TNF-induced NF-κB Activation Research Articles

Influence of Race and High Laminar Shear Stress on TNFR1 Signaling in Endothelial Cells.

Tumor necrosis factor (TNF) binding to endothelial TNF receptor-I (TNFR-I) facilitates monocyte recruitment and chronic inflammation, leading to the development of atherosclerosis. In vitro data show a heightened inflammatory response and atherogenic potential in endothelial cells (ECs) from African American (AA) donors. High laminar shear stress (HSS) can mitigate some aspects of racial differences in endothelial function at the cellular level. We examined possible racial differences in TNF-induced monocyte adhesion and TNFR1 signaling complex expression/activity, along with the effects of HSS. Tohoku Hospital Pediatrics-1 (THP-1) monocytes were used in a co-culture system with human umbilical vein ECs (HUVECs) from Caucasian American (CA) and AA donors to examine racial differences in monocyte adhesion. An in vitro exercise mimetic model was applied to investigate the potential modulatory effect of HSS. THP-1 adherence to ECs and TNF-induced nuclear factor kappa B (NF-κB) DNA binding were elevated in AA ECs compared to CA ECs, but not significantly. We report no significant racial differences in the expression of the TNFR-I signaling complex. Application of HSS significantly increased the expression and shedding of TNFR-I and the expression of TRAF3, and decreased the expression of TRAF5 in both groups. Our data does not support TNF-induced NF-κB activation as a potential mediator of racial disparity in this model. Other pathways and associated factors activated by the TNFR1 signaling complex are recommended targets for future research.

Discovery of a Non-competitive TNFR1 Antagonist Affibody with Picomolar Monovalent Potency That Does Not Affect TNFR2 Function.

Tumor necrosis factor (TNF) is a key regulator of immune responses and plays a significant role in the initiation and maintenance of inflammation. Upregulation of TNF expression leads to several inflammatory diseases, such as Crohn's, ulcerative colitis, and rheumatoid arthritis. Despite the clinical success of anti-TNF treatments, the use of these therapies is limited because they can induce adverse side effects through inhibition of TNF biological activity, including blockade of TNF-induced immunosuppressive function of TNFR2. Using yeast display, we identified a synthetic affibody ligand (ABYTNFR1-1) with high binding affinity and specificity for TNFR1. Functional assays showed that the lead affibody potently inhibits TNF-induced NF-κB activation (IC50 of 0.23 nM) and, crucially, does not block the TNFR2 function. Additionally, ABYTNFR1-1 acts non-competitively─it does not block TNF binding or inhibit receptor-receptor interactions in pre-ligand-assembled dimers─thereby enhancing inhibitory robustness. The mechanism, monovalent potency, and affibody scaffold give this lead molecule uniquely strong potential as a therapeutic candidate for inflammatory diseases.

Abstract 3708: OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates tumors in combination with PD-1 inhibition

Abstract Post-translational modifications of DNA and histones can improve the intrinsic antitumor capacity of the immune system. Using the iOTarg screening platform, salt-inducible kinase 3 (SIK3) was identified as a novel epigenetic modulator in cancer therapy. SIK3, a serine/threonine kinase of the AMP-activated protein kinase family, is known for regulating the NF-κB driven gene landscape through phosphorylation of class IIa histone deacetylases (HDACs) and CREB-regulated transcriptional coactivators causing the tumor to evade death receptor-mediated killing. Moreover, salt inducible kinases were recently described to regulate cell cycle checkpoints and thereby promote cancer cell proliferation. We demonstrate that SIK3 knockdown abates downstream pro-survival signaling and induces cell death in a distinct panel of tumor cell lines. OMX-0407, an orally available, single-digit nanomolar inhibitor of SIK3 was shown to effectively reduce TNF-induced HDAC4 phosphorylation and downstream NF-κB activity in a dose-dependent manner, thereby enhancing apoptosis in murine and human tumor cell lines. OMX-0407 dose-dependent suppression of intratumoral NF-κB activity was shown in vivo in an MC38 NF-κB-luc reporter cell line. Using OMX-0407 monotherapy, this translated to significant tumor growth inhibition as well as prolonged survival in the highly immune infiltrated syngeneic murine colorectal carcinoma model MC38. Besides its direct inhibitory effects on cancer cells, OMX-0407 repolarizes the tumor microenvironment (TME) by strongly decreasing regulatory T cells (T-regs) and M2-polarized macrophages in the tumor bed, while not affecting the peripheral T cell compartment. Thus, exposure to OMX-0407 leads to a distinct pro-inflammatory TME, characterized by an expansion of activated cytotoxic T lymphocytes (CTL) and an increased CTL-to-T-reg ratio. Using an immune-excluded tumor phenotype as seen in the breast cancer mouse model EMT6, we demonstrated that OMX-0407 and anti-PD-1 act synergistically by combining the sensitization towards cell death with a reduction in immunosuppressive TME and an increase in cytotoxic T cell activity, despite having only minimal anti-tumor efficacy as monotherapy. Thereby, partial or complete tumor remission in 60% of the animals and extension of overall survival were achieved. In summary, OMX-0407, a first-in-class oral SIK3 inhibitor, demonstrates potent monotherapy efficacy in a pro-inflammatory tumor setting by reshaping the immune compartment and accelerating tumor cell death. The ability of OMX-0407 to remodel an immunosuppressed TME in a generally cold tumor setting, harbors great clinical potential for OMX-0407 combination therapy with anti-PD-1/PD-L1 immune checkpoint blockade, specifically in patients with high unmet medical need who are resistant to current immune checkpoint inhibitor monotherapy. Citation Format: Christina Hartl, Ilona-Petra Maser, Tillmann Michels, Ronny Milde, Vanessa Klein, Philipp Beckhove, Nisit Khandelwal, Hannes Loferer, Stefan Bissinger. OMX-0407, a highly potent SIK3 inhibitor, sensitizes tumor cells to cell death and eradicates tumors in combination with PD-1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3708.

Salt-inducible kinase 3 protects tumor cells from cytotoxic T-cell attack by promoting TNF-induced NF-κB activation

BackgroundCancer immunotherapeutic strategies showed unprecedented results in the clinic. However, many patients do not respond to immuno-oncological treatments due to the occurrence of a plethora of immunological obstacles, including tumor...

Roburic Acid Targets TNF to Inhibit the NF-κB Signaling Pathway and Suppress Human Colorectal Cancer Cell Growth.

Tumor necrosis factor (TNF)-stimulated nuclear factor-kappa B (NF-κB) signaling plays very crucial roles in cancer development and progression, and represents a potential target for drug discovery. Roburic acid is a newly discovered tetracyclic triterpene acid isolated from oak galls and exhibits anti-inflammatory activity. However, whether roburic acid exerts antitumor effects through inhibition of TNF-induced NF-κB signaling remains unknown. Here, we demonstrated that roburic acid bound directly to TNF with high affinity (K D = 7.066 μM), blocked the interaction between TNF and its receptor (TNF-R1), and significantly inhibited TNF-induced NF-κB activation. Roburic acid exhibited antitumor activity in numerous cancer cells and could effectively induce G0/G1 cell cycle arrest and apoptosis in colorectal cancer cells. Importantly, roburic acid inhibited the TNF-induced phosphorylation of IKKα/β, IκBα, and p65, degradation of IκBα, nuclear translocation of p65, and NF-κB-target gene expression, including that of XIAP, Mcl-1, and Survivin, in colorectal cancer cells. Moreover, roburic acid suppressed tumor growth by blocking NF-κB signaling in a xenograft nude mouse model of colorectal cancer. Taken together, our findings showed that roburic acid directly binds to TNF with high affinity, thereby disrupting its interaction with TNF-R1 and leading to the inhibition of the NF-κB signaling pathway, both in vitro and in vivo. The results indicated that roburic acid is a novel TNF-targeting therapeutics agent in colorectal cancer as well as other cancer types.

Impact of Interleukin 10 Deficiency on Intestinal Epithelium Responses to Inflammatory Signals.

Interleukin 10 (IL-10) is a pleiotropic, anti-inflammatory cytokine that has a major protective role in the intestine. Although its production by cells of the innate and adaptive immune system has been extensively studied, its intrinsic role in intestinal epithelial cells is poorly understood. In this study, we utilised both ATAC sequencing and RNA sequencing to define the transcriptional response of murine enteroids to tumour necrosis factor (TNF). We identified that the key early phase drivers of the transcriptional response to TNF within intestinal epithelium were NFκB transcription factor dependent. Using wild-type and Il10−/− enteroid cultures, we showed an intrinsic, intestinal epithelium specific effect of IL-10 deficiency on TNF-induced gene transcription, with significant downregulation of identified NFκB target genes Tnf, Ccl20, and Cxcl10, and delayed overexpression of NFκB inhibitor encoding genes, Nfkbia and Tnfaip3. IL-10 deficiency, or immunoblockade of IL-10 receptor, impacted on TNF-induced endogenous NFκB activity and downstream NFκB target gene transcription. Intestinal epithelium-derived IL-10 appears to play a crucial role as a positive regulator of the canonical NFκB pathway, contributing to maintenance of intestinal homeostasis. This is particularly important in the context of an inflammatory environment and highlights the potential for future tissue-targeted IL-10 therapeutic intervention.

Cucurbitacin D Inhibits NF-κB Activation, Thereby Inducing Apoptosis and Suppressing Tumor Growth

Recently, more and more researchers are investigating the effectiveness of using traditional Chinese medicine as a treatment to treat cancer. This work investigates the Cucurbitacin D, an active component found in the traditional Chinese medicine Trichosanthes kirilowii tuber. Its abilities to inhibit NF-κB activation and to induce apoptosis are studied. This work also examines the potential ability of Cucurbitacin D to inhibit TNF-induced NF-κB activation, to inhibit TNF-dependent IκBα degradation and TNF-dependent IκBα phosphorylation, to repress the TNF-induced NF-κB-dependent reporter gene expression, to potentiate the apoptosis effect, to down regulates the expression of anti-apoptotic proteins regulated by the NF-κB pathway, and to inhibits the expression of gene products involved in cell proliferation using methods such as the EMSA, western blotting, NF-κB-dependent reporter gene expression assay, TUNEL assay, live and dead assay, and the MTT assay. There are several possible results that can be derived from each testing. In this work, the final conclusion is that Cucurbitacin D could have the potential ability to inhibit NF-kB activation and therefore to induce apoptosis, so it could have the potential ability to reduce cancer cell progression.

Regulation of inflammatory signaling by the ST6Gal-I sialyltransferase.

The ST6Gal-I sialyltransferase, an enzyme that adds α2-6-linked sialic acids to N-glycosylated proteins, regulates multiple immunological processes. However, the contribution of receptor sialylation to inflammatory signaling has been under-investigated. In the current study, we uncovered a role for ST6Gal-I in promoting sustained signaling through two prominent inflammatory pathways, NFκB and JAK/STAT. Using the U937 monocytic cell model, we determined that knockdown (KD) of ST6Gal-I expression had no effect on the rapid activation of NFκB by TNF (≤ 30 min), whereas long-term TNF-induced NFκB activation (2–6 hr) was diminished in ST6Gal-I-KD cells. These data align with prior work in epithelial cells showing that α2–6 sialylation of TNFR1 prolongs TNF-dependent NFκB activation. Similar to TNF, long-term, but not short-term, LPS-induced activation of NFκB was suppressed by ST6Gal-I KD. ST6Gal-I KD cells also exhibited reduced long-term IRF3 and STAT3 activation by LPS. Given that ST6Gal-I activity modulated LPS-dependent signaling, we conducted pull-down assays using SNA (a lectin specific for α2–6 sialic acids) to show that the LPS receptor, TLR4, is a substrate for sialylation by ST6Gal-I. We next assessed signaling by IFNγ, IL-6 and GM-CSF, and found that ST6Gal-I-KD had a limited effect on STAT activation induced by these cytokines. To corroborate these findings, signaling was monitored in bone marrow derived macrophages (BMDMs) from mice with myeloid-specific deletion of ST6Gal-I (LysMCre/ST6Gal-Ifl/fl). In agreement with data from U937 cells, BMDMs with ST6Gal-I knockout displayed reduced long-term activation of NFκB by both TNF and LPS, and diminished long-term LPS-dependent STAT3 activation. However, STAT activation induced by IFNγ, IL-6 and GM-CSF was comparable in wild-type and ST6Gal-I knockout BMDMs. These results implicate ST6Gal-I-mediated receptor sialylation in prolonging the activity of select signaling cascades including TNF/NFκB, LPS/NFκB, and LPS/STAT3, providing new insights into ST6Gal-I’s role in modulating the inflammatory phenotype of monocytic cells.

Abstract 6698: A first-in-class SIK3 inhibitor, OMX-0370, effectively inhibits tumor growth in syngeneic tumor models, as single agent, by abolishing tumor resistance to immune-derived TNF

Abstract SIK3 is an intracellular serine/threonine kinase belonging to the AMPK superfamily. We recently discovered a novel role of SIK3 in conferring TNF resistance to tumor cells. Resistance to TNF is an emerging mode of immune evasion in multiple solid tumors. In fact, while treating a broad panel of tumor cell lines with TNF we observed that almost 70% were either resistant or even proliferative in response to TNF. SIK3 knockout using CRISPR re-sensitized human PANC-1 and murine MC38 tumor cells to TNF-mediated death. Intratumoral SIK3 induces TNF resistance by retaining HDAC4 in the cytoplasm thus keeping the chromatin open and potentiating the TNF-driven pro-tumorigenic activity of NF-κB. To translate these findings into the clinic, we have developed a potent (low nM range) inhibitor of SIK3, OMX-0370. Firstly, OMX-0370 exhibits dose-dependent inhibition of HDAC4 phosphorylation and nuclear NF-κB activity in response to TNF, thereby effectively neutralizing the SIK3-HDAC4-NF-κB axis. Consequently, treatment of human as well as murine tumor cells with OMX-0370 induces significant TNF-mediated apoptosis while sparing non-TNF treated cells. In mouse DMPK studies, OMX-0370 was found to be orally bioavailable with a favorable pharmacokinetic profile and was well tolerated at 100 mg/kg twice daily dosing in wild type C57BL/6 mice. Having shown a potent and favorable biochemical, functional as well as pharmacokinetic profile of the molecule, we next investigated the ability of OMX-0370 to inhibit the growth of established tumors in multiple syngeneic tumor models, including MC38, EMT6 and RENCA. Notably, OMX-0370 showed significant tumor growth inhibition as a single agent, which was found to be even superior to anti-PD-1 antibody treatment in RENCA and EMT-6 models. Immune profiling showed enhanced activation of intratumoral T cells, improved ratio of CTLs to Tregs and depletion of tumor-associated M2 macrophages, while no effect on peripheral leukocyte counts was observed. To monitor target engagement and pharmacodynamics of OMX-0370 in vivo, we developed a reporter MC38 cell line expressing luciferase under a NF-κB promotor. Using this reporter cell line, we could show that OMX-0370 inhibits TNF-induced NF-κB activation in a dose-dependent manner. Encouraged by the strong single-agent activity of OMX-0370 in solid tumor models, we have developed follow-on variants that exhibit higher potency as well as improved exposure in vivo. In summary, we here report that OMX-0370, a first-in-class inhibitor of SIK3 kinase, is an active immunotherapeutic drug in vivo which effectively abolishes the TNF-driven NF-κB activity in tumors and re-sensitize them to TNF-induced apoptosis. OMX-0370 and its follow-on improved variants address the high unmet medical need of effective immunotherapeutic agents that neutralize clinically relevant and key orthogonal immune evasion axes in solid tumors as a monotherapy regimen. Citation Format: Tillmann Michels, Stefan Bissinger, Peter Sennhenn, Hannes Loferer, Catarina Martins Freire, Olivia Reidell, Sebastian Meier-Ewert, Apollon Papadimitriou, Philipp Beckhove, Nisit Khandelwal. A first-in-class SIK3 inhibitor, OMX-0370, effectively inhibits tumor growth in syngeneic tumor models, as single agent, by abolishing tumor resistance to immune-derived TNF [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6698.

USP20 (Ubiquitin-Specific Protease 20) Inhibits TNF (Tumor Necrosis Factor)-Triggered Smooth Muscle Cell Inflammation and Attenuates Atherosclerosis.

Objective- Signaling that activates NFκB (nuclear factor κB) in smooth muscle cells (SMCs) is integral to atherosclerosis and involves reversible ubiquitination that activates proteins downstream of proatherogenic receptors. Deubiquitination of these proteins is mediated by USP20 (ubiquitin-specific protease 20), among other deubiquitinases. We sought to determine whether USP20 activity in SMCs decreases atherosclerosis. Approach and Results- To address this question, we used male Ldlr-/- mice without (control) or with SMC-specific expression of murine USP20 (SMC-USP20-transgenic) or its dominant-negative (DN; C154S/H643Q) mutant (SMC-DN-USP20-transgenic). Before the appearance of intimal macrophages, NFκB activation in aortic medial SMCs was greater in SMC-DN-USP20-transgenic than in control mice. After 16 weeks on a Western diet, SMC-DN-USP20-transgenic mice had 46% greater brachiocephalic artery atheroma area than control mice. Congruently, aortic atherosclerosis assessed en face was 21% greater than control in SMC-DN-USP20-transgenic mice and 13% less than control in SMC-USP20-transgenic mice. In response to TNF (tumor necrosis factor), SMCs from SMC-DN-USP20-transgenic mice showed ≈3-fold greater NFκB activation than control SMCs. Silencing USP20 in SMCs with siRNA (small interfering RNA) augmented NFκB activation by ≈50% in response to either TNF or IL-1β (interleukin-1β). Coimmunoprecipitation experiments revealed that USP20 associates with several components of the TNFR1 (TNF receptor-1) signaling pathway, including RIPK1 (receptor-interacting protein kinase 1), a critical checkpoint in TNF-induced NFκB activation and inflammation. TNF evoked ≈2-fold more RIPK1 ubiquitination in SMC-DN-USP20-transgenic than in control SMCs, and RIPK1 was deubiquitinated by purified USP20 in vitro. Conclusions- USP20 attenuates TNF- and IL-1β-evoked atherogenic signaling in SMCs, by deubiquitinating RIPK1, among other signaling intermediates.

An Update on Autoinflammatory Diseases: Relopathies.

The nuclear factor κB (NF-κB) pathway is tightly regulated through multiple posttranslational mechanisms including ubiquitination. Mutations in these regulatory pathways can cause disease and are the focus of this review. The linear ubiquitin chain assembly complex (LUBAC) is a trimer made up of HOIL-1L, SHARPIN, and the catalytic subunit HOIP. Loss of function mutations in HOIL-1L and HOIP result in largely overlapping phenotypes, characterized by multi-organ autoinflammation, immunodeficiency, and amylopectinosis. Interestingly, patient fibroblasts exhibited diminished IL-1β- and TNF-induced NF-κB activation, yet monocytes were hyper-responsive to IL-1β, hinting at cell type or target specific roles of LUBAC-mediated ubiquitination. Ubiquitin-driven signaling is counterbalanced by deubiquitinase enzymes (DUBs), such as OTULIN and A20. Hypomorphic mutations in OTULIN result in elevated NF-κB signaling causing an autoinflammatory syndrome. Similarly, patients with high-penetrance heterozygous mutations in the gene encoding A20 (haploinsufficiency of A20 (HA20)) display excessive ubiquitination and increased activity of NF-κB and of NLRP3 inflammasome activation. HA20 patients present with Behçet-like characteristics or an autoimmune lymphoproliferative syndrome (ALPS)-like phenotype, indicating diverse protein functions. This review summarizes recent discoveries in the field of NF-kB-related autoinflammatory diseases (relopathies) within the past 3years and points to several questions that still remain unanswered.

TNF-driven adaptive response mediates resistance to EGFR inhibition in lung cancer.

Although aberrant EGFR signaling is widespread in cancer, EGFR inhibition is effective only in a subset of non-small cell lung cancer (NSCLC) with EGFR activating mutations. A majority of NSCLCs express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. TNF is a major mediator of inflammation-induced cancer. We find that a rapid increase in TNF level is a universal adaptive response to EGFR inhibition in NSCLC, regardless of EGFR status. EGFR signaling actively suppresses TNF mRNA levels by inducing expression of miR-21, resulting in decreased TNF mRNA stability. Conversely, EGFR inhibition results in loss of miR-21 and increased TNF mRNA stability. In addition, TNF-induced NF-κB activation leads to increased TNF transcription in a feed-forward loop. Inhibition of TNF signaling renders EGFRwt-expressing NSCLC cell lines and an EGFRwt patient-derived xenograft (PDX) model highly sensitive to EGFR inhibition. In EGFR-mutant oncogene-addicted cells, blocking TNF enhances the effectiveness of EGFR inhibition. EGFR plus TNF inhibition is also effective in NSCLC with acquired resistance to EGFR inhibition. We suggest concomitant EGFR and TNF inhibition as a potentially new treatment approach that could be beneficial for a majority of lung cancer patients.

Chelidonine inhibits TNF-α-induced inflammation by suppressing the NF-κB pathways in HCT116 cells.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a complex that regulates several hundreds of genes, including those involved in immunity and inflammation, survival, proliferation, and the negative feedback of NF-κB signaling. Chelidonine, a major bioactive, isoquinoline alkaloid ingredient in Chelidonium majus, exhibits antiinflammatory pharmacological properties. However, its antiinflammatory molecular mechanisms remain unclear. In this work, we explored the effect of chelidonine on TNF-induced NF-κB activation in HCT116 cells. We found chelidonine inhibited the phosphorylation and degradation of the inhibitor of NF-κB alpha and nuclear translocation of RELA. Furthermore, by inhibiting the activation of NF-κB, chelidonine downregulated target genes involved in inflammation, proliferation, and apoptosis. Chelidonine also inhibited mitogen-activated protein kinase pathway activation by blocking c-Jun N-terminal kinase and p38 phosphorylation. These results suggest that chelidonine may be a potential therapeutic agent against inflammatory diseases in which inhibition of NF-κB activity plays an important role.

Integrin beta 1 inhibition alleviates the chronic hyperproliferative dermatitis phenotype of SHARPIN-deficient mice.

SHARPIN (Shank-Associated RH Domain-Interacting Protein) is a component of the linear ubiquitin chain assembly complex (LUBAC), which enhances TNF-induced NF-κB activity. SHARPIN-deficient (Sharpincpdm/cpdm) mice display multi-organ inflammation and chronic proliferative dermatitis (cpdm) due to TNF-induced keratinocyte apoptosis. In cells, SHARPIN also inhibits integrins independently of LUBAC, but it has remained enigmatic whether elevated integrin activity levels in the dermis of Sharpincpdm/cpdm mice is due to increased integrin activity or is secondary to inflammation. In addition, the functional contribution of increased integrin activation to the Sharpincpdm/cpdm phenotype has not been investigated. Here, we find increased integrin activity in keratinocytes from Tnfr1-/- Sharpincpdm/cpdm double knockout mice, which do not display chronic inflammation or proliferative dermatitis, thus suggesting that SHARPIN indeed acts as an integrin inhibitor in vivo. In addition, we present evidence for a functional contribution of integrin activity to the Sharpincpdm/cpdm skin phenotype. Treatment with an integrin beta 1 function blocking antibody reduced epidermal hyperproliferation and epidermal thickness in Sharpincpdm/cpdm mice. Our data indicate that, while TNF-induced cell death triggers the chronic inflammation and proliferative dermatitis, absence of SHARPIN-dependent integrin inhibition exacerbates the epidermal hyperproliferation in Sharpincpdm/cpdm mice.

A20 regulates NF-κB activation through K48 linked polyubiquitination of NEMO

Abstract Activation of the NF-κB family of transcription factors is related to diverse biological processes, such as development, immune responses, and inflammation. As a result, mutations affecting the NF-κB signaling pathway cause syndromes characterized by immunodeficiency and autoinflammaotory disease. Recently, we identified the C-terminus of NEMO to be a regulator of NF-κB signaling, as a class of NEMO mutations lacking the C-terminus (ΔCT-NEMO) confer gain-of-function properties to the IKK complex due to impaired recruitment of A20 to the TNFR. Here, we investigated details by which A20 recruitment to the TNFR suppresses NF-κB signaling. The C-terminus of NEMO has been previously identified to preferentially recognize K63-linked polyubiquitin that in its unanchored form can mediate NEMO/A20 interaction. We found that the IKK complex containing ΔCT-NEMO forms but not other mutant forms of NEMO exhibit impaired K63 polyubiquitin binding ability. In addition to unanchored K63-lilnked polyubiquitin, we hypothesized that direct polyubiquitination of A20 could enable a functional interaction between A20 and NEMO. Using mass spectroscopy, we identified 4 lysine residues on A20 to be ubiquitination sites. Mutation of these lysines to arginine impaired A20’s ability to suppress TNF induced NF-κB activation. We noted stabilization of NEMO in A20 deficient cells following TNF stimulation. Biochemical studies in Jurkat and 293 cells revealed A20 dependent K48 linked polyubiquitination of NEMO at the TNFR following receptor stimulation. Our data suggest a mechanism of NF-κB regulation in which A20 is directly K63 polyubiquitinated to functionally interacts with NEMO which it targets by K48 linkages proteasomal degradation.

Augmented Serum Amyloid A1/2 Mediated by TNF-induced NF-κB in Human Serous Ovarian Epithelial Tumors.

Tumor necrosis factor-α (TNF) is well known to be involved in the immune system and ovarian inflammation. Ovarian cancer is an inflammation-related malignancy that lacks early screening strategies, resulting in late diagnosis followed by high mortality. Based on our previous data, TNF induced abundant serum amyloid A (SAA), an acute phase protein linked to inflammation, in ovarian granulosal cells. To date, the regulation and expression of SAA in ovarian cancer is not fully elucidated. Here, we investigated the relationship between TNF and SAA by comparing human normal ovarian tissues and serous ovarian tumors. We found that SAA1/2 was significantly expressed in tumor tissues, but no or trace expression levels in normal tissues. TNF was also significantly upregulated in ovarian tumor tissues compared to normal tissues. Moreover, TNF significantly increased SAA1/2 levels in human ovarian cancer cell lines, OVCAR-3 and SKOV-3, in a time-dependent manner. Since the SAA1 promoter contains two nuclear factor (NF)-κB sites, we examined whether TNF regulates SAA1 promoter activity. Deletion analysis revealed that the proximal NF-κB site (−95/−85) played a critical role in regulating TNF-induced SAA1 promoter activity. Within 2 h after intraperitoneal injection of lipopolysaccharide, a product known to stimulate release of TNF, SAA preferably localized to ovarian epithelial cells and the thecal-interstitial layers compared to granulosal cell layers. Based on Gene Expression Omnibus (GEO) database, SAA1/2 and TNF were dominantly expressed in advanced grade ovarian cancer. Taken together, the accumulation of SAA1/2 in ovarian cancer could be mediated by TNF-induced NF-κB activation.

Doxycycline is an NF-κB inhibitor that induces apoptotic cell death in malignant T-cells.

Cutaneous T-cell Lymphoma (CTCL) is a rare non-Hodgkin's lymphoma that can affect the skin, blood, and lymph nodes, and can metastasize at late stages. Novel therapies that target all affected disease compartments and provide longer lasting responses while being safe are needed. One potential therapeutic target is NF-λB, a regulator of immune responses and an important participant in carcinogenesis and cancer progression. As a transcription factor, NF-λB targets genes that promote cell proliferation and survival. Constitutive or aberrant activation of NF-λB is encountered in many types of cancer, including CTCL.Recently, while analyzing gene-expression profiles of a variety of small molecule compounds that target NF-λB, we discovered the tetracycline family of antibiotics, including doxycycline, to be potent inhibitors of the NF-λB pathway. Doxycycline is well-tolerated, safe, and inexpensive; and is commonly used as an antibiotic and anti-inflammatory for the treatment a multitude of medical conditions.In our current study, we show that doxycycline induces apoptosis in a dose dependent manner in multiple different cell lines from patients with the two most common subtypes of CTCL, Mycosis Fungoides (MF) and Sézary Syndrome (SS). Similar results were found using primary CD4+ T cells from a patient with SS. Doxycycline inhibits TNF induced NF-λB activation and reduces expression of NF-λB dependent anti-apoptotic proteins, such as BCL2α. Furthermore, we have identified that doxycycline induces apoptosis through reactive oxygen species.

NEMO regulates a cell death switch in TNF signaling by inhibiting recruitment of RIPK3 to the cell death-inducing complex II.

Incontinentia Pigmenti (IP) is a rare X-linked disease characterized by early male lethality and multiple abnormalities in heterozygous females. IP is caused by NF-κB essential modulator (NEMO) mutations. The current mechanistic model suggests that NEMO functions as a crucial component mediating the recruitment of the IκB-kinase (IKK) complex to tumor necrosis factor receptor 1 (TNF-R1), thus allowing activation of the pro-survival NF-κB response. However, recent studies have suggested that gene activation and cell death inhibition are two independent activities of NEMO. Here we describe that cells expressing the IP-associated NEMO-A323P mutant had completely abrogated TNF-induced NF-κB activation, but retained partial antiapoptotic activity and exhibited high sensitivity to death by necroptosis. We found that robust caspase activation in NEMO-deficient cells is concomitant with RIPK3 recruitment to the apoptosis-mediating complex. In contrast, cells expressing the ubiquitin-binding mutant NEMO-A323P did not recruit RIPK3 to complex II, an event that prevented caspase activation. Hence NEMO, independently from NF-κB activation, represents per se a key component in the structural and functional dynamics of the different TNF-R1-induced complexes. Alteration of this process may result in differing cellular outcomes and, consequently, also pathological effects in IP patients with different NEMO mutations.

Abstract 3564: MUC13 protects colorectal cancer cells from death by activating the NF-κb pathway and is a potential therapeutic target

Abstract MUC13 is a transmembrane mucin glycoprotein that is overexpressed by many cancers, although its functions are not fully understood. NF-κB is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently up-regulating BCL-XL. MUC13 promoted TNF-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NEMO. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signalling in response to both TNF and DNA damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to death in response to cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers. Citation Format: Yong H. Sheng, Yaowu He, sumaira Z. hasnain, Ran Wang, Hui Tong, Daniel T. Clarke, Rohan Lourie, Iulia Oancea, kuanyau wong, John W. Lumley, Timothy H. Florin, Philip Sutton, John. D. Hooper, Nigel A. Mcmillan, Michael A. Mcguckin. MUC13 protects colorectal cancer cells from death by activating the NF-κb pathway and is a potential therapeutic target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3564.

The Chemopreventive Phytochemical Moringin Isolated from Moringa oleifera Seeds Inhibits JAK/STAT Signaling.

Sulforaphane (SFN) and moringin (GMG-ITC) are edible isothiocyanates present as glucosinolate precursors in cruciferous vegetables and in the plant Moringa oleifera respectively, and recognized for their chemopreventive and medicinal properties. In contrast to the well-studied SFN, little is known about the molecular pathways targeted by GMG-ITC. We investigated the ability of GMG-ITC to inhibit essential signaling pathways that are frequently upregulated in cancer and immune disorders, such as JAK/STAT and NF-κB. We report for the first time that, similarly to SFN, GMG-ITC in the nanomolar range suppresses IL-3-induced expression of STAT5 target genes. GMG-ITC, like SFN, does not inhibit STAT5 phosphorylation, suggesting a downstream inhibitory event. Interestingly, treatment with GMG-ITC or SFN had a limited inhibitory effect on IFNα-induced STAT1 and STAT2 activity, indicating that both isothiocyanates differentially target JAK/STAT signaling pathways. Furthermore, we showed that GMG-ITC in the micromolar range is a more potent inhibitor of TNF-induced NF-κB activity than SFN. Finally, using a cellular system mimicking constitutive active STAT5-induced cell transformation, we demonstrated that SFN can reverse the survival and growth advantage mediated by oncogenic STAT5 and triggers cell death, therefore providing experimental evidence of a cancer chemopreventive activity of SFN. This work thus identified STAT5, and to a lesser extent STAT1/STAT2, as novel targets of moringin. It also contributes to a better understanding of the biological activities of the dietary isothiocyanates GMG-ITC and SFN and further supports their apparent beneficial role in the prevention of chronic illnesses such as cancer, inflammatory diseases and immune disorders.