NF-κB-inducing Kinase Expression Research Articles

PUM1-TRAF3 fusion protein activates non-canonical NF-κB signaling via rescued NIK in biliary tract cancer

Discovery and verification of diagnostic or therapeutic biomarkers for biliary tract cancer (BTC) is challenging owing to the low prevalence of the disease. Here, we identified and investigated the clinical impact of a fusion gene, Pumilio1-tumor necrosis factor receptor-associated factor 3 (PUM1-TRAF3), caused by 1;14 chromosomal translocation in BTC. PUM1-TRAF3 was initially identified in the RNA-sequencing of five BTC surgical tissues and confirmed by fluorescence in situ hybridization. Expression of the fusion gene was validated in an expanded cohort (5/55, 9.1%). Establishment and molecular assessment of PUM1-TRAF3 expressing BTC cells revealed that PUM1-TRAF3 activates non-canonical NF-κB signaling via NF-κB-inducing kinase (NIK). Abnormal TRAF3 activity, driven by competitive binding of PUM1-TRAF3 and TRAF3 to NIK, led to NIK rescue followed by P52/RelB nuclear translocation, all of which were reverted by an NIK inhibitor. The elevated expression of NIK and activated NF-κB signaling was observed in the PUM1-TRAF3-expressing regions of patient tissues. Expression of the PUM1-TRAF3 fusion was significantly correlated with strong NIK expression, which is associated with a poorer prognosis for patients with BTC. Overall, our study identifies a new fusion gene, PUM1-TRAF3, that activates NIK and non-canonical NF-κB signaling, which may be beneficial for developing precise treatment strategies for BTC.

Grass carp (Ctenopharyngodon idella) NIK up-regulates the expression of IL-8 by activating the NF-κB canonical pathway

NIK (NF-κB inducing kinase) belongs to the mitogen-activated protein kinase family, which activates NF-κB and plays a vital role in immunology, inflammation, apoptosis, and a series of pathological responses. In NF-κB noncanonical pathway, NIK and IKKα have been often studied in mammals and zebrafish. However, few have explored the relationship between NIK and other subunits of the IKK complex. As a classic kinase in the NF-κB canonical pathway, IKKβ has never been researched with NIK in fish. In this paper, the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) NIK (CiNIK) was first cloned and identified. The expression level of CiNIK in grass carp cells was increased under GCRV stimuli. Under the stimulation of GCRV, poly (I:C), and LPS, the expression of NIK in various tissues of grass carp was also increased. This suggests that CiNIK responds to viral stimuli. To study the relationship between CiNIK and CiIKKβ, we co-transfected CiNIK-FLAG and CiIKKB-GFP into grass carp cells in coimmunoprecipitation and immunofluorescence experiments. The results revealed that CiNIK interacts with CiIKKβ. Besides, the degree of autophosphorylation of CiNIK was enhanced under poly (I:C) stimulation. CiIKKβ was phosphorylated by CiNIK and then activated the activity of p65. The activity change of p65 indicates that NF-κB downstream inflammatory genes will be functioning. CiNIK or CiIKKβ up-regulated the expression of IL-8. It got higher when CiNIK and CiIKKβ coexisted. This paper revealed that NF-κB canonical pathway and noncanonical pathway are not completely separated in generating benefits.

Abstract B049: Uncovering sex-biased immunometabolic roles for NIK in the GBM tumor microenvironment

Abstract The interconnected relationship between the central nervous system (CNS) and the immune system is crucial for brain function. Astrocytes, microglia and infiltrating macrophages perform diverse functions for the maintenance of CNS homeostasis, including inflammatory responses to injury or infection, tissue repair and immune surveillance of cancers such as glioblastoma (GBM). Indeed, these cells comprise up to 50% of GBM tumor mass, yet the impact of their interactions in regulating neuroinflammation and immunosuppression in the GBM tumor microenvironment is not well understood. We have recently found that NF-κB-Inducing Kinase (NIK), an essential activator of the noncanonical NF-κB pathway, governs the mitochondrial metabolic reprogramming that is required for the acquisition of pro-repair bone-marrow-derived macrophage (BMDM) immune effector functions. Although NIK is highly expressed in brain immune cells, including resident macrophages/microglia and astrocytes, little is known regarding NIK-dependent regulatory pathways controlling their immune cell effector functions in brain health and systemic responses to disease. Our preliminary data demonstrate that similar to BMDMs, NIK knockout (KO) microglia and astrocytes have impaired mitochondrial oxidative metabolism and skew towards pro-inflammatory phenotypes. Moreover, NIK KO mice harboring orthotopic syngeneic brain tumors exhibit improved survival and reduced microglia and macrophage tumor infiltration, suggesting that NIK functions in the TME to support immune cell interactions that promote GBM growth. Notably these effects were sex-biased, with male NIK KO mice having a more pronounced survival benefit. Additionally, we observe whole-body metabolic dysfunction in myeloid- and astrocyte-specific conditional NIK knockout mice, implying a potential NIK-dependent feedback relationship between CNS immune/glial cell functions and control of systemic metabolism. Overall, these results are consistent with poorer survival of patients with elevated NIK expression and provides a possible rationale for the higher incidence of GBM in males. Coupled with our previous work demonstrating that NIK has several GBM cell-intrinsic roles to promote tumor invasion and adaptive growth through control of mitochondrial fission and cellular metabolism, these new findings highlight the importance of understanding NIK-dependent immunometabolic functions in the brain. Therapeutic strategies targeting NIK may not only attenuate GBM cell invasion and render them vulnerable to metabolic stress, but also modulate metabolic rewiring of immune cells in the tumor microenvironment to improve patient outcomes. Citation Format: Justin N. Keeney, Caren Stuebe, Kathryn M. Pflug, L. Gerard Toussaint, Raquel Sitcheran. Uncovering sex-biased immunometabolic roles for NIK in the GBM tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr B049.

Transcriptional induction of NF-κB-inducing kinase by E2F4/5 facilitates collective invasion of GBM cells

The prognosis of high-grade gliomas, such as glioblastoma multiforme (GBM), is extremely poor due to the highly invasive nature of these aggressive cancers. Previous work has demonstrated that TNF-weak like factor (TWEAK) induction of the noncanonical NF-κB pathway promotes the invasiveness of GBM cells in an NF-κB-inducing kinase (NIK)-dependent manner. While NIK activity is predominantly regulated at the posttranslational level, we show here that NIK (MAP3K14) is upregulated at the transcriptional level in invading cell populations, with the highest NIK expression observed in the most invasive cells. GBM cells with high induction of NIK gene expression demonstrate characteristics of collective invasion, facilitating invasion of neighboring cells. Furthermore, we demonstrate that the E2F transcription factors E2F4 and E2F5 directly regulate NIK transcription and are required to promote GBM cell invasion in response to TWEAK. Overall, our findings demonstrate that transcriptional induction of NIK facilitates collective cell migration and invasion, thereby promoting GBM pathogenesis.

High expression of NF-κB inducing kinase in the bulge region of hair follicle induces tumor

The bulge region, a reservoir of multipotent stem cells, is possibly responsible for tumorigenesis. NF-κB-inducing kinase (NIK) is a kinase involved in the activation of the noncanonical NF-κB pathway and exhibits positive staining in tumor cells. However, whether high expression of NIK can result in tumorigenesis has not been reported in published papers. By establishing Nik-coe (Nik-stopF/F crossed with Chat-cre) and Nik-soe (Nik-stopF/F crossed with Sox9-cre) mice, we found that overexpression of Nik in the bulge region of hair follicles induced hair follicle loss and tumorigenesis. Furthermore, RNA sequencing, proteomic and phosphopeptide analyses revealed that multiple cancer pathways are involved in tumor formation. Taken together, these findings indicate that constitutive activation of Nik in the bulge region induces tumorigenesis.

NF-κB-Inducing Kinase Governs the Mitochondrial Respiratory Capacity, Differentiation, and Inflammatory Status of Innate Immune Cells.

NF-κB-inducing kinase (NIK), which is essential for the activation of the noncanonical NF-κB pathway, regulates diverse processes in immunity, development, and disease. Although recent studies have elucidated important functions of NIK in adaptive immune cells and cancer cell metabolism, the role of NIK in metabolic-driven inflammatory responses in innate immune cells remains unclear. In this study, we demonstrate that murine NIK-deficient bone marrow-derived macrophages exhibit defects in mitochondrial-dependent metabolism and oxidative phosphorylation, which impair the acquisition of a prorepair, anti-inflammatory phenotype. Subsequently, NIK-deficient mice exhibit skewing of myeloid cells characterized by aberrant eosinophil, monocyte, and macrophage cell populations in the blood, bone marrow, and adipose tissue. Furthermore, NIK-deficient blood monocytes display hyperresponsiveness to bacterial LPS and elevated TNF-α production ex vivo. These findings suggest that NIK governs metabolic rewiring, which is critical for balancing proinflammatory and anti-inflammatory myeloid immune cell function. Overall, our work highlights a previously unrecognized role for NIK as a molecular rheostat that fine-tunes immunometabolism in innate immunity, and suggests that metabolic dysfunction may be an important driver of inflammatory diseases caused by aberrant NIK expression or activity.

BMP-2 functional polypeptides relieve osteolysis via bi-regulating bone formation and resorption coupled with macrophage polarization

Osteolysis caused by wear debris around the prosthesis is the main reason for aseptic loosening. Extending prosthetic service life is still challenging. In this study, we first synthesized a bone morphogenetic protein-2 (BMP-2) functional polypeptide (BMP2pp), and evaluated the effects of BMP2pp on macrophage polarization and impaired osteogenesis caused by titanium (Ti) particles in vitro. Then, we delineated the impact of BMP2pp on bone formation and resorption in a mouse calvarial bone osteolysis model induced by Ti particles. The results showed that BMP2pp not only alleviated the Ti-induced inhibition of osteoblastic differentiation in human placenta-derived mesenchymal stem cells (hPMSCs) but also prevented Ti-induced M1 macrophage polarization and promoted M2 macrophage differentiation in mice. Conditioned medium from BMP2pp-activated macrophages increased the osteogenesis of hPMSCs. The western blot results indicated a significant decrease in the expression of NF-κB inducing kinase (NIK) and phospho-NF-κB p65 in bone marrow-derived macrophages treated with BMP2pp. Furthermore, we clarified the protective effect of BMP2pp on bone formation and the reduction in bone resorption coupled with the immunomodulatory properties of calvarial osteolysis in mice. In summary, BMP2pp ameliorated the Ti-mediated impairment in osteogenic potential of hPMSCs, suppressed the M1 polarization of macrophages by inhibiting the activation of the NF-κB signaling pathway, and ameliorated Ti-induced bone osteolysis. Our research suggests that BMP2pp may be a potential option for treating prosthetic loosening induced by wear debris from prostheses.

TNF receptor-related factor 3 inactivation promotes the development of intrahepatic cholangiocarcinoma through NF-κB-inducing kinase-mediated hepatocyte transdifferentiation.

Intrahepatic cholangiocarcinoma (ICC) is a deadly but poorly understood disease, and its treatment options are very limited. The aim of this study was to identify the molecular drivers of ICC and search for therapeutic targets. We performed a Sleeping Beauty transposon-based in vivo insertional mutagenesis screen in liver-specific Pten -deficient mice and identified TNF receptor-related factor 3 ( Traf3 ) as the most significantly mutated gene in murine ICCs in a loss-of-function manner. Liver-specific Traf3 deletion caused marked cholangiocyte overgrowth and spontaneous development of ICC in Pten knockout and KrasG12D mutant mice. Hepatocyte-specific, but not cholangiocyte-specific, Traf3 -deficient and Pten -deficient mice recapitulated these phenotypes. Lineage tracing and single-cell RNA sequencing suggested that these ICCs were derived from hepatocytes through transdifferentiation. TRAF3 and PTEN inhibition induced a transdifferentiation-like phenotype of hepatocyte-lineage cells into proliferative cholangiocytes through NF-κB-inducing kinase (NIK) up-regulation in vitro. Intrahepatic NIK levels were elevated in liver-specific Traf3 -deficient and Pten -deficient mice, and NIK inhibition alleviated cholangiocyte overgrowth. In human ICCs, we identified an inverse correlation between TRAF3 and NIK expression, with low TRAF3 or high NIK expression associated with poor prognosis. Finally, we showed that NIK inhibition by a small molecule inhibitor or gene silencing suppressed the growth of multiple human ICC cells in vitro and ICC xenografts in vivo. TRAF3 inactivation promotes ICC development through NIK-mediated hepatocyte transdifferentiation. The oncogenic TRAF3-NIK axis may be a potential therapeutic target for ICC.

NIK-SIX1 signalling axis regulates high glucose-induced endothelial cell dysfunction and inflammation

Endothelial dysfunction and inflammation are the main manifestations of diabetes-associated atherosclerosis. This paper studied the roles of NF-κB-inducing kinase (NIK) and sine oculis homeobox homolog 1 (SIX1) in regulating high glucose-induced endothelial dysfunction and inflammation. The expression of NIK and SIX1 in human umbilical vein endothelial cells (HUVECs) was silenced by transfection with the specific shRNAs. HUVECs exposed to high glucose were considered as a cell model of endothelial dysfunction. Expression of NIK and SIX1 following transfection was measured by qRT-PCR and western blotting analysis. The proliferation, migration, and inflammation of HUVECs were evaluated by EdU staining, scratch test, ELISA, and western blotting. High glucose (30 mM) significantly decreased the proliferation and migration of HUVECs. High glucose-induced the expression of adhesion molecules VCAM-1 and ICAM-1. Moreover, high glucose increased the release of IL-1β, IL-6, TNF-α, and MCP-1. Transfection of cells with NIK shRNA significantly reversed the toxic effects of high glucose on HUVECs. Of contrast, SIX1 shRNA accelerated the effects of high glucose on HUVECs. NIK shRNA inhibited the accumulation of RelA, RelB, and p52. Meanwhile, NIK shRNA led to SIX1 downregulation which further induced the activation of the NF-κB pathway. NIK-SIX1 signalling axis was suggested to be critical in the regulation of high glucose-induced endothelial dysfunction and inflammation. SIX1 may function as an immunological gatekeeper to control the excessive inflammation mediated by NIK in diabetes-associated atherosclerosis.

BYHWD Alleviates Inflammatory Response by NIK-Mediated Repression of the Noncanonical NF-κB Pathway During ICH Recovery.

Intracerebral hemorrhage (ICH) is a life-threatening type of stroke that lacks effective treatments. The inflammatory response following ICH is a vital response that affects brain repair and organism recovery. The nuclear factor κB (NF-κB) signaling pathway is considered one of the most important inflammatory response pathways and one of its response pathways, the noncanonical NF-κB signaling pathway, is known to be associated with persistent effect and chronic inflammation. NF-κB–inducing kinase (NIK) via the noncanonical NF-κB signaling plays a key role in controlling inflammation. Here, we investigated potential effects of the traditional Chinese medicine formula Buyang Huanwu Decoction (BYHWD) on inflammatory response in a rat model of ICH recovery by inhibiting the NIK-mediated the noncanonical NF-κB signaling pathway. In the first part, rats were randomly divided into three groups: the sham group, the ICH group, and the BYHWD group. ICH was induced in rats by injecting collagenase (type VII) into the right globus pallidus of rats' brain. For the BYHWD group, rats were administered BYHWD (4.36 g/kg) once a day by intragastric administration until they were sacrificed. Neurological function was evaluated in rats by a modified neurological severity score (mNSS), the corner turn test, and the foot-fault test. The cerebral edema showed the degree of inflammatory response by sacrificed brain water content. Western blot and real-time quantitative reverse transcription PCR tested the activity of inflammatory response and noncanonical NF-κB signaling. In the second part, siRNA treatment and assessment of inflammation level as well as alterations in the noncanonical NF-κB signaling were performed to determine whether the effect of BYHWD on inflammatory response was mediated by suppression of NIK via the noncanonical NF-κB signaling pathway. We show that BYHWD treated rats exhibited: (i) better health conditions and better neural functional recovery; (ii) decreased inflammatory cytokine and the edema; (iii) reduced expression of NIK, a key protein in unregulated the noncanonical NF-κB signaling pathways; (iv) when compared with pretreated rats with NIK targeting (NIK siRNAs), showed the same effect of inhibiting the pathway and decreased inflammatory cytokine. BYHWD can attenuate the inflammatory response during ICH recovery in rats by inhibiting the NIK-mediated noncanonical NF-κB signaling pathway.

NF-κB-inducing kinase maintains T cell metabolic fitness in antitumor immunity.

Metabolic reprograming towards aerobic glycolysis is a pivotal mechanism shaping immune responses. Here we show deficiency in NF-κB-inducing kinase (NIK) impairs glycolysis induction, rendering CD8+ effector T cells hypofunctional in tumor microenvironment. Conversely, ectopic expression of NIK promotes CD8+ T cell metabolism and effector function, thereby profoundly enhancing antitumor immunity and improving the efficacy of T cell adoptive therapy. NIK regulates T cell metabolism via an NF-κB-independent mechanism that involves stabilization of hexokinase 2 (HK2), a rate-limiting enzyme of the glycolytic pathway. NIK prevents autophagic degradation of HK2 through controlling cellular ROS levels, which in turn involves modulation of glucose-6-phosphate dehydrogenase (G6PD), an enzyme mediating production of the antioxidant NADPH. We show that the G6PD-NADPH redox system is important for HK2 stability and metabolism in activated T cells. These findings establish NIK as a pivotal regulator of T cell metabolism and highlight a posttranslational mechanism of metabolic regulation.

Targeting NF-κB-Inducing Kinase (NIK) in Immunity, Inflammation, and Cancer.

NF-κB-inducing kinase (NIK), the essential upstream kinase, which regulates activation of the noncanonical NF-κB pathway, has important roles in regulating immunity and inflammation. In addition, NIK is vital for maintaining cellular health through its control of fundamental cellular processes, including differentiation, growth, and cell survival. As such aberrant expression or regulation of NIK is associated with several disease states. For example, loss of NIK leads to severe immune defects, while the overexpression of NIK is observed in inflammatory diseases, metabolic disorders, and the development and progression of cancer. This review discusses recent studies investigating the therapeutic potential of NIK inhibitors in various diseases.

HuR Affects Proliferation and Apoptosis of Chronic Lymphocytic Leukemia Cells via NF-κB Pathway.

Objective To investigate the effects of HuR protein on the treatment of chronic lymphocytic leukemia (CLL). Methods LCL lymphoblast cells and B lymphocytes were subjected to HuR overexpression (OV) or interference (IV). Western blot was used to observe the protein expression of human tumor necrosis factor-associated factor 1 (TRAF1), human inhibitor of nuclear factor kappa-B kinase α (IKK-α), NF-κB-inducing kinase (NIK), and p52. Flow cytometry was performed to evaluate apoptosis, and the mRNA expression of TRAF1 was examined by quantitative reverse transcription polymerase chain reaction. Immunofluorescence was carried out to visualize the expression of HuR, and the relationship between HuR and TRAF1 was observed by pull-down test. Cell sensitivity to chlorambucil (CLB) and fludarabine (Flu) was assessed by Cell Counting Kit-8. Results The expression of HuR and TRAF1 in LCLs was significantly increased compared to that in B lymphocytes. Compared with the control, HuR OV significantly increased the expression of TRAF1 (P < 0.05), whereas it was significantly decreased in the IV group (P < 0.05). HuR can bind to TRAF1 directly, and the binding rate is positively correlated with HuR expression. After inhibiting HuR, the expression of TRAF1, IKK-α, NIK, p52, pro-Caspase 3, and PARP was significantly upregulated in LCLs and B lymphocytes (P < 0.05), while Caspase 3 was downregulated (P < 0.05). Compared with the control, the proliferation of LCLs and B lymphocytes treated by CLB and Flu decreased significantly after HuR blockade (P < 0.05). Conclusion HuR may be a key protein regulating CLL resistance. After inhibiting HuR, inflammatory response and apoptosis were significantly increased, and the cell sensitivity to CLB and Flu increased, suggesting that inhibiting HuR activity may be a potential strategy to solve the problem of drug resistance in CLL cells.

Zebrafish NIK Mediates IFN Induction by Regulating Activation of IRF3 and NF-κB.

Type I IFN mediates the innate immune system to provide defense against viral infections. NF-κB-inducing kinase (NIK) potentiates the basal activation of endogenous STING, which facilitates the recruitment of TBK1 with the ectopically expressed IRF3 to induce IFN production. Moreover, NIK phosphorylates IKKα and confers its ability to phosphorylate p100 (also known as NF-κB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKα in the cytoplasm and that IKKα phosphorylates the NIK at the residue Thr432, which is different from the mammals. Overexpression of NIK caused the activation of IRF3 and NF-κB, which in turn led to the production of IFN and IFN-stimulated genes (ISGs). Furthermore, the ectopic expression of NIK was observed to be associated with a reduced replication of the fish virus, whereas silencing of endogenous NIK had an opposite effect in vitro. Furthermore, NIK knockdown significantly reduced the expression of IFN and key ISGs in zebrafish larvae after spring viremia of carp virus infection. Additionally, the replication of spring viremia of carp virus was enhanced in NIK knockdown zebrafish larvae, leading to a lower survival rate. In summary, our findings revealed a previously undescribed function of NIK in activating IFN and ISGs as a host antiviral response. These findings may facilitate the establishment of antiviral therapy to combat fish viruses.

Potential mechanism of transcription factor peroxisome proliferator-activated receptor-gamma coactivator-1 beta on promoting osteoclastogenesis

Objective: To investigate the role of transcription factor peroxisome proliferator-activated receptor-gamma coactivator-1 beta (PGC-1β) on osteoclastogenesis and related regulatory mechanism in the mouse monocyte-macrophage cell line (RAW264.7). Methods: PGC-1β expression and location in RAW264.7 cells was detected by immunofluorescence, flow cytometry and western blot analysis with nuclear protein extraction. RAW264.7 cells were transfected with lentivirus for gene silencing or over-expression of PGC-1β and cultured with macrophage colony-stimulating factor and receptor activator for nuclear factor-κB ligand. Cell viability was detected by cell counting kit-8. Cell apoptosis and cell cycle were detected by flow cytometry. Mature osteoclasts and their bone resorption activity were determined by tartrate-resistant acid phosphatase (TRAP) expression and toluidine blue staining. Western blot analysis was performed for detecting dendritic cell-specific transmembrane protein (DC-STAMP), cathepsin K, TRAP and matrix metalloproteinase (MMP)-9 expression, as well as cytoplasmic NF-κB-inducing kinase (NIK) and nuclear RelB. Results: PGC-1β expression was observed in the nuclei of RAW264.7 cells. Down-regulation or overexpression of PGC-1β in RAW264.7 cells did not affect cell viability, apoptosis or cell cycle. Down-regulation of PGC-1β decreased the count of mature osteoclasts (49±21 cells vs. 147±42 cells, P=0.004) and the pit area of bone resorption lacunae (42.11μm(2)±11.30 μm(2) vs. 204.80μm(2)±31.09 μm(2), P<0.001), as well as the expression of cathepsin K, TRAP and MMP-9, but not DC-STAMP. Overexpression of PGC-1β promoted osteoclast differentiation and bone resorption activity, as well as the expression of cathepsin K, TRAP and MMP-9. Down-regulation of PGC-1β suppressed the protein expression of cytoplasmic NIK and nuclear RelB in RAW264.7 cells. Conclusion: PGC-1β can promote the differentiation of RAW264.7 into osteoclasts and improve the bone resorption ability of the cells via activation of NIK/RelB pathway, which might be a promising therapeutic target for osteoporosis.

Lymphotoxin‐β receptor‐NIK signaling induces alternative RELB/NF‐κB2 activation to promote metastatic gene expression and cell migration in head and neck cancer

Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-β (LTβ), receptor LTβR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTβR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTβ, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTβR, NIK, and RELB proteins. Recombinant LTβ, and siRNA depletion of endogenous LTβR and NIK, modulated expression of LTβR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTβ induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTβ-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTβ is expressed. Our findings show that LTβ/LTβR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.

Abstract 3906: Inhibition ofNF-kB Inducing Kinase (NIK) to supressNF-kB alternative pathway and cell migration in HNSCC

Abstract Nuclear factor-κB (NF-κB) activation through the alternative pathway has been implicated in cancer progression and survival, however the regulatory signaling and biological significances are not well understood, especially in head and neck squamous cell carcinoma (HNSCC). NF-κB-inducing kinase (NIK) is a key molecule in the alternative NF-κB pathway, which receives signals from Lymphotoxin beta/Lymphotoxin beta receptor (LTβ/LTβR) to activate Inhibitor-κB kinase α (IKKα) and transcription factors RELB/NF-κB2. In a panel of HNSCC cell lines, we observed that dysregulation of the alternative NF-κB pathway occurred primarily through increased LTβ receptor signaling via NIK and the non-canonical NF-κB subunits. We identified a NIK inhibitor 1, 3[2H, 4H]-Iso-Quinoline Dione, which inhibited cell migration at a low dose range, but exhibited minimal anti-proliferative effect in HPV(-) HNSCC cell lines. The biological effects of the inhibitor were through suppression of the alternative NF-κB signaling. Using Western blot and immunofluorescence staining, we showed that the inhibitor reduced the protein expression and/or phosphorylation of NIK, IKKα, and RELB/NF-κB2 (p52) in the cytoplasm, and/or the protein translocation in the nucleus. The NIK inhibitor also blocked LTβ induced translocation of RELB to the nucleus. The effects of NIK inhibitor were validated by knockdown of NIK using siRNA, which inhibited cell migration and LTβ-induced expression of MET. Met is a hepatocyte growth factor receptor implicated in promoting metastasis, and knockdown of MET slowed down HNSCC cell migration. We further extended the study on a murine oral metastasis cell line MOC2, which is a syngeneic mouse model suitable for testing drug effects on tumor growth and migration in vivo. We found that the NIK inhibitor decreased cell proliferation, migration, and invasion through an extracellular matrix barrier at low concentration ranges. Currently, we have identified a potent and selective small molecule NIK inhibitor, which is orally bioavailable and active in vivo. We plan to test the effects of this NIK inhibitor on HNSCC tumor growth and metastasis in the syngeneic MOC2 model. Our study may shed light on how HNSCC tumors migrate and metastasize to local lymph nodes, where the microenvironment promotes alternative NF-κB activation by LTβ through NIK signaling. Targeting NIK mediated alternative NF-κB activation could be a potential therapeutic strategy for preventing tumor migration and metastasis of HNSCC. (Supported by NIDCD intramural projects ZIA-DC-000016, 74) Citation Format: Rita Das, Paul E. Clavijo, Clint Allen, Anthony Saleh, Zhong Chen, Carter Van Waes. Inhibition ofNF-kB Inducing Kinase (NIK) to supressNF-kB alternative pathway and cell migration in HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3906.

Silencing NIK potentiates anti-VEGF therapy in a novel 3D model of colorectal cancer angiogenesis.

Angiogenesis is essential for colorectal cancer (CRC) progression, as demonstrated by the beneficial clinical effects of therapeutics inhibiting VEGF signaling. However, alternative mechanisms of neovascularization can develop, resulting in treatment failure. Previously we demonstrated NF-κB-inducing kinase (NIK) contributes to pathological angiogenesis. Here, we investigate NIK as a therapeutic target in endothelial cells (EC) in CRC. To determine NIK expression levels in CRC tissues, we immunostained both primary colorectal tumors and tumors metastasized to the liver. Additionally, a 3D tumor-stromal cell interaction model was developed including EC, fibroblasts and CRC cells to study tumor angiogenesis. This model tested efficacy of NIK-targeting siRNA (siNIK) in EC alone or in combination with the anti-VEGF antibody, bevacizumab. Both primary CRC and liver metastases contained blood vessels expressing NIK. In patients receiving chemotherapy plus bevacizumab, immature NIK+ vessels (p < 0.05) were increased as compared to chemotherapy alone. Activation of NIK by lymphotoxin-beta receptor (LTβR) induced increases in pro-angiogenic mediators, including interleukin (IL)-6, IL-8, chemokine (C-X-C motif) ligand (CXCL)1 and CXCL5 in EC and fibroblasts, accompanied by sprouting in the 3D model, which was blocked by siNIK in EC. Treatment with bevacizumab plus siNIK in EC resulted in a synergistic effect and reduced VEGF and bFGF-induced sprouting (p < 0.05). Here, we demonstrate a role for NIK in CRC-associated angiogenesis. Targeting NIK in EC in combination with anti-VEGF antibody bevacizumab may hold therapeutic potential to increase efficiency in blocking tumor neovascularization, either to prevent treatment failure due to activation of accessory pathways such as NF-κB signaling or as a rescue treatment.

Noncanonical NF-κB signaling in microvessels of atherosclerotic lesions is associated with inflammation, atheromatous plaque morphology and myocardial infarction

Background and aimsNeovascularization is associated with atherosclerotic plaque instability and increased chance of myocardial infarction (MI). Patients with chronic inflammatory diseases (CID) have increased risk of atherosclerosis, and evidence demonstrates that NF-κB inducing kinase (NIK)-mediated noncanonical NF-κB signaling in endothelial cells (EC) is linked to inflammation and angiogenesis. Here, we hypothesized NIK may also be activated in EC of atherosclerotic lesion microvessels. MethodsUsing cohorts of atherosclerotic lesions from coronary and carotid arteries, we quantified NIK expression in plaque microvessels and compared it to pathological markers, including inflammatory cell content, plaque characteristics and MI. Differences in gene transcripts were evaluated between stable and ruptured lesions. ResultsNIK+EC were present in both coronary and carotid lesions. In CID patients, plaques with stenosis >40% had an increased number of NIK+EC and higher content of immune cells (p < .05) as compared to controls. Immune cells per NIK+EC were also greater in CID patients (p < .05), with pronounced differences as stenosis increased. In unstable lesions, NIK+EC were elevated as were EC expressing CXCL12 (p < .05). NIK+EC were increased in lesions with lipid content >40% (p < .05) and more abundant in coronary artery lesions implicated in MI (p < .05). These vessels also associated with atheromatous rather than fibrous plaque morphology (p < .05). Transcriptomic profiling demonstrated components of noncanonical NF-κB pathway were also upregulated in ruptured plaques (p < .05). ConclusionsNIK+EC associate with chronic inflammation in advanced lesions and are linked to markers of local inflammation, lipid content, unstable plaque phenotype and development of MI. Therefore, targeting noncanonical NF-κB signaling may hold therapeutic potential for patients with atherosclerosis and cardiovascular disease.

Helicobacter pylori induces direct activation of the lymphotoxin beta receptor and non-canonical nuclear factor-kappa B signaling

The pathogen Helicobacter pylori, which infects half of the world's population, is a major risk factor for the development of gastric diseases including chronic gastritis and gastric cancer. Among H. pylori's virulence factors is the cytotoxin-associated gene pathogenicity island (cagPAI), which encodes for a type IV secretion system (T4SS). The T4SS induces fast canonical nuclear factor-kappa B (NF-κB) signaling, a major factor increasing inflammation, supressing apoptotic cell death and thereby promoting the development of neoplasia. However, H. pylori's capability to mediate fast non-canonical NF-κB signaling is unresolved, despite a contribution of non-canonical NF-κB signaling to gastric cancer has been suggested.We analyzed signaling elements within non-canonical NF-κB in response to H. pylori in epithelial cell lines by immunoprecipitation, immunoblot, electrophoretic mobility shift assay and RNA interference knockdown. In addition, tissue samples of H. pylori-infected patients were investigated by immunohistochemistry.Here, we provide evidence for a T4SS-dependent direct activation of non-canonical NF-κB signaling. We identified the lymphotoxin beta receptor (LTβR) to elicit the fast release of NF-κB inducing kinase (NIK) from the receptor complex leading to non-canonical NF-κB signaling. Further, NIK expression was increased in human biopsies of H. pylori-associated gastritis. Thus, NIK could represent a novel target to reduce Helicobacter pylori-induced gastric inflammation and pathology.