the reduction of IFNβ mRNA expression. We found that miR-302c prevented the translocation of NF-κB from the cytosol to the nucleus. Furthermore, IAV infection downregulated miR-302c expression, leading to the activation of IFNβ expression and the inhibition of viral replication. Compared to miR-302c, miR-520e cannot promote viral replication and production, although the two microRNAs target the same site of the NIK 3′UTR. Collectively, our work defines a novel signaling pathway implicated in the control of IFNβ mRNA expression during IAV infection.

Inhibitor of NFκB Kinase Subunit 2 Blockade Hinders the Initiation but Aggravates the Progression of Crescentic GN.

The NFκB transcription factor family facilitates the activation of dendritic cells (DCs) and CD4(+) T helper (Th) cells, which are important for protective adaptive immunity. Inappropriate activation of these immune cells may cause inflammatory disease, and NFκB inhibitors are promising anti-inflammatory drug candidates. Here, we investigated whether inhibiting the NFκB-inducing kinase IKK2 can attenuate crescentic GN, a severe DC- and Th cell-dependent kidney inflammatory disease. Prophylactic pharmacologic IKK2 inhibition reduced DC and Th cell activation and ameliorated nephrotoxic serum-induced GN in mice. However, therapeutic IKK2 inhibition during ongoing disease aggravated the nephritogenic immune response and disease symptoms. This effect resulted from the renal loss of regulatory T cells, which have been shown to protect against crescentic GN and which require IKK2. In conclusion, although IKK2 inhibition can suppress the induction of nephritogenic immune responses in vivo, it may aggravate such responses in clinically relevant situations, because it also impairs regulatory T cells and thereby, unleashes preexisting nephritogenic responses. Our findings argue against using IKK2 inhibitors in chronic GN and perhaps, other immune-mediated diseases.

Dendritic cells require NIK for CD40-dependent cross-priming of CD8+ T cells

Dendritic cells (DCs) link innate and adaptive immunity and use a host of innate immune and inflammatory receptors to respond to pathogens and inflammatory stimuli. Although DC maturation via canonical NF-κB signaling is critical for many of these functions, the role of noncanonical NF-κB signaling via the serine/threonine kinase NIK (NF-κB-inducing kinase) remains unclear. Because NIK-deficient mice lack secondary lymphoid organs, we generated transgenic mice with targeted NIK deletion in CD11c(+) cells. Although these mice exhibited normal lymphoid organs, they were defective in cross-priming naive CD8(+) T cells following vaccination, even in the presence of anti-CD40 or polyinosinic:polycytidylic acid to induce DC maturation. This impairment reflected two intrinsic defects observed in splenic CD8(+) DCs in vitro, namely antigen cross-presentation to CD8(+) T cells and secretion of IL-12p40, a cytokine known to promote cross-priming in vivo. In contrast, antigen presentation to CD4(+) T cells was not affected. These findings reveal that NIK, and thus probably the noncanonical NF-κB pathway, is critical to allow DCs to acquire the capacity to cross-present antigen and prime CD8 T cells after exposure to licensing stimuli, such as an agonistic anti-CD40 antibody or Toll-like receptor 3 ligand.

Induction of IκB-ζ by Epstein-Barr virus latent membrane protein-1 and CD30.

Activation of nuclear factor-κB (NF-κB) in Burkitt's lymphoma (BL) and Hodgkin's lymphoma (HL) cells is important in the transformation and development process of these lymphomas. Epstein-Barr virus (EBV) latent membrane protein-1 (LMP-1) and ligand-independent signaling by overexpressed CD30 are known to cause permanent activation of NF-κB in lymphomas. However, hyperactivation of NF-κB triggers cellular senescence and apoptosis. Here, we show that IκB-ζ, an inducible regulator of NF-κB, is constitutively expressed in BL and HL cell lines. In addition, immunohistochemical staining identified nuclear IκB-ζ‑positive BL cells, and Hodgkin and Reed-Sternberg cells in lymph nodes. Expression of LMP-1 and CD30 increased IκB-ζ expression at the transcriptional level. IκB-ζ promoter was regulated by activation of the NF-κB‑inducing kinase (NIK)/IκB kinase/NF-κB pathway via the carboxyl‑terminal tumor necrosis factor (TNF) receptor‑associated factor (TRAF)-interacting regions of LMP-1 and CD30. Interestingly, IκB-ζ inhibited NF-κB activation by LMP-1 and CD30. The results suggest that NF-κB-induced IκB-ζ negatively modulates NF-κB hyperactivation, resulting in a fine balance that ultimately endows a net evolutionary benefit to the survival of BL and HL cells.

Desflurane preconditioning protects human umbilical vein endothelial cells against anoxia/reoxygenation by upregulating NLRP12 and inhibiting non-canonical nuclear factor-κB signaling.

Volatile anesthetics modulate endothelial cell apoptosis and inhibit nuclear factor-κB (NF-κB) signaling. In this study, we aimed to assess whether desflurane preconditioning protects human umbilical vein endothelial cells (HUVECs) agaist anoxia/reoxygenation (A/R) injury. HUVECs were pre-conditioned with desflurane (1.0 MAC) for 30 min, followed by a 15-min washout, then exposed to 60 min anoxia and 60 min reoxygenation (A/R), and incubated with 10 ng/ml tumor necrosis factor (TNF)-α for 60 min. HUVEC viability and apoptosis were measured by MTT assay and annexin V staining, and immunoblot analysis was used to measure the levels of Smac and cellular inhibitor of apoptosis 1 (cIAP1). NF-κB activation was assessed using the NF-κB signaling pathway real‑time PCR array, and the levels of NF-κB inducing kinase (NIK), p52, IκB kinase (IKK)α, p100, RelB and NLR family, pyrin domain containing 12 (NLRP12) were assessed by immunoblot analysis. Desflurane preconditioning attenuated the effects of A/R and/or A/R plus TNF-α on cell viability, decreasing the levels of Smac and enhancing the levels of of cIAP1 (P<0.05). Preconditioning with desflurane also enhanced the mRNA levels of interleukin (IL)-10 and NLRP12 in the cells exposed to A/R by 2.40- and 2.16‑fold, respectively. The HUVECs exposed to A/R had greater levels of NIK and p100 and reduced levels of p52 and IKKα. Desflurance preconditioning further increased p100 levels, decreased the level of NIK, further decreased p52 levels and further reduced IKKα levels. A/R in combination with TNF-α increased the NIK, IKKα, p100 and RelB levels, and this increase was significantly attenuated by desflurance preconditioning (all P<0.05). Desflurane preconditioning enhanced HUVEC survival and protected the cells against A/R injury, and our results suggested that this process involved the upregulation of NLRP12 and the inhibition of non-canonical NF-κB signaling.

Loss of NDRG2 enhanced activation of the NF-κB pathway by PTEN and NIK phosphorylation for ATL and other cancer development.

The activation of nuclear factor kappa B (NF-κB) signaling has a central role in the development of adult T-cell leukemia/lymphoma (ATL) and many other cancers. However, the activation mechanism of the NF-κB pathways remains poorly understood. Recently, we reported that N-myc downstream-regulated gene 2 (NDRG2) is a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT pathway by promoting the active dephosphorylated form of PTEN at its C-terminus via the recruitment of PP2A. Additionally, the down-regulation of NDRG2 expression promotes the inactive phosphorylated form of PTEN, which results in constitutively active PI3K/AKT signaling in various cancer cell types. Here, we investigated the involvement of NDRG2 in modulating NF-κB signaling. The forced expression of NDRG2 in ATL cells down-regulates not only the canonical pathway by inhibiting AKT signaling but also the non-canonical pathway by inducing NF-κB-inducing kinase (NIK) dephosphorylation via the recruitment of PP2A. Therefore, NDRG2 works as a PP2A recruiter to suppress not only PI3K/AKT signaling but also NF-κB signaling, which is particularly important in host defenses or immune responses to Human T-cell leukemia virus type 1 (HTLV-1) infection. Furthermore, the loss of NDRG2 expression might play an important role in the progression of tumor development after HTLV-1 infection.

Conditional Deletion of NF-κB-Inducing Kinase (NIK) in Adult Mice Disrupts Mature B Cell Survival and Activation.

NF-κB-inducing kinase (NIK) is a primary regulator of the noncanonical NF-κB signaling pathway, which plays a vital role downstream of BAFF, CD40L, lymphotoxin, and other inflammatory mediators. Germline deletion or inactivation of NIK in mice results in the defective development of B cells and secondary lymphoid organs, but the role of NIK in adult animals has not been studied. To address this, we generated mice containing a conditional allele of NIK. Deletion of NIK in adult mice results in decreases in B cell populations in lymph nodes and spleen, similar to what is observed upon blockade of BAFF. Consistent with this, B cells from mice in which NIK is acutely deleted fail to respond to BAFF stimulation in vitro and in vivo. In addition, mice with induced NIK deletion exhibit a significant decrease in germinal center B cells and serum IgA, which is indicative of roles for NIK in additional pathways beyond BAFF signaling. Our conditional NIK-knockout mice may be broadly useful for assessing the postdevelopmental and cell-specific roles of NIK and the noncanonical NF-κB pathway in mice.

Abstract 2353: Noncanonical NF-κB activation mediates STAT3-stimulated IDO up-regulation in myeloid-derived suppressor cells in breast cancer

Abstract Immunotherapy for cancer treatment is achieved through the activation of competent immune effector cells and the inhibition of immunosuppressive cells, such as myeloid-derived suppressor cells (MDSCs). While MDSCs have been shown to contribute to breast cancer development, the mechanism underlying MDSCs-mediated immunosuppression is unclear. We have identified a poorly differentiated MDSC subset in breast cancer suppressing T-cell function through signal transducer and activator of transcription (STAT) 3-dependent indoleamine 2,3 dioxygenase (IDO) up-regulation. Here we investigated the mechanisms underlying aberrant expression of IDO in MDSCs. MDSCs were induced by co-culturing human CD33+ myeloid progenitors with MDA-MB-231 breast cancer cells. Increased STAT3 activation in MDSCs was correlated with activation of the noncanonical nuclear factor-κB (NF-κB) pathway, including increased NF-κB-inducing kinase (NIK) protein level, phosphorylation of cytoplasmic inhibitor of NF-κB kinase (IKK) α and p100, and RelB-p52 nuclear translocation. Blocking STAT3 activation with the small molecule inhibitor JSI-124 significantly inhibited the accumulation of NIK and IDO expression in MDSCs. Knock-down of NIK in MDSCs suppressed IDO expression, but not STAT3 activation. RelB-p52 dimers were found to directly bind to the IDO promoter, leading to IDO expression in MDSCs. IL-6 was found to stimulate STAT3-dependent, NF-κB-mediated IDO up-regulation in MDSCs. Furthermore, significant positive correlation between the numbers of pSTAT3+ MDSCs, IDO+ MDSCs, and NIK+ MDSCs was observed in human breast cancers. These results demonstrate a STAT3-NF-κB-IDO pathway in breast cancer-derived MDSCs, which provides insight into understanding immunosuppressive mechanisms of MDSCs in breast cancer. Citation Format: Jinpu Yu, Yue Wang, Hui Li, Xiubao Ren. Noncanonical NF-κB activation mediates STAT3-stimulated IDO up-regulation in myeloid-derived suppressor cells in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2015-2353