Activation Of NF-kB Signaling Pathway Research Articles

Adipose tissue-derived small extracellular vesicles modulate macrophages to improve the homing of adipocyte precursors and endothelial cells in adipose tissue regeneration.

Rapid infiltration of endogenous cells induced by cell-free biomaterials is the first and crucial step in tissue regeneration and macrophage is largely involved. Our previous study reported adipose tissue-derived small extracellular vesicles (sEV-AT) could successfully promote adipose tissue regeneration. However, the role of macrophages in this process was unknown. In this study, we isolated sEV-AT and subcutaneously implanted it into the back of SD rats. The results showed sEV-AT increased macrophage infiltration significantly, which was followed by improving homing of adipocyte precursors (APs) and endothelial cells (ECs). However, when macrophages were depleted by clodronate liposome within 1 week, the homing of APs and ECs, and adipose tissue regeneration were destroyed. In vitro, sEV-AT showed the ability to promote the migration of macrophages directly. Besides, sEV-AT-pretreated macrophages improved the migration of APs and ECs, accompanied by the increase of chemokines (MCP-1, SDF-1, VEGF, and FGF) and the activation of NF-kB signaling pathway. These findings indicated sEV-AT might regulate the secretion of chemokines via activating NF-kB signaling pathway to improve homing of APs and ECs and facilitate adipose tissue regeneration. These findings deepened our understanding of small extracellular vesicle-induced tissue regeneration and laid a theoretical foundation for the clinical application of sEV-AT.

Comparative assessment on the probable mechanisms underlying the hepatorenal toxicity of commercial imidacloprid and hexaflumuron formulations in rats

Pesticides are viewed as a major wellspring of ecological contamination and causing serious risky consequences for people and animals. Imidacloprid (IM) and hexaflumuron (HFM) are extensively utilized insect poisons for crop assurance on the planet. A few investigations examined IM harmfulness in rodents, but its exact mechanism hasn’t been mentioned previously as well as the toxicity of HFM doesn’t elucidate yet. For this reason, the present study was designed to explore the mechanism of each IM and HFM–evoked rat liver and kidney toxicity and to understand its molecular mechanism. 21 male Wistar albino rats were divided into 3 groups, as follows: group (1), normal saline; group (2), IM; and group (3), HFM. Both insecticides were orally administered every day for 28 days at a dose equal to 1/10 LD50 from the active ingredient. After 28 days postdosing, rats were anesthetized to collect blood samples then euthanized to collect liver and kidney tissue specimens. The results showed marked changes in walking, body tension, alertness, and head movement with a significant reduction in rats’ body weight in both IM and HFM receiving groups. Significant increases in MDA levels and decrease of GHS levels were recorded in liver and kidney homogenates of either IM or HFM groups. Liver and kidney tissues obtained from both pesticide receiving groups showed extensive histopathological alterations with a significant increase in the serum levels of ALT, AST, urea, and creatinine and a decrease in total proteins, albumin, and globulin levels. In addition, there was upregulation of the transcript levels of casp-3, JNK, and HO-1 genes with strong immunopositivity of casp-3, TNF-ὰ, and NF-KB protein expressions in the liver and kidneys of rats receiving either IM or HFM compared with the control group. In all studied parameters, HFM caused hepatorenal toxicity more than those induced by IM. We can conclude that each IM and HFM provoked liver and kidneys damage through overproduction of ROS, activation of NF-KB signaling pathways and mitochondrial/JNK-dependent apoptosis pathway.

MiR-142a-3p Enhances FlaA N/C Protection Against Radiation-Mediated Intestinal Injury by Modulating the IRAK1/NF-κB Signaling Pathway

Our purpose was to investigate the role of recombinant protein flagellin A N/C (FlaA N/C) protein-mediated pyroptosis inhibition and related miRNA in radiation protection. Mice received 10 Gy irradiation after FlaA N/C pretreatment, IRAK-1/4 Inhibitor I treatment, or pyrrolidine dithiocarbamate treatment. Human intestinal epithelial cells (HIEC) received 10 Gy irradiation after FlaA N/C pretreatment, overexpressed miR-142a-3p with miR-142a-3p mimics, or inhibited miR-142a-3p with miR-142a-3p inhibitor. Mouse & Rat miRNA OneArray determined the change in relevant miRNA after FlaA N/C pretreatment; real-time polymerase chain reaction detected IRAK1 and miR-142a-3p expression; a CCK-8 assay evaluated cell viability; LDH release analyzed cytotoxicity; caspase-1 activity assay, interleukin-1β level, and flow cytometry analyzed pyroptosis in cells; hematoxylin-eosin staining evaluated the damage to intestinal tissue; CO-IP detected the inflammation activation; immunohistochemistry, Western blot analysis, and immunofluorescence analyzed activation of pyroptosis-related proteins and the activation of NF-kB signaling pathways; and luciferase reporter assay and fluorescence in situ hybridization detected the interaction between miR-142a-3p and IRAK1. FlaA N/C reduced radiation-induced pyroptosis in vivo and in vitro, and miR-142a-3p expression increased after FlaA N/C pretreatment. Upregulating the expression of miR-142a-3p inhibited radiation-induced pyroptosis in HIEC, and downregulating the expression of miR-142a-3p led to radiation-induced pyroptosis in HIEC after FlaA N/C pretreatment. IRAK1 was a direct target of miR-142a-3p and played an important role in radiation-induced pyroptosis in HIEC. Inhibiting IRAK1 reduced radiation-mediated pyroptosis in mice intestines. miR-142a-3p downregulated IRAK1 and suppressed the NF-kB pathway. Inhibiting the NF-kB signaling pathway can reduce radiation-mediated pyroptosis in mice intestines. Our findings indicated this new radioprotectant protein regulates miR-142a-3p, effectively inhibiting radiation-induced pyroptosis mediated by the IRAK1/NF-κB signaling pathway in intestinal cells.

Chemerin facilitates intervertebral disc degeneration via TLR4 and CMKLR1 and activation of NF-kB signaling pathway

Now days, obesity is a major risk factor for intervertebral disc degeneration (IDD). However, adipokine, such as chemerin is a novel cytokine, which is secreted by adipose tissue, and are thought to be played major roles in various degenerative diseases. Obese individuals are known to have high concentration of serum chemerin. Our purpose was to study whether chemerin acts as a biochemical relationship between obesity, and IDD. In this study, we found that the expression level of chemerin was significantly increased in the human degenerated nucleus pulposus (NP) tissues, and had higher level in the obese people than the normal people. Chemerin significantly increased the inflammatory mediator level, contributing to ECM degradation in nucleus pulposus cells (NPCs). Furthermore, chemerin overexpression aggravates the puncture-induced IVDD progression in rats, while knockdown CMKLR1 reverses IVDD progression. Chemerin activates the NF-kB signaling pathway via its receptors CMKLR1, and TLR4 to release inflammatory mediators, which cause matrix degradation, and cell aging. These findings generally provide novel evidence supporting the causative role of obesity in IDD, which is essentially important to literally develop novel preventative or generally therapeutic treatment in the disc degenerative disorders.

Oral glutamine supplementation attenuates inflammation and oxidative stress-mediated skeletal muscle protein content degradation in immobilized rats: Role of 70 kDa heat shock protein

Skeletal muscle disuse results in myofibrillar atrophy and protein degradation, via inflammatory and oxidative stress-mediated NF-kB signaling pathway activation. Nutritional interventions, such as l-glutamine (GLN) supplementation have shown antioxidant properties and cytoprotective effects through the modulation on the 70-kDa heat shock protein (HSP70) expression. However, these GLN-mediated effects on cell signaling pathways and biochemical mechanisms that control the myofibrillar protein content degradation in muscle disuse situations are poorly known yet. This study investigated the effects of oral GLN plus l-alanine (ALA; GLN ​+ ​ALA-solution) supplementation, either in their free or dipeptide (L-alanyl-l-glutamine-DIP) form, on GLN-glutathione (GSH) axis and cytoprotection mediated by HSP70 protein expression in the slow-twitch soleus and fast-twitch gastrocnemius skeletal muscle of rats submitted to 14-days of hindlimb immobilization-induced disuse muscle atrophy. Forty-eight Wistar rats were distributed into 6 groups: hindlimb immobilized (IMOB group) and hindlimb immobilized orally supplemented with either GLN (1 g kg−1) plus ALA (0.61 g kg−1) ​(GLN ​+ ​ALA-IMOB group) or 1.49 ​g ​kg−1 of DIP (DIP-IMOB group) and; no-immobilized (CTRL) and no-immobilized supplemented GLN ​+ ​ALA and DIP baselines groups. All animals, including CTRL and IMOB rats (water), were supplemented via intragastric gavage for 14 days, concomitantly to immobilization period. Plasma and muscle GLN levels, lipid (thiobarbituric acid reactive substances-TBARS) and protein (carbonyl) peroxidation, erythrocyte concentration of reduced GSH and GSH disulfide (GSSG), plasma and muscle pro-inflammatory TNF-α levels, muscle IKKα/β-NF-kB signaling pathway and, the myofibrillar protein content (MPC) were measured. The MPC was significantly lower in IMOB rats, compared to CTRL, GLN ​+ ​ALA, and DIP animals (p ​< ​0.05). This finding was associated with reduced plasma and muscle GLN concentration, equally in IMOB animals. Conversely, both GLN ​+ ​ALA and DIP supplementation restored plasma and muscle GLN levels, which equilibrated GSH and intracellular redox status (GSSG/GSH ratio) in erythrocytes and skeletal muscle even as, increased muscle HSP70 protein expression; attenuating oxidative stress and TNF-α-mediated NF-kB pathway activation, fact that reverberated on reduction of MPC degradation in GLN ​+ ​ALA-IMOB and DIP-IMOB animals (p ​< ​0.05). In conclusion, the findings shown herein support the oral GLN ​+ ​ALA and DIP supplementations as a therapeutic and effective nutritional alternative to attenuate the deleterious effects of the skeletal muscle protein degradation induced by muscle disuse.

Abstract 140: Neuronal Prorenin Receptor Deletion Attenuates Astrogliosis and Inflammation in the Arcuate Nucleus of Mice Fed With High Fat Diet

The (pro)renin receptor (PRR) is a key component of the brain renin-angiotensin system and plays a pivotal role in the development of hypertension. However, its importance in high fat diet (HFD)-induced metabolic physiology is not well understood. We recently showed that neuron-specific PRR deletion (PRRKO) improves fasting blood glucose and glucose tolerance in mice under 16 weeks of HFD. However, the molecular mechanisms by which PRR acts remain unclear. Previous research showed that astrogliosis, characterized by enhanced glial fibrillary acid protein (GFAP) expression, is a result of HFD treatment. NF-kB signaling pathway activation leads to hypothalamic inflammation and astrogliosis. Accordingly, we hypothesized that PRR deletion in the neurons improves type II diabetes by regulating astrogliosis and inflammation in the arcuate nucleus (ARC) during HFD. To test our hypothesis, PRRKO and wildtype (WT) littermates were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) with matching calories for 16 weeks. Brain tissues were immunolabeled with GFAP and NF-kB p65 and presented as relative fluorescence unit, RFU. We found that in the NFD groups, the GFAP immunolabelling was similar in the ARC (3.9 ± 1.1 vs. 2.2 ± 0.7) between WT and PRRKO mice, suggesting that PRRKO has no effect on astrocyte status in a NFD condition. Following 16 weeks of HFD, the GFAP intensity (7.6 ± 2.2, p=0.04) was significantly increased in the WT mice. PRR deletion in the neurons significantly reduced the HFD-induced astrogliosis in the ARC (4.0 ± 0.6, p=0.02) compared with the WT mice fed HFD. The NF-ĸB p65 level was elevated in the ARC of WT mice following 16 weeks of HFD compared with the NFD (13.1 ± 1.6 vs. 5.4 ± 0.6, p&lt;0.0001). Interestingly, PRR deletion prevented the elevation of NF-ĸB p65 levels induced by HFD (6.7 ± 0.7, p&lt;0.0001). Furthermore, astrocyte specific NF-ĸB p65 levels were higher in the WT mice fed a HFD compared to NFD (77.6 ± 1.6 vs. 14.1 ± 2.9, p&lt;0.0001). Notably, under HFD treatment, PRRKO significantly reduced the astrocytic NF-ĸB p65 levels compared with the WT mice (33.1 ± 6.3, p&lt;0.0001). Our data indicate that PRRKO attenuates HFD-induced type II diabetes and is associated with reduction in astrogliosis and NF-kB activation in the ARC.

669 Paeoniflorin suppresses keratin 17 expression via NF-kB pathway and exerts anti-psoriatic effect

Psoriasis, affecting 2%-3% of the population worldwide, is an autoimmune inflammatory skin disease characterized by hyperproliferation of epidermal keratinocytes. Regarding the complexity of disease pathogenesis, there is still no curative therapy for psoriasis. Paeoniflorin (PF), a traditional Chinese herbal medicine, which is the main bioactive component of the total glucosides of peony, is extracted from Paeonia lactiflora Pall (Family: Ranunculaceae). This agent has been shown to have various pharmacological effects, including anti-inflammatory, anti-phlogistic, and anti-apoptotic effects. In the present study, we aimed to illustrate the effect and mechanism of PF on keratinocyte proliferation and immunological activity. In IMQ-induced psoriasis-like lesions, epidermal thickness was reducing to 50% as compared with control groups after topical application of PF. Consistently, dermal infiltrated lymphocytes was markedly reduce. Here we showed that PF could inhibit the proliferation of human keratinocyte cell line HaCaT in a dose-dependent manner. To further clarify the related mechanism, we evaluated the effect of PF on the expression of psoriasis-related hyperproliferation marker K17. Interestingly, K17 protein and mRNA levels were significantly downregulated in HaCaT cells after application of PF. Besides, PF showed inhibitory effect on the inflammatory function of keratinocytes, representing by reduced activation of NF-kB signaling pathways, such as phosphorylated NF-kB p65 and phosphoryalated-IkB. Together, our findings uncovers a previous unrecognized role of PF on inhibiting the proliferation and inflammation of keratinocytes by targeting K17, suggesting that PF might be a potential target in the treatment of psoriasis.

UCHL1 regulates Interleukin‐6 expression in skeletal muscles

Chronic low grade inflammation is a hallmark of aging and has been linked to aging related dysfunction and disease. As the largest tissue mass, skeletal muscles express and release several inflammatory cytokines including interleukin‐6 (IL‐6). With altered function and metabolism, aged muscles can be a major source of inflammatory cytokine in aging. Ubiquitin C‐terminal Hydrolase L1 (UCHL1) was originally found in the brain. Our recent study revealed that UCHL1 is inducible and upregulated in skeletal muscles in mice by denervation and lipopolysaccharide (LPS). In differentiated C2C12 cells. UCHL1 gene knockdown using siRNA significantly increased Interleukin 6 (IL‐6) level in the media. Consistent with this in vitro data, the plasma level of IL‐6 was also higher in mice with global or muscle specific UCHL1 gene knockout. Moreover, UCHL1 gene knockdown in C2C12 significantly reduced the protein level of IκB, a key inhibitory molecule for the activation of NF‐kB signaling pathway. Reduced protein level of IκB was also observed in muscles from UCHL1 knockout mice. These data suggest a potential novel mechanism by which UCHL1 controls IL‐6 expression in skeletal muscle by stabilizing IκB and thus inhibiting activation of NF‐kB pathway. Given the impaired UCHL1 function in the brain with aging, it is plausible to hypothesize that aging may also reduce the UCHL1 control of NF‐kB activation in skeletal muscle, leading to muscle‐derived chronic inflammation in aging.Support or Funding InformationThis study is supported by NIH grant 1R15HL118696 and 1R03AG051926, the USD BBS graduate program, USD CBBRe pilot grant, and USD PQCD pilot grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Upregulation of Pro-inflammatory Cytokine Expression Following Chronic Paracetamol Treatment in Astrocyte.

The present study aimed to investigate the effect of APAP treatment on the expression of pro-inflammatory cytokines in the astrocytes. The mouse astrocyte cells (C8-D1A) were treated with APAP at the concentration of 100μM for 24h, 16 and 28days. The expressions of pro-inflammatory cytokines and NF-kB were determined using western blot analysis. Furthermore, the expression and localization of phosphorylation of NF-kB were detected by immunohistochemical and immunofluorescent analysis. The ultrastructure of C8-D1A cells was as well monitored. The results revealed that acute APAP treatment (24h) had no effect on the expression of pro-inflammatory cytokines and pNF-kB. This treatment did not alter the ultrastructure of C8-D1A cells when compared with those in the control cells. However, the results obtained from the study on chronic APAP-treated cells (16 and 28days) showed the different effect of APAP treatment. The results obtained from western blot analysis showed the increment of pro-inflammatory cytokine (IL-1β and TNF-α) expressions and the activation of NF-kB signaling pathway. Nuclear translocation of pNF-kB and alteration of several cell structures were well observed in the C8-D1A cells with chronic APAP treatment. The results obtained from this study suggest that chronic APAP treatment can induce an upregulation of pro-inflammatory cytokines (IL-1β and TNFα) in astrocytes. This alteration implies the involvement of the activation of NF-kB signaling pathway.

Baicalin Alleviates Lipopolysaccharide-Induced Liver Inflammation in Chicken by Suppressing TLR4-Mediated NF-κB Pathway.

As a kind of potent stimulus, lipopolysaccharide (LPS) has the ability to cause cell damage by activating toll-like receptor(TLR)4, then nuclear factor kappa B (NF-κB) translocates into the nucleus and changes the expression of related inflammatory genes. Baicalin is extracted from Radix Scutellariae, which possesses anti-inflammation, antioxidant and antibacterial properties. However, the effects of it on LPS-induced liver inflammation have not been fully elucidated. This study aims to investigate the anti-inflammatory effects of Baicalin on the LPS-induced liver inflammation and its underlying molecular mechanisms in chicken. The results of histopathological changes, serum biochemical analysis, NO levels and myeloperoxidase activity showed that Baicalin pretreatment ameliorated LPS-induced liver inflammation. ELISA and qPCR assays showed that Baicalin dose-dependently suppressed the production of IL-1β, IL-6, and TNF-α. Furthermore, the mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were significantly decreased by Baicalin. TLR4 is an important sensor in LPS infection. Molecular studies showed that the expression of TLR4 was inhibited by Baicalin pretreatment. In addition, Baicalin pretreatment inhibited NF-kB signaling pathway activation. All results demonstrated the protective effects of Baicalin pretreatment against LPS-induced liver inflammation in chicken via negative regulation of inflammatory mediators through the down-regulation of TLR4 expression and the inhibition of NF-kB activation.

P771 The recombinant NZ9000SHD-5 attenuates the inflammation and mucosal lesions in dextran sodium sulfate-induced colitis

Background: Defensin, as a family member of antimicrobial peptide, plays an important role in host immunity and maintaining the gut barrier function. Meanwhile, probiotics were reported to be a protective role in patients of IBD. Methods: In this study, we optimized a defensin mHD-5 mainly consisted by the mature peptide of human defensin 5 (HD-5) and constructed vector-pN8148-SHD-5 and transfected into Lactococcus lactis to build the recombinant NZ9000SHD-5 based on the nisin-controlled gene expression and gene splicing technology. Results: We found that recombinant NZ9000SHD-5 attenuated imflammatory cells infiltration, histopathological changes in colonic gland, and protected the intensity of epitheal cells in DSS-induced colitis. Recombinant NZ9000SHD-5 remarkably suppressed the production of inflammatory cytokine, such as interleukin-1B (IL-1B), IL-6 and tumor necrosis factor-alpha (TNF-α). Meamwhile, NZ9000SHD-5 increased the expression of zonula occcludens-1 and occluding mRNA and proteins, and it decreased the permeability of FITC-D while preserving the structure and function of Tight junctions in the DSS-induced models. We tested the mechanism of protective effects of NZ9000SHD-5 on LPS-induced model of mouse macrophage RAW264.7 cell. Pretreated with the supernatant of NZ9000SHD-5 for 4h before exposed to LPS significantly reduce the concentrations of IL-6, IL-1B and TNF-α, inhibited the expression of LPS-induced phosphorylation nuclear transcription factor-kappa B (NF-κB) p65 protein and its inhibitor IκBα of NF-κB signaling pathways. In accordance with the protective effect of NZ9000SHD-5 on gut barrier of DSS-induced colitis, we observed the positive effect of NZ9000SHD-5 in mucosal injury and the disruption of epithelial barrier on Caco-2 cells. We found that NZ9000SHD-5 increased the transepithelial electrical resistance, and it decreased the permeability of FITC-D and the damage the of skeleton of TJs against the dextran sodium sulfate Conclusions: The results indicate that the NZ9000SHD-5 relieves the changes in DSS-induced mucosal damage and paracellular permeability possibly through attenuating the colon inflammation by downregulating the activation of NF-KB signaling pathway, and therefore the administration of NZ9000SHD-5 may be a possible option for colitis.

A Fermented Whole Grain Prevents Lipopolysaccharides-Induced Dysfunction in Human Endothelial Progenitor Cells.

Endogenous and exogenous signals derived by the gut microbiota such as lipopolysaccharides (LPS) orchestrate inflammatory responses contributing to development of the endothelial dysfunction associated with atherosclerosis in obesity, metabolic syndrome, and diabetes. Endothelial progenitor cells (EPCs), bone marrow derived stem cells, promote recovery of damaged endothelium playing a pivotal role in cardiovascular repair. Since healthy nutrition improves EPCs functions, we evaluated the effect of a fermented grain, Lisosan G (LG), on early EPCs exposed to LPS. The potential protective effect of LG against LPS-induced alterations was evaluated as cell viability, adhesiveness, ROS production, gene expression, and NF-kB signaling pathway activation. Our results showed that LPS treatment did not affect EPCs viability and adhesiveness but induced endothelial alterations via activation of NF-kB signaling. LG protects EPCs from inflammation as well as from LPS-induced oxidative and endoplasmic reticulum (ER) stress reducing ROS levels, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defense. Moreover, LG pretreatment prevented NF-kB translocation from the cytoplasm into the nucleus caused by LPS exposure. In human EPCs, LPS increases ROS and upregulates proinflammatory tone, proapoptotic factors, and antioxidants. LG protects EPCs exposed to LPS reducing ROS, downregulating proinflammatory and proapoptotic factors, and strengthening antioxidant defenses possibly by inhibiting NF-κB nuclear translocation.

Smoothened (SMO) Is an Adaptor Protein That Recruits TRAF6 and Phospholipase C Gamma 2 (PLCg2) to Enhance the Activation of NF-Kb Signaling Pathway

Background: Constitutive activation of NF-κB signaling is a hallmark of DLBCL. Activation of NF-κB is a multifactorial process resulting from oncogenic mutations (CARD11, MYD88…), chromosomal abnormalities, chronic activation of B-cell receptor signaling (BCR) as well as stimuli from the microenvironment. Chronic activation of BCR is not only the result of gene mutations (e.g.CD79B) but also is the result of stimuli generated from the lymphoma microenvironment.We previously found that smoothened (SMO), transducer of hedgehog (Hh) signaling, enhanced NF-κB activation in lymphoma cells, independently of the presence of oncogenic mutations (Changju et al., Blood 2013). Hh ligands are provided by the lymphoma microenvironment (e.g. stromal cells) to activate SMO in the lymphoma cells. Activated SMO, recruits and activates trimeric-G-coupled proteins to activate PKCβ/CARMA1/TRAF6/NEMO axis, followed by assembling of the CARMA1/BCL10/MALT1/TRAF6 complex to SMO resulting in NF-κB activation. Now, we reveal an additional mechanism by which SMO further contributes to the activation of NF-κB in DLBCL.Summary of results: We explored if recombinant Hh ligand can activate NF-κB signaling in the presence of the BTK inhibitor (PCI-32765). Hh stimulation resulted in phosphorylation of PLCg2 (downstream of BTK) and partially rescued the inhibitory effect of the BTK inhibitor on phosphorylation of PLCg2 suggesting a crosstalk between SMO and BCR receptor signaling. We identified that SMO forms a complex with TRAF-6 and PLCg2 using immunoprecipitation and Duolink in situ proximity ligation assays. We found that SMO stabilizes and protects TRAF-6 from proteosomal degradation. SMO-dependent TRAF6 stabilization is mediated by the ubiquitin-specific protease-8 (USP-8) by removing ubiquitin from K48-linked lysine of TRAF6.Conclusions: Collectively, our data reveals multilayer crosstalk between Hh and BCR/NF-κB pathways and provide significant insights into how SMO contributes to chemotolerance and progression of DLBCL. We expect that our results will delineate novel molecular mechanisms involved in the pathobiology of DLBCL that may serve as therapeutic targets. DisclosuresVega:Seatle Genetics: Honoraria; NIH: Research Funding.

Abstract LB-186: Twist-1 overexpression modulates cancer progression according to the microsatellite instability status in colorectal cancer cell lines

Abstract Background: The Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal cells. EMT has also been shown to occur in the initiation of metastasis for cancer progression. Twist-1 has driving effects in cancer metastasis and is involved in the process of EMT. Overexpression of Twist-1 is common in metastatic carcinomas. Moreover, Twist-1 plays an important role in some physiological processes involved in metastasis, like tumor cell migration, angiogenesis, invasion, chromosomal instability and maintenance of cancer stem cells via the induction of EMT. In this study, we evaluated if Twist-1 and its downstream signaling cascades induced activation of EMT according to the microsatellite instability status in colon cancer cell lines. Methods: To understand this mechanism, LS513 (MSS) and LoVo (MSI) cells were transfected with Twist-1-pCMV plasmids and GFP-pCMV plasmid was used as negative control. Western blots were used to detect expression of EMT related proteins. Cell proliferation assay, cell migration assay, cell invasion assay, and colony forming assay were used to confirm the metastatic effect by Twist-1 overexpression. Also, we examined tumorigenesis assay in vivo to evaluate cancer growth induced by Twist-1 overexpression. Results: The expression of E-cadherin was down-regulated, but that of vimentin was up-regulated in both Twist overexpressed-LS513 and LoVo cell lines. There were mesenchymal morphological changes such as spindle like shape in both cell lines. We found that overexpression of Twist-1 induced downregulation of E-cadherin and upregulation of β-catenin via AKT phosphorylation in these cells. Also, we identified the upregulation of NF-kB as a mediator of the cancer progression in Twist-1 overexpressed LS513 cells. However, the expression of NF-kB decreased in Twist-1 overexpressed LoVo cell lines. Cell proliferation, migration, invasion and in vivo tumorigenecity were increased more in Twist-1 activated cells than negative control cells in LS513 cell line. However, there were no differences of colony forming assay and in vivo tumor growth between Twist-1 activated cells and negative control cells in LoVo cell line although cell proliferation, cell migration, and cell invasion were increased in Twist-1 activated cell lines. Conclusion: Twist-1 overexpression could induce activation of EMT in colon cancer cell lines. NF-kB played as a mediator of cancer progression in Twist-1 overexpressed LS513 (MSS) cells. But, NF-kB expression was down-regulated in Twist-1 overexpressed-LoVo (MSI) cells. These results suggest that upregulation of Twist-1 is associated with colon cancer progression via activation of NF-kB signaling pathway in MSS colon cancers but not in MSI colon cancers. Citation Format: Sungwon Jung, Hye Kyung Hong, Bo Young Oh, So Young Kim, Heunmin Song, Heewon Park, Woo Yong Lee, Yong Beom Cho. Twist-1 overexpression modulates cancer progression according to the microsatellite instability status in colorectal cancer cell lines. [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 LB-186. doi:10.1158/1538-7445.AM2014-LB-186

Examination of Genetic Aberrations in Chlamydophila Psittaci negative MALT Lymphomas of the Ocular Adnexa

AbstractAbstract 1569Ocular adnexal mucosa associated lymphoid tissue lymphomas (OAMALTL) are the most common lymphomas of the eye. The etiology and pathogenesis of ocular adnexa MALT lymphomas (OAMALTL) are still unknown and the association with Chlamydophila psittaci (C. psittaci) has been shown in only some geographic regions. Only few small studies specifically examined for the presence of t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH, t(14;18)(q32;q21)/IGH-MALT1, t(3;14)(p13;q32)/FOXP1-IGH translocations or for TNFAIP3 (A20) mutations/deletions, which may contribute to the activation of canonical nuclear factor (NF)-kB pathway in OAMALTL. Herein we sought to comprehensively examine the frequency of these translocations as well as CARD11 and MYD88 (L265P) mutations, in addition to A20 mutations /deletions in a large cohort of C. psittaci negative OAMALTL. A total of 47 OAMALTL originating in the orbit (22), conjunctiva (19) and lacrimal gland (6) were used for analysis. Dual color fusion probes for t(14;18)(q32;q21) IGH-MALT1, t(11;18)(q21;q21) BIRC3-MALT1 and dual color break apart probes for MALT1, BCL6, and CEP18 (chromosome 18 centromere) were used for FISH analysis in all the analyzed tumors. Dual color break apart probe for IGH was used for selected tumors. Extracted DNA was used for PCR amplification and sequencing of the coiled-coil domain of CARD11, exon 5 of MYD88, in which the L265P mutation was previously reported in diffuse large B-cell and MALT lymphomas, and all the coding exons of the A20 gene. A20 gene copy number variation was analyzed by TaqMan Copy Number Assay (Applied Biosystems). DNA extracted from peripheral blood lymphocytes of 3 healthy volunteers and OCI-LY8 and Jeko-1 cell lines served as calibrator and positive controls for A20 deletion. CARD11 mutations were not found in all the analyzed tumors. The MYD88 L265P mutation was detected in 3 (6.4%) tumors. A total of 9 A20 mutations were identified in 7 (14.9%) tumors. One tumor harbored 3 distinct mutations (F149C; 1bp deletion in exon 3 and 2bp deletion in exon 5). Among the 9 detected A20 mutations, the majority (89%) would produce truncated proteins due to out-frame insertion/deletion (7), while one of these deletions was located in a known splicing site. Only 2 missense mutations were observed, including one in a tumor in which 2 concomitant deletions were also present (described above). A20 heterozygous deletions were detected in 7 (14.9%) tumors. There was no association between A20 mutations and heterozygous deletion in any of the analyzed tumors. None of the tumors harbored a concomitant A20 mutation/deletion and MYD88 L265P mutation.A total of 5 tumors harbored chromosomal alterations: additional copy of BCL6, most probably due to trisomy 3, in 2 tumors, an additional copy of IGH in 1, extra copies of both IGH and MALT1 in 1, and extra copies of IGH, MALT1, BIRC3 together with IGH rearrangement to unidentified partner in 1. t(14;18)(q32;q21) IGH-MALT1 and t(11;18)(q21;q21) BIRC3-MALT1 were not detected in any of the analyzed tumors. A20 mutations were detected in a tumor with extra signals of both IGH and MALT1 genes as well as in one of the tumors with an additional copy of BCL6. The tumor with complex chromosomal aberrations including the IGH rearrangement to an unidentified partner also harbored A20 deletion. 26 of the analyzed tumors previously were shown to exhibit evidence of antigen selection based on the analysis of IGHV mutation pattern (PLoS One. 2011;6(12):e29114). There was no association between antigen selection and A20 mutations/deletions, MYD88 (L265P) mutation and chromosomal alterations. Overall, our data suggest that translocations characteristic for MALT lymphomas are rarely observed in OAMALTL. The MYD88 (L265P) mutation is also uncommon, while most of the A20 mutations/deletions affect only one allele and thus most probably do not lead to NF-kB activation. This raises a question on what are the mechanisms for canonical NF-kB signaling pathway activation in OAMALTL and if it is indeed activated in these tumors. It is possible that NF-kB signaling pathway activation maybe due to B cell receptor signaling, as may be reflected by association with presence of antigen selection that is observed in major fraction of these tumors. Immunohistochemical studies addressing these questions are underway and will be presented at the meeting. Disclosures:No relevant conflicts of interest to declare.

AML1/Runx1 Is a Cytoplasmic Attenuator of NF-Kb Signaling: Implication in Pathogenesis and Targeted Therapy of AML1-Related Leukemia.

Abstract 1962Poster Board I-985 Introduction:AML1/Runx1 is one of the most frequent targets of chromosomal abnormalities in human leukemia. Functional impairment of AML1 caused by point mutation is also reported in patients with leukemia or myelodysplastic syndrome (MDS). However, molecular basis for leukemogenesis caused by functional impairment of AML1 is still elusive. In this study, we clarified the deregulated signaling pathway induced by loss of AML1. Results:To find the direct target of AML1, we compared gene expression profile between AML1-conditionally deleted and normal KSL cells using Cre-ER system. Gene set enrichment analysis (GSEA) using molecular signature database (MSigDB) clarified enhanced expression of NF-kB target genes in AML1 deficient cells. In addition, NF-kB inhibitor attenuated the enhanced colony forming activity of bone marrow cells from AML1 conditional knockout (cKO) mice. These data indicate the aberrant activation of NF-kB signaling pathway in stem/progenitor cells of AML1 deficient mice. NF-kB is a transcription factor which is involved in many physiological phenomena including proliferation, survival, and inflammation. Because deregulated activation of NF-kB signaling has been reported to be responsible for many types of tumors including hematological malignancies, we assumed that lack of AML1-mediated suppression of NF-kB signaling lead to malignant transformation of hematopoietic cells. p65, one of the major components of NF-kB stays in cytoplasm with IkB in a steady state. Once receiving stimulating signals from cell surface receptors such as TNF-a receptor, IkB is phosphorylated by IKK complex and subsequently degraded through the ubiquitin-proteasome pathway, resulting in nuclear translocation of p65 and transactivation of NF-kB target genes. First, we found that AML1 inhibits nuclear translocation of p65 and that nuclear localization of p65 is enhanced in AML1 deficient cells, which is cancelled by NF-kB inhibitors. In addition, AML1 inhibited p65 phosphorylation at serine 536, which is important for its activation. We found that AML1 physically interacts with IKK complex and thus suppresses its kinase activity, which accounts for a mechanistic basis for inhibition of NF-kB signaling by AML1. Suppression of IKK kinase activity by AML1 results in inhibition of both nuclear translocation of p65 and activation of NF-kB target genes. Next, we examined how leukemia-related AML1 mutants affect NF-kB signaling. Remarkably, AML1 D171N mutant found in MDS neither inhibited nuclear translocation of p65 nor attenuated the kinase activity of IKK complex. Similar results were obtained with AML1/ETO generated in leukemia with t(8;21). Mouse bone marrow cells immortalized by AML1/ETO showed enhanced nuclear localization of p65 compared with those immortalized by MLL/ENL, another leukemia-related fusion protein. Indeed, AML1/ETO immortalized cells are more sensitive to NF-kB inhibitor-mediated growth suppression, indicating a critical role of NF-kB signaling in transformation by AML1/ETO. To verify the activation of NF-kB signaling by AML1/ETO in human hematopoietic cells, we analyzed the gene expression data reported by Valk et al. in silico. We found that NF-kB signaling is distinctly activated in AML1-related leukemia patients. These results suggest that aberrant activation of NF-kB signaling induced by functional impairment of AML1 may contribute to the development of leukemia via proliferation signals. Conclusions:We found that AML1 is a cytoplasmic attenuator of NF-kB signaling pathway. Functional impairment of AML1 caused by genetic disruption results in distinct activation of NF-kB signaling by altering IKK kinetic activity. This aberrant activation may play a central role in pathogenesis of AML1-related leukemia and MDS. Therefore, NF-kB signaling is one of the attractive candidates for molecular targeted therapy against AML1-related hematological disorders. Disclosures:No relevant conflicts of interest to declare.