NF-κB Inducing Kinase Attenuates Colorectal Cancer by Regulating Noncanonical NF-κB Mediated Colonic Epithelial Cell Regeneration.

BACKGROUND & AIMS: Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-κB-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/differentiation via noncanonical NF-κB signaling that is unique from canonical NF-kB signaling. METHODS: Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis were initiated in whole-body NIK knockout mice (Nik-/-) and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium. RESULTS: Human transcriptomic data revealed dysregulated noncanonical NF-kB signaling. In vitro studies evaluating Nik-/- crypts and organoids derived from mature, nondividing CECs, and colonic stem cells exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik-/- cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed in vivo, Nik-/- mice exhibited more severe colitis with dextran sulfate sodium administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammation-induced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (NikΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (NikΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelAΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelAΔCEC). CONCLUSIONS: Dysregulated noncanonical NF-κB signaling is associated with the development of colorectal cancer in a tissue-dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.

Modulating inflammation through the negative regulation of NF-κB signaling.

Immune system activation is essential to thwart the invasion of pathogens and respond appropriately to tissue damage. However, uncontrolled inflammation can result in extensive collateral damage underlying a diverse range of auto-inflammatory, hyper-inflammatory, and neoplastic diseases. The NF-κB signaling pathway lies at the heart of the immune system and functions as a master regulator of gene transcription. Thus, this signaling cascade is heavily targeted by mechanisms designed to attenuate overzealous inflammation and promote resolution. Mechanisms associated with the negative regulation of NF-κB signaling are currently under intense investigation and have yet to be fully elucidated. Here, we provide an overview of mechanisms that negatively regulate NF-κB signaling through either attenuation of signal transduction, inhibition of posttranscriptional signaling, or interference with posttranslational modifications of key pathway components. While the regulators discussed for each group are far from comprehensive, they exemplify common mechanistic approaches that inhibit this critical biochemical signaling cascade. Despite their diversity, a commonality among these regulators is their selection of specific targets at key inflection points in the pathway, such as TNF-receptor-associated factor family members or essential kinases. A better understanding of these negative regulatory mechanisms will be essential to gain greater insight related to the maintenance of immune system homeostasis and inflammation resolution. These processes are vital elements of disease pathology and have important implications for targeted therapeutic strategies.

Noncanonical NF-κB signaling and the essential kinase NIK modulate crucial features associated with eosinophilic esophagitis pathogenesis.

Eosinophilic esophagitis (EoE) is an allergic disease of the esophagus driven by T cell and eosinophil responses to dietary allergens, resulting in chronic mucosal inflammation. Few spontaneous animal models of esophageal eosinophilia exist, with most studies relying on artificial sensitization procedures. NF-κB-inducing kinase (NIK; MAP3K14) is a key signaling molecule of the noncanonical NF-κB (NFKB1) pathway, an alternative signaling cascade producing chemokines involved in lymphoid stroma development and leukocyte trafficking. Nik-/- mice have been shown to develop a hypereosinophilic syndrome in peripheral blood and major filtering organs; however, the gastrointestinal mucosa of these mice has not been well characterized. We show that Nik-/- mice develop significant, localized eosinophilic esophagitis that mimics human EoE, including features such as severe eosinophil accumulation, degranulation, mucosal thickening, fibrosis and basal cell hyperplasia. The remainder of the GI tract, including the caudal stomach, small intestine and colon, in mice with active EoE are unaffected, also similar to human patients. Gene expression patterns in esophageal tissue of Nik-/- mice mimics human EoE, with thymic stromal lymphopoetin (TSLP) in particular also elevated at the protein level. In gene expression data sets from human biopsy specimens, we further show that many genes associated with noncanonical NF-κB signaling are significantly dysregulated in EoE patients, most notably a paradoxical upregulation of NIK itself with concurrent upregulation of powerful protein-level destabilizers of NIK. These findings suggest that Nik-/- mice could be useful as a spontaneous model of specific features of EoE and highlight a novel role for noncanonical NF-κB signaling in human patients.

ERpS294 is a biomarker of ligand or mutational ERα activation and a breast cancer target for CDK2 inhibition.

ERα phosphorylation at hinge site S294 (pS294) was recently shown to be essential for ER-dependent gene transcription and mediated by an unknown cyclin-dependent kinase (CDK). This study was undertaken to identify the exact CDK pathway mediating pS294 formation, and to determine if this phosphorylation event occurs with, and can be targeted to treat, the ligand-independent growth of breast cancers expressing endocrine-refractory ESR1 mutations. Using a newly developed anti-pS294 monoclonal antibody, a combination of CDK specific siRNA knockdown studies and a broad panel of CDK selective inhibitors against ligand (E2)-stimulated MCF7 cells, we first identified CDK2 as the primary mediator of pS294 formation and showed that CDK2-selective inhibitors like Dinaciclib, but not CDK4/6 inhibitors like Palbociclib, can selectively prevent pS294 formation and repress ER-dependent gene expression. We then expressed the ER-activating mutations ERmut(Y537S) and ERmut(D538G) in MCF7 cells, and demonstrated their ability to induce ligand-independent and tamoxifen-resistant growth, associated with constitutive and CDK2-dependent pS294 expression. Following robust growth of E2-independent and TAM-resistant MCF7mutER(Y537S) tumors in vivo, nude mice were also treated with either Dinaciclib or Palbociclib at doses and injection schedules unable to retard tumor growth as single agents; the TAM plus Palbociclib combination arrested further tumor growth without affecting pS294 formation, while the TAM plus Dinaciclib combination produced tumor regression associated with loss of pS294 expression. These findings, and our proposed mechanistic model, provide new rationale for the clinical evaluation of CDK2 inhibitors given in combination with endocrine agents as a new treatment strategy against ESR1 mutation expressing breast cancers.

TIPS pentacene loaded PEO-PDLLA core-shell nanoparticles have similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in vivo microenvironments.

Nanoparticle based drug delivery platforms have the potential to transform disease treatment paradigms and therapeutic strategies, especially in the context of pulmonary medicine. Once administered, nanoparticles disperse throughout the lung and many are phagocytosed by macrophages. However, there is a paucity of knowledge regarding cellular up-take dynamics of nanoparticles due largely to macrophage heterogeneity. To address this issue, we sought to better define nanoparticle up-take using polarized M1 and M2 macrophages and novel TIPS-pentacene loaded PEO-PDLLA nanoparticles. Our data reveal that primary macrophages polarized to either M1 or M2 phenotypes have similar levels of nanoparticle phagocytosis. Similarly, M1 and M2 polarized macrophages isolated from the lungs of mice following either acute (Th1) or allergic (Th2) airway inflammation also demonstrated equivalent levels of nanoparticle up-take. Together, these studies provide critical benchmark information pertaining to cellular up-take dynamics and biodistribution of nanoparticles in the context of clinically relevant inflammatory microenvironments.

Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.

Aberrant inflammation is a hallmark of inflammatory bowel disease (IBD) and colorectal cancer. IRAK-M is a critical negative regulator of TLR signaling and overzealous inflammation. Here we utilize data from human studies and Irak-m-/- mice to elucidate the role of IRAK-M in the modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M expression is up-regulated during IBD and colorectal cancer. Further functional studies in mice revealed that Irak-m-/- animals are protected against colitis and colitis associated tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune system of Irak-m-/- mice is highly efficient at eliminating microbial translocation following epithelial barrier damage. This attenuation of pathogenesis is associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil migration, and enhanced T-cell recruitment. Further evaluation of Irak-m-/- mice revealed a splice variant that robustly activates NF-κB signaling. Together, these data identify IRAK-M as a potential target for future therapeutic intervention.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

Nonessential Role for the NLRP1 Inflammasome Complex in a Murine Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) elicits the immediate production of proinflammatory cytokines which participate in regulating the immune response. While the mechanisms of adaptive immunity in secondary injury are well characterized, the role of the innate response is unclear. Recently, the NLR inflammasome has been shown to become activated following TBI, causing processing and release of interleukin-1ß (IL-1ß). The inflammasome is a multiprotein complex consisting of nucleotide-binding domain and leucine-rich repeat containing proteins (NLR), caspase-1, and apoptosis-associated speck-like protein (ASC). ASC is upregulated after TBI and is critical in coupling the proteins during complex formation resulting in IL-1ß cleavage. To directly test whether inflammasome activation contributes to acute TBI-induced damage, we assessed IL-1ß, IL-18, and IL-6 expression, contusion volume, hippocampal cell death, and motor behavior recovery in Nlrp1(-/-), Asc(-/-), and wild type mice after moderate controlled cortical impact (CCI) injury. Although IL-1ß expression is significantly attenuated in the cortex of Nlrp1(-/-) and Asc(-/-) mice following CCI injury, no difference in motor recovery, cell death, or contusion volume is observed compared to wild type. These findings indicate that inflammasome activation does not significantly contribute to acute neural injury in the murine model of moderate CCI injury.

The NLRP1 inflammasome attenuates colitis and colitis-associated tumorigenesis.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a diverse family of pattern recognition receptors that are essential mediators of inflammation and host defense in the gastrointestinal system. Recent studies have identified a subgroup of inflammasome forming NLRs that modulate the mucosal immune response during inflammatory bowel disease (IBD) and colitis associated tumorigenesis. To better elucidate the contribution of NLR family members in IBD and cancer, we conducted a retrospective analysis of gene expression metadata from human patients. These data revealed that NLRP1, an inflammasome forming NLR, was significantly dysregulated in IBD and colon cancer. To better characterize the function of NLRP1 in disease pathogenesis, we used Nlrp1b(-/-) mice in colitis and colitis-associated cancer models. In this paper, we report that NLRP1 attenuates gastrointestinal inflammation and tumorigenesis. Nlrp1b(-/-) mice demonstrated significant increases in morbidity, inflammation, and tumorigenesis compared with wild-type animals. Similar to data previously reported for related inflammasome forming NLRs, the increased inflammation and tumor burden was correlated with attenuated levels of IL-1ß and IL-18. Further mechanistic studies using bone marrow reconstitution experiments revealed that the increased disease pathogenesis in the Nlrp1b(-/-) mice was associated with nonhematopoietic-derived cells and suggests that NLRP1 functions in the colon epithelial cell compartment to attenuate tumorigenesis. Taken together, these data identify NLRP1 as an essential mediator of the host immune response during IBD and cancer. These findings are consistent with a model whereby multiple NLR inflammasomes attenuate disease pathobiology through modulating IL-1ß and IL-18 levels in the colon.

Caspase-11 attenuates gastrointestinal inflammation and experimental colitis pathogenesis.

Nucleotide-binding domain and leucine-rich repeat containing protein inflammasome formation plays an essential role in modulating immune system homeostasis in the gut. Recently, a caspase-11 noncanonical inflammasome has been characterized and appears to modulate many biological functions that were previously considered to be solely dependent on caspase-1 and the canonical inflammasome. To better elucidate the function of this noncanonical inflammasome during inflammatory bowel disease, experimental colitis was induced in wild-type and Casp11(-/-) mice utilizing dextran sulfate sodium (DSS). Here, we report that caspase-11 attenuates acute experimental colitis pathogenesis. Casp11(-/-) mice showed significantly increased morbidity and colon inflammation following DSS exposure. Subsequent cytokine analysis revealed that IL-1ß and IL-18 levels in the colon were significantly reduced in the Casp11(-/-) mice compared with the wild-type animals. Additional mechanistic studies utilizing IL-1ß and IL-18 reconstitution revealed that Casp11(-/-) hypersensitivity was associated with the loss of both of these cytokines. Bone marrow reconstitution experiments further revealed that caspase-11 gene expression and function in both hematopoietic- and nonhematopoietic-derived cells contribute to disease attenuation. Interestingly, unlike caspase-1, caspase-11 does not appear to influence relapsing remitting disease progression or the development of colitis-associated tumorigenesis. Together, these data identify caspase-11 as a critical factor protecting the host during acute DSS-induced colonic injury and inflammation but not during chronic inflammation and tumorigenesis.

Sources of superoxide/H2O2 during mitochondrial proline oxidation.

p53 Inducible gene 6 (PIG6) encodes mitochondrial proline dehydrogenase (PRODH) and is up-regulated several fold upon p53 activation. Proline dehydrogenase is proposed to generate radicals that contribute to cancer cell apoptosis. However, there are at least 10 mitochondrial sites that can produce superoxide and/or H2O2, and it is unclear whether proline dehydrogenase generates these species directly, or instead drives production by other sites. Amongst six cancer cell lines, ZR75-30 human breast cancer cells had the highest basal proline dehydrogenase levels, and mitochondria isolated from ZR75-30 cells consumed oxygen and produced H2O2 with proline as sole substrate. Insects use proline oxidation to fuel flight, and mitochondria isolated from Drosophila melanogaster were even more active with proline as sole substrate than ZR75-30 mitochondria. Using mitochondria from these two models we identified the sites involved in formation of superoxide/H2O2 during proline oxidation. In mitochondria from Drosophila the main sites were respiratory complexes I and II. In mitochondria from ZR75-30 breast cancer cells the main sites were complex I and the oxoglutarate dehydrogenase complex. Even with combinations of substrates and respiratory chain inhibitors designed to minimize the contributions of other sites and maximize any superoxide/H2O2 production from proline dehydrogenase itself, there was no significant direct contribution of proline dehydrogenase to the observed H2O2 production. Thus proline oxidation by proline dehydrogenase drives superoxide/H2O2 production, but it does so mainly or exclusively by providing anaplerotic carbon for other mitochondrial dehydrogenases and not by producing superoxide/H2O2 directly.

RPL24: a potential therapeutic target whose depletion or acetylation inhibits polysome assembly and cancer cell growth.

Partial loss of large ribosomal subunit protein 24 (RPL24) function is known to protect mice against Akt or Myc-driven cancers, in part via translational inhibition of a subset of cap(eIF4E)-dependently translated mRNAs. The role of RPL24 in human malignancies is unknown. By analyzing a public dataset of matched human breast cancers and normal mammary tissue, we found that breast cancers express significantly more RPL24 than matched normal breast samples. Depletion of RPL24 in breast cancer cells by >70% reduced cell viability by 80% and decreased protein expression of the eIF4E-dependently translated proteins cyclin D1 (75%), survivin (46%) and NBS1 (30%) without altering GAPDH or beta-tubulin levels. RPL24 knockdown also reduced 80S subunit levels relative to 40S and 60S levels. These effects on expression of eIF4E-dependent proteins and ribosome assembly were mimicked by 2-24 h treatment with the pan-HDACi, trichostatin A (TSA), which induced acetylation of 15 different polysome-associated proteins including RPL24. Furthermore, HDAC6-selective inhibition or HDAC6 knockdown induced ribosomal protein acetylation. Via mass spectrometry, we found that 60S-associated, but not, polysome-associated, RPL24 undergoes HDACi-induced acetylation on K27. Thus, RPL24 K27 acetylation may play a role in ribosome assembly. These findings point toward a novel acetylation-dependent polysome assembly mechanism regulating tumorigenesis.

mTORC1/C2 and pan-HDAC inhibitors synergistically impair breast cancer growth by convergent AKT and polysome inhibiting mechanisms.

Resistance of breast cancers to targeted hormone receptor (HR) or human epidermal growth factor receptor 2 (HER2) inhibitors often occurs through dysregulation of the phosphoinositide 3-kinase, protein kinase B/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Presently, no targeted therapies exist for breast cancers lacking HR and HER2 overexpression, many of which also exhibit PI3K/AKT/mTOR hyper-activation. Resistance of breast cancers to current therapeutics also results, in part, from aberrant epigenetic modifications including protein acetylation regulated by histone deacetylases (HDACs). We show that the investigational drug MLN0128, which inhibits both complexes of mTOR (mTORC1 and mTORC2), and the hydroxamic acid pan-HDAC inhibitor TSA synergistically inhibit the viability of a phenotypically diverse panel of five breast cancer cell lines (HR-/+, HER2-/+). The combination of MLN0128 and TSA induces apoptosis in most breast cancer cell lines tested, but not in the non-malignant MCF-10A mammary epithelial cells. In parallel, the MLN0128/TSA combination reduces phosphorylation of AKT at S473 more than single agents alone and more so in the 5 malignant breast cancer cell lines than in the non-malignant mammary epithelial cells. Examining polysome profiles from one of the most sensitive breast cancer cell lines (SKBR3), we demonstrate that this MLN0128/TSA treatment combination synergistically impairs polysome assembly in conjunction with enhanced inhibition of 4eBP1 phosphorylation at S65. Taken together, these data indicate that the synergistic growth inhibiting consequence of combining a mTORC1/C2 inhibitor like MLN0128 with a pan-HDAC inhibitor like TSA results from their mechanistic convergence onto the PI3K/AKT/mTOR pathway, profoundly inhibiting both AKT S473 and 4eBP1 S65 phosphorylation, reducing polysome formation and cancer cell viability.