P100 Processing Research Articles

Quantitative Dissection and Modeling of the NF-κB p100-p105 Module Reveals Interdependent Precursor Proteolysis

The mechanisms that govern proteolytic maturation or complete destruction of the precursor proteins p100 and p105 are fundamental to homeostasis and activation of NF-κB; however, they remain poorly understood. Using mass-spectrometry-based quantitative analysis of noncanonical LTβR-induced signaling, we demonstrate that stimulation induces simultaneous processing of both p100 and p105. The precursors not only form hetero-oligomers but also interact with the ATPase VCP/p97, and their induced proteolysis strictly depends on the signal response domain (SRD) of p100, suggesting that the SRD-targeting proteolytic machinery acts in cis and in trans. Separation of cellular pools by isotope labeling revealed synchronous dynamics of p105 and p100 proteolysis. The generation of p50 and p52 from their precursors depends on functional VCP/p97. We have developed quantitative mathematical models that describe the dynamics of the system and predict that p100-p105 complexes are signal responsive.

P100/IκBδ sequesters and inhibits NF-κB through kappaBsome formation

Degradation of I kappaB (κB) inhibitors is critical to activation of dimeric transcription factors of the NF-κB family. There are two types of IκB inhibitors: the prototypical IκBs (IκBα, IκBβ, and IκBε), which form low-molecular-weight (MW) IκB:NF-κB complexes that are highly stable, and the precursor IκBs (p105/IκBγ and p100/IκBδ), which form high-MW assemblies, thereby suppressing the activity of nearly half the cellular NF-κB [Savinova OV, Hoffmann A, Ghosh G (2009) Mol Cell 34(5):591-602]. The identity of these larger assemblies and their distinct roles in NF-κB inhibition are unknown. Using the X-ray crystal structure of the C-terminal domain of p100/IκBδ and functional analysis of structure-guided mutants, we show that p100/IκBδ forms high-MW (IκBδ)4:(NF-κB)4 complexes, referred to as kappaBsomes. These IκBδ-centric "kappaBsomes" are distinct from the 2:2 complexes formed by IκBγ. The stability of the IκBδ tetramer is enhanced upon association with NF-κB, and hence the high-MW assembly is essential for NF-κB inhibition. Furthermore, weakening of the IκBδ tetramer impairs both its association with NF-κB subunits and stimulus-dependent processing into p52. The unique ability of p100/IκBδ to stably interact with all NF-κB subunits by forming kappaBsomes demonstrates its importance in sequestering NF-κB subunits and releasing them as dictated by specific stimuli for developmental programs.

Nuclear Factor-κB-inducing Kinase (NIK) Contains an Amino-terminal Inhibitor of Apoptosis (IAP)-binding Motif (IBM) That Potentiates NIK Degradation by Cellular IAP1 (c-IAP1)

Activation of the noncanonical NF-κB pathway hinges on the stability of the NF-κB-inducing kinase (NIK), which is kept at low levels basally by a protein complex consisting of the E3 ubiquitin ligases cellular inhibitor of apoptosis 1 and 2 (c-IAP1/2) proteins and the tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2/3). NIK is brought into close proximity to the c-IAPs through a TRAF2-TRAF3 bridge where TRAF2 recruits c-IAP1/2 and TRAF3 binds to NIK. However, it is not clear how the c-IAPs specifically recognize and ubiquitylate NIK in the complex. We have identified an IAP-binding motif (IBM) at the amino terminus of NIK. IBMs are utilized by a number of proapoptotic proteins to antagonize IAP function. Here, we utilize mutational studies to demonstrate that wild-type NIK is destabilized in the presence of c-IAP1, whereas the NIK IBM mutant is stable. NIK interacts with the second baculovirus IAP repeat (BIR2) domain of c-IAP1 via the IBM, and this interaction, in turn, provides substrate recognition for c-IAP1 mediated ubiquitylation and degradation of NIK. Furthermore, in the presence of the NIK IBM mutant, we observed an elevated processing of p100 to p52 followed by increased expression of NF-κB target genes. Together, these findings reveal the novel identification and function of the NIK IBM, which promotes c-IAP1-dependent ubiquitylation of NIK, resulting in optimal NIK turnover to ensure that noncanonical NF-κB signaling is off in the absence of an activating signal.

Abstract 5119: Smac mimetic inhibits tumorigenicity and growth of glioblastoma by promoting differentiation of glioblastoma cancer stem-like cells

Abstract Inhibitors of apoptosis (IAP) proteins are often overexpressed in human cancers including glioblastoma (GBM) and are well characterized for their antiapoptotic properties. Recently, a broader role of IAP proteins in tumor biology has been described, as they also regulate several non-apoptotic signal transduction pathways. Therefore, we investigated the effect of non-toxic concentrations of small-molecule Smac mimetics, which antagonize IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary GBM specimens. Since CSLCs have been implicated in resistance to chemo- and radiotherapy and tumor recurrence, they are considered as critical therapeutic targets in GBM. Here, we identify a novel function of Smac mimetic in regulating differentiation of GBM CSLCs by activating NF-κB. Smac mimetic at non-lethal doses stimulates morphological changes associated with differentiation of GBM CSLCs. Smac mimetic increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker β-III-tubulin, indicating that Smac mimetic induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that Smac mimetic triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor (IκBα-SR) blocks the Smac mimetic-stimulated increase in GFAP and differentiation. Interestingly, this Smac mimetic-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, Smac mimetic does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, Smac mimetic treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models of GBM and significantly increases the survival of mice bearing GBM. By identifying a novel role of Smac mimetic in promoting the differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs. Citation Format: Aurelie Tchoghandjian, Claudia Jennewein, Ines Eckhardt, Stefan Momma, Dominique Figarella-Branger, Simone Fulda. Smac mimetic inhibits tumorigenicity and growth of glioblastoma by promoting differentiation of glioblastoma cancer stem-like cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5119. doi:10.1158/1538-7445.AM2014-5119

Abstract SY06-04: Regulation of cancer cell proliferation and survival by NF-κB

Abstract SY06-04: Regulation of cancer cell proliferation and survival by NF-κB

Regulation of p53 and Rb links the alternative NF-κB pathway to EZH2 expression and cell senescence.

There are two major pathways leading to induction of NF-κB subunits. The classical (or canonical) pathway typically leads to the induction of RelA or c-Rel containing complexes, and involves the degradation of IκBα in a manner dependent on IκB kinase (IKK) β and the IKK regulatory subunit NEMO. The alternative (or non-canonical) pathway, involves the inducible processing of p100 to p52, leading to the induction of NF-κB2(p52)/RelB containing complexes, and is dependent on IKKα and NF-κB inducing kinase (NIK). Here we demonstrate that in primary human fibroblasts, the alternative NF-κB pathway subunits NF-κB2 and RelB have multiple, but distinct, effects on the expression of key regulators of the cell cycle, reactive oxygen species (ROS) generation and protein stability. Specifically, following siRNA knockdown, quantitative PCR, western blot analyses and chromatin immunoprecipitation (ChIP) show that NF-κB2 regulates the expression of CDK4 and CDK6, while RelB, through the regulation of genes such as PSMA5 and ANAPC1, regulates the stability of p21WAF1 and the tumour suppressor p53. These combine to regulate the activity of the retinoblastoma protein, Rb, leading to induction of polycomb protein EZH2 expression. Moreover, our ChIP analysis demonstrates that EZH2 is also a direct NF-κB target gene. Microarray analysis revealed that in fibroblasts, EZH2 antagonizes a subset of p53 target genes previously associated with the senescent cell phenotype, including DEK and RacGAP1. We show that this pathway provides the major route of crosstalk between the alternative NF-κB pathway and p53, a consequence of which is to suppress cell senescence. Importantly, we find that activation of NF-κB also induces EZH2 expression in CD40L stimulated cells from Chronic Lymphocytic Leukemia patients. We therefore propose that this pathway provides a mechanism through which microenvironment induced NF-κB can inhibit tumor suppressor function and promote tumorigenesis.

T cell-intrinsic function of the noncanonical NF-κB pathway in the regulation of GM-CSF expression and experimental autoimmune encephalomyelitis pathogenesis.

The noncanonical NF-κB pathway induces processing of the NF-κB2 precursor protein p100, and thereby mediates activation of p52-containing NF-κB complexes. This pathway is crucial for B cell maturation and humoral immunity, but its role in regulating T cell function is less clear. Using mutant mice that express a nonprocessible p100, NF-κB2(lym1), we show that the noncanonical NF-κB pathway has a T cell-intrinsic role in regulating the pathogenesis of a T cell-mediated autoimmunity, experimental autoimmune encephalomyelitis (EAE). Although the lym1 mutation does not interfere with naive T cell activation, it renders the Th17 cells defective in the production of inflammatory effector molecules, particularly the cytokine GM-CSF. We provide evidence that p52 binds to the promoter of the GM-CSF-encoding gene (Csf2) and cooperates with c-Rel in the transactivation of this target gene. Introduction of exogenous p52 or GM-CSF to the NF-κB2(lym1) mutant T cells partially restores their ability to induce EAE. These results suggest that the noncanonical NF-κB pathway mediates induction of EAE by regulating the effector function of inflammatory T cells.

Pre-conditioning with high-mobility group box 1 (HMGB1) attenuates excessive TLR-mediated inflammation. (INM9P.456)

Abstract Activation of innate immunity via TLRs plays a pivotal role in host defense against pathogens through the recognition of PAMPs. However, since uncontrolled immune response leads to various kinds of autoimmune and inflammatory diseases, TLR signaling should be tightly regulated. Recent studies have shown that HMGB1 could be critical for the control of TLR signaling. In this study, we demonstrated that HMGB1 mediated negative regulating signals of TLR activation. Our data indicated that HMGB1 induced NF-κB activation via non-canonical pathway by processing of p100 to p52 and translocation of p52 into the nucleus. Also, HMGB1 induced expression of TRIM30α which is a regulator of TLR signaling. Preconditioned HMGB1 attenuated TLR activation by LPS in murine, human macrophage cell lines and mouse BMDM. Treatment of HMGB1 followed by LPS stimulation decreased TNF-α and IL-6 level in mouse BMDM when compared with LPS stimulation alone. Furthermore, HMGB1 pretreatment in the presence of polymyxin B, an endotoxin inactivator, induced HMGB1-mediated tolerance. Pretreatment of HMGB1 promoted rapid termination of signaling in macrophage cells through transcription factor NF-κB by augmenting negative regulators of TLRs such as TRIM30α and SHIP-1. Knockdown experiments using TRIM30α-specific small interfering RNA impaired the HMGB1-mediated TLR tolerance. Collectively, our results demonstrate that HMGB1 mediated the mechanism for prevention of excessive TLR-mediated inflammation.

RelB-induced Expression of Cot, an MAP3K Family Member, Rescues RANKL-induced Osteoclastogenesis in Alymphoplasia Mice by Promoting NF-κB2 Processing by IKKα

The alternative nuclear factor-κB (NF-κB) pathway, mainly the RelB-p52 heterodimer, plays important roles in bone metabolism through an unknown mechanism. We have previously reported that alymphoplasia (aly/aly) mice, which lack active NF-κB-inducing kinase (NIK), show mild osteopetrosis due to the inhibition of osteoclastogenesis. p100 retains RelB in the cytoplasm and inhibits RANKL-induced osteoclastogenesis in aly/aly cells. Furthermore, the overexpression of RelB in aly/aly cells rescues RANKL-induced osteoclastogenesis by inducing p100 processing. In contrast, the overexpression of p65 in aly/aly cells has no effect. However, the overexpression of RelB fails to rescue RANKL-induced osteoclastogenesis in the presence of p100ΔGRR, which cannot be processed to p52, suggesting that p100 processing is a key step in RelB-rescued, RANKL-induced osteoclastogenesis in aly/aly cells. In this study, Cot (cancer Osaka thyroid), an MAP3K, was up-regulated by RelB overexpression. Analysis of the Cot promoter demonstrated that p65 and RelB bound to the distal NF-κB-binding site and that RelB but not p65 bound to the proximal NF-κB-binding site in the Cot promoter. The knocking down of Cot expression significantly reduced the RANKL-induced osteoclastogenesis induced by RelB overexpression. The phosphorylation of IKKα at threonine 23 and its kinase activity were indispensable for the processing of p100 and osteoclastogenesis by RelB-induced Cot. Finally, constitutively activated Akt enhanced osteoclastogenesis by RelB-induced Cot, and a dominant-negative form of Akt significantly inhibited it. Taken together, these results indicate that the overexpression of RelB restores RANKL-induced osteoclastogenesis by activation of Akt/Cot/IKKα-induced p100 processing.

Loss of the HPV-infection resistance EVER2 protein impairs NF-κB signaling pathways in keratinocytes.

Homozygous mutations in EVER genes cause epidermodysplasia verruciformis (EV), characterized by an immune defect and the development of skin cancers associated with β- human papillomavirus (HPV) infections. The effects of EVER protein loss on the keratinocyte immune response remain unknown. We show here that EVER2 plays a critical role in the interplay between the NF-κB and JNK/AP-1 signaling pathways. EVER2-deficient cells overproduce IL-6 following the upregulation of JNK activation. They respond poorly to phorbol ester and TNF via the NF-κB pathway. They have lower levels of IKKα subunit, potentially accounting for impairments of p100 processing and the alternative NF-κB pathway. The loss of EVER2 is associated with an unusual TRAF protein profile. We demonstrate that EVER2 deficiency sustains TRAF2 ubiquitination and decreases the pool of TRAF2 available in the detergent-soluble fraction of the cell. Finally, we demonstrate that EVER2 loss induces constitutive PKCα-dependent c-jun phosphorylation and facilitates activation of the HPV5 long control region through a JNK-dependent pathway. These findings indicate that defects of the EVER2 gene may create an environment conducive to HPV replication and the persistence of lesions with the potential to develop into skin cancer.

Smac mimetic promotes glioblastoma cancer stem-like cell differentiation by activating NF-κB

Recently, a broader role of inhibitor of apoptosis (IAP) proteins besides their antiapoptotic functions has been described. Therefore, we investigated the effect of non-toxic concentrations of the small-molecule Smac mimetic BV6, which antagonizes IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary glioblastoma (GBM) specimens. Here, we identify a novel function of BV6 in regulating differentiation of GBM CSLCs by activating NF-κB. BV6 at non-lethal doses stimulates morphological changes associated with the differentiation of GBM CSLCs. BV6 increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker β-III-tubulin, indicating that BV6 induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that BV6 triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα super-repressor (IκBα-SR) blocks the BV6-stimulated increase in GFAP and differentiation. Interestingly, this BV6-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, BV6 does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, BV6 treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice. By identifying a novel role of BV6 in promoting differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs.

Activation of alternative NF-κB signaling during recovery of disuse-induced loss of muscle oxidative phenotype

Physical inactivity-induced loss of skeletal muscle oxidative phenotype (OXPHEN), often observed in chronic disease, adversely affects physical functioning and quality of life. Potential therapeutic targets remain to be identified, since the molecular mechanisms involved in reloading-induced recovery of muscle OXPHEN remain incompletely understood. We hypothesized a role for alternative NF-κB, as a recently identified positive regulator of muscle OXPHEN, in reloading-induced alterations in muscle OXPHEN. Markers and regulators (including alternative NF-κB signaling) of muscle OXPHEN were investigated in gastrocnemius muscle of mice subjected to a hindlimb suspension/reloading (HLS/RL) protocol. Expression levels of oxidative phosphorylation subunits and slow myosin heavy chain isoforms I and IIA increased rapidly upon RL. After an initial decrease upon HLS, mRNA levels of peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC) molecules PGC-1α and PGC-1β and mRNA levels of mitochondrial transcription factor A (Tfam) and estrogen-related receptor α increased upon RL. PPAR-δ, nuclear respiratory factor 1 (NRF-1), NRF-2α, and sirtuin 1 mRNA levels increased during RL although expression levels were unaltered upon HLS. In addition, both Tfam and NRF-1 protein levels increased significantly during the RL period. Moreover, upon RL, IKK-α mRNA and protein levels increased, and phosphorylation of P100 and subsequent processing to P52 were elevated, reflecting alternative NF-κB activation. We conclude that RL-induced recovery of muscle OXPHEN is associated with activation of alternative NF-κB signaling.

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

A common single nucleotide polymorphism impairs B-cell activating factor receptor's multimerization, contributing to common variable immunodeficiency

Abstract A204: Flow cytometric assay of proteasome inhibition in whole blood.

Abstract Introduction: Two proteasome inhibitors are currently approved by the US FDA for the treatment of multiple myeloma: bortezomib and carfilzomib. Both drugs make a covalent bond with a Thr in the active site of the 20S catalytic β5 subunit. While the covalent bond is hydrolyzable in the case of bortezomib, its binding is characterized by a long residency time with a half-life of close to 2 hours. The current clinical surrogate assay measures the suppression of proteasome activity in pooled cell lysates from patients’ blood or tissue following the administration of inhibitor. As such, this assay depends upon the long residency time of current inhibitors. Second-generation proteasome inhibitors that are in development, such as ixazomib, have a much faster off-rate, making accurate measurement of inhibition in diluted cell lysates problematic. Our objective was to develop a surrogate proteasome assay for use in whole blood, without requiring cell lysis, thus preventing sample dilution. Methods: Whole-blood samples were pre-incubated with proteasome inhibitors for 1-2 hours at 37°C. Subsequently, samples were stimulated with LPS at 37°C, then fixed and permeabilized using PerFix-P (Beckman Coulter). Washed white blood cells were stained with IκBα-AF488 (Cell Signaling Technologies), CD45-KrO, and CD14-PC7 monoclonal antibodies (Beckman Coulter) and analyzed using a Gallios™ flow cytometer. Results and Discussion: During the classical activation of NF-κB signaling, the NF-κB inhibitor, IκBα, undergoes phosphorylation and ubiquitination, followed by rapid proteasomal degradation. Released NF-κB complexes can then translocate to the nucleus, where they initiate transcriptional activation. In this study, we analyzed the LPS-induced degradation of IκBα in whole-blood monocytes by flow cytometry. We found a 70-80% decrease in the median fluorescent intensity (MFI) of IκBα-AF488 after 10 min of LPS stimulation. The kinetic profile and magnitude of the MFI reduction were highly reproducible in whole-blood monocytes from healthy donors, as well as in a limited number of blood samples obtained from multiple-myeloma patients. The MFI reduction was inhibited in a dose-dependent manner by different proteasome inhibitors, including bortezomib, carfilzomib, and MG132. To increase the specificity of the assay, ERK phosphorylation can also be monitored simultaneously within the same cells. ERK phosphorylation in whole-blood monocytes peaks at ∼10 min after LPS stimulation, and is caused by the release of TPL2 from its complex with p105 (unprocessed NF-κB1) following the proteasomal processing of p105 into p50. Because of this, ERK phosphorylation is also inhibited by proteasome inhibitors in a dose-dependent manner, similar to IκBα. This assay can be optimized for different cell types within whole blood by using different activators, although the kinetics of IκBα degradation varies. Conclusions: We have established a fast and convenient assay to measure proteasome activity and inhibition in whole blood leukocytes. This assay can be used for investigation of the PK/PD relationships of new-generation proteasome inhibitors with fast off-rates. Disclaimer: PerFix-P and Gallios™ are for research use only. Not for use in diagnostic procedures. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A204. Citation Format: Sergei Gulnik, T. Vincent Shankey, Lidice L. Lopez, David W. Hedley, Sue Chow. Flow cytometric assay of proteasome inhibition in whole blood. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A204.

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs(∗)7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853(∗)), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.

273 : Antibody deficiency associated with an inherited autosomal dominant mutation in TWEAK

Mutations in the TNF family of proteins have been associated with inherited forms of immune deficiency. Using an array-based sequencing assay, we identified an autosomal-dominant deficiency in TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) in a kindred with recurrent infection and impaired antibody responses to protein and polysaccharide vaccines. This mutation occurs in the sixth exon of TWEAK and results in the amino acid substitution R145C within the conserved TNF-homology domain of the full-length protein. TWEAK mutant protein formed high molecular weight aggregates under nonreducing conditions, suggesting an increased propensity for intermolecular interactions. As a result, mutant TWEAK associated with B-cell–activating factor (BAFF) protein and down-regulated the BAFF-mediated activation of the noncanonical NF-κB pathway through inhibition of p100 processing to p52, resulting in inhibition of BAFF-dependent B-cell survival and proliferation. As BAFF mediates T-cell–independent isotype switching and B-cell survival, our data implicate TWEAK as a disease-susceptibility gene for a humoral immunodeficiency.

Extracellular Tissue Transglutaminase Activates Noncanonical NF-κB Signaling and Promotes Metastasis in Ovarian Cancer

Tissue transglutaminase (TG2) is a multifunctional protein that binds to fibronectin and exerts protein transamidating activity in the presence of Ca2+. We previously reported that TG2 is upregulated in ovarian tumors and enhances intraperitoneal (i.p.) metastasis. TG2 is secreted abundantly in ovarian cancer (OC) ascites as an active enzyme, yet its function in the extracellular compartment remains unknown. To study the distinct functions of secreted TG2, we used recombinant His6-tagged TG2 and catalytically inactive enzyme in vitro and in vivo. By using i.p. and orthotopic ovarian xenografts, we show that extracellular transglutaminase promoted OC peritoneal metastasis. The main pathway activated by extracellular TG2 was noncanonical nuclear factor-kappa B (NF-κB) signaling, and the enzymatic function of the protein was required to induce phosphorylation of IκBkinaseá and processing of the precursor protein p100 into the active p52 subunit. A specific target of TG2-activated p52/RelB complex is the hyaluronan receptor, CD44. Noncanonical NF-κB activation by extracellular TG2 induced CD44 up-regulation and epithelial-to-mesenchymal transition, contributing to increased cancer cell invasiveness and OC peritoneal dissemination. Taken together, our data support that noncanonical NF-κB activation is the pathway through which extracellular TG2 promotes OC metastasis.

Effective treatment of aggressive B-cell lymphomas by downregulated NIK-induced noncanonical NF-κB pathway activation through inhibition of BAFF.

e13554 Background: We recently reported that autocrine BAFF (B cell–activating factor belonging to the TNF family) signal transduction pathway contributes to H. pylori-independent growth of gastric diffuse large B-cell lymphoma (DLBCL) (Blood 2008;112:2927-34; Ann Hematol 2010;89:431-6). In this study, we sought to investigate whether activation of BAFF signaling pathway can promote the survival and proliferation of aggressive B-cell lymphoma. Methods: Seven aggressive NHL cell lines (EBV-negative Burkitt’s lymphoma (Ramos), EBV-positive Burkitt’s lymphoma (Raji), EBV-negative undifferentiated lymphoma (MC116), activated B cell (ABC)-like DLBCL (OCI-Ly3, OCI-Ly10), and germinal center B cell (GCB)-like DLBCL (OCI-Ly7, and Pfeiffer) were used in this study. Cell cycle was analyzed by flow cytometry. The DNA-binding activity of NF-kB was determined by the luciferase assay. Expression of non-canonical NF-κB signatures-related proteins (BAFF, BAFF-R, NIK, cIAP1, TRAF2, cIAP1/2, TRAF3, IKKa, p100, p52 and RelB, BCL10, BCL3, and STAT3) was assessed by immunoblotting. Results: Our results showed that in GCB-DLBCL cell lines, activation of BAFF induced recruitment and degradation of TRAF3, which resulted in NIK kinase accumulation, BCL10 Ser138 phosphorylation, IKKa phosphorylation, and NF-kB p100 processing, thereby resulting in continuous activation of non-canonical NF-kB pathway. This phenomenon also resulted in BCL3 nuclear translocation and STAT3 activation, and subsequently activated STAT3 downstream-regulated genes (BCL2, survivin, and cyclin D1). Furthermore, we found that inhibition of BAFF by short hairpin RNA (shRNA) suppressed the growth of ABC-DLBCL cells and Burkitt lymphoma cells through the down-regulation of BAFF/BAFF-R/TRAF3/NIK/BCL3/NF-kB signaling pathway. Conclusions: Our results indicate that constitutive BAFF signaling activates NIK-induced non-canonical NF-kB signaling pathway in aggressive B-cell lymphoma, and inhibition of BAFF is particularly effective in the treatment of this subgroup of tumors.

Inducible Tumor Necrosis Factor (TNF) Receptor-associated Factor-1 Expression Couples the Canonical to the Non-canonical NF-κB Pathway in TNF Stimulation

The NF-κB transcription factor mediates the inflammatory response through distinct (canonical and non-canonical) signaling pathways. The mechanisms controlling utilization of either of these pathways are largely unknown. Here we observe that TNF stimulation induces delayed NF-κB2/p100 processing and investigate the coupling mechanism. TNF stimulation induces TNF-associated factor-1 (TRAF-1) that directly binds NF-κB-inducing kinase (NIK) and stabilizes it from degradation by disrupting its interaction with TRAF2·cIAP2 ubiquitin ligase complex. We show that TRAF1 depletion prevents TNF-induced NIK stabilization and reduces p52 production. To further examine the interactions of TRAF1 and NIK with NF-κB2/p100 processing, we mathematically modeled TRAF1·NIK as a coupling signaling complex and validated computational inference by siRNA knockdown to show non-canonical pathway activation is dependent not only on TRAF1 induction but also NIK stabilization by forming TRAF1·NIK complex. Thus, these integrated computational-experimental studies of TNF-induced TRAF1 expression identified TRAF1·NIK as a central complex linking canonical and non-canonical pathways by disrupting the TRAF2-cIAP2 ubiquitin ligase complex. This feed-forward kinase pathway is essential for the activation of non-canonical pathway.

IKKalpha is critical for the maintenance of a normal pool of regulatory T cells and for the expansion of both regulatory and effector T cells (P1095)

Abstract Our previous studies show that TNF activates and expands Tregs through TNFR2. It was reported that TNF-TNFR2 interaction results in the activation of alternative NFκB pathway through p100 processing, in IKKα-dependent manner. We therefore hypothesize that IKKα may play an important role in determining the size of Treg pool. To this end, we generated a mouse strain with conditional KO of IKKα in CD4 cells (CD4-IKKα-/-) which showed a marked reduction in the number of Tregs and in the proportion of Tregs in CD4 cells, indicating that IKKα KO had more profound impact on Tregs than Teffs. In vitro, naïve CD4 cells deficient in IKKα could be differentiated as usual into Foxp3-expressing cells by TGFβ, suggesting that reduction of Tregs in CD4-IKKα-/- mice was likely due to a defect in the maintenance of the Treg pool, rather than a malfunction in the induction of Foxp3 expression. The function of Tregs deficient in IKKα was examined by co-transfer to Rag1-/- mice together with naïve CD4 cells. Although WT Tregs was able to inhibit colitis induced by transfer of WT naïve CD4 cells, IKKα-deficient Tregs failed to inhibit colitis which was associated with their incapacity to reconstitute in Rag1-/- mice. Nevertheless, naïve CD4 cells deficient in IKKα also failed to reconstitute Rag1-/- mice. Therefore, our study indicate that IKKα is important for the maintenance of normal Treg pool and is critical for the expansion of Treg cells as well as Teff cells.