IKKβ Inhibitor Research Articles

Tumour necrosis factor α induces neuroinflammation and insulin resistance in immortalised hypothalamic neurones through independent pathways.

The links between obesity, inflammation and insulin resistance, which are all key characteristics of type 2 diabetes mellitus, are yet to be delineated in the brain. One of the key neuroinflammatory proteins detected in the hypothalamus with over-nutrition is tumour necrosis factor (TNF)α. Using immortalised embryonic rat and mouse hypothalamic cell lines (rHypoE-7 and mHypoE-46) that express orexigenic neuropeptide Y and agouti-related peptide, we investigated changes in insulin signalling and inflammatory gene marker mRNA expression after TNFα exposure. A quantitative polymerase chain reaction array of 84 inflammatory markers (cytokines, chemokines and receptors) demonstrated an increase in the expression of multiple genes encoding inflammatory markers upon exposure to 100ngmL-1 TNFα for 4hours. Furthermore, neurones pre-exposed to TNFα (50ngmL-1 ) for 6 or 16hours exhibited a significant reduction in phosphorylated Akt compared to control after insulin treatment, indicating the attenuation of insulin signalling. mRNA expression of insulin signalling-related genes was also decreased with exposure to TNFα. TNFα significantly increased mRNA expression of IκBα, Tnfrsf1a and IL6 at 4 and 24hours, activating a pro-inflammatory state. An inhibitor study using an inhibitor of nuclear factor kappa B kinase subunit β (IKK-β) inhibitor, PS1145, demonstrated that TNFα-induced neuroinflammatory marker expression occurs through the IKK-β/nuclear factor-kappa B pathway, whereas oleate, a monounsaturated fatty acid, had no effect on inflammatory markers. To test the efficacy of anti-inflammatory treatment to reverse insulin resistance, neurones were treated with TNFα and PS1145, which did not significantly restore the TNFα-induced changes in cellular insulin sensitivity, indicating that an alternative pathway may be involved. In conclusion, exposure to the inflammatory cytokine TNFα causes cellular insulin resistance and inflammation marker expression in the rHypoE-7 and mHypoE-46 neurones, consistent with effects seen with TNFα in peripheral tissues. It also mimics insulin- and palmitate-induced insulin resistance in hypothalamic neurones. The present study provides further evidence that altered central energy metabolism may be caused by obesity-induced cytokine expression.

In silico drug design of inhibitor of nuclear factor kappa B kinase subunit beta inhibitors from 2-acylamino-3-aminothienopyridines based on quantitative structure–activity relationships and molecular docking

Inhibitor of nuclear factor kappa B kinase subunit beta (IKK-β), a specific regulator of nuclear factor-κB (NF-κB), is considered a valid target to design novel candidate drugs to treat rheumatoid arthritis and various cancers. In the present study, quantitative structure–activity relationships (QSAR) and molecular docking techniques were used to screen for new IKK-β inhibitors from a series of 2-acylamino-3-aminothienopyridine analogs. During the two-dimensional QSAR phase, the statistical model partial least square was selected from among two alternatives (r2 = 0.868, q2 (cross-validation) = 0.630). Descriptors with positive or negative contributions were derived from the created model. To build of three-dimensional QSAR models, we used three different fingerprints as analysis precepts for molecular clustering and the subsequent division of training sets and test sets. The best model, which used fingerprint model definition language public keys, was selected for further prediction of the compounds’ activities. Favorable physicochemical, structural, electrostatic, and steric properties were derived from the created QSAR models and then used for drug design with an in-house library. Amongst the designed compounds, compounds B01 and B02 showed good predicted activities. Furthermore, after a selecting the protein structure and docking method, docking studies were carried out to reveal the detailed interactions between the ligands and the target protein. Binding affinity was measured and sorted using the value of “-CDOCKER_ENERGY”. The high -CDOCKER_ENERGY values of compounds B01 (41.6134 kcal/mol) and B02 (40.1366 kcal/mol) indicated their prominent docking affinities.

Co-targeting EGFR and IKK\u03b2/NF-\u03baB signalling pathways in head and neck squamous cell carcinoma: a potential novel therapy for head and neck squamous cell cancer

BackgroundEpidermal growth factor receptor (EGFR) plays an important role in head and neck squamous cell carcinoma (HNSCC) proliferation and therapy resistance, but the efficacy of targeting of EGFR for therapy has been limited. Here, we explore the molecular link between EGFR and inhibitor of κB kinase β/nuclear factor-κB (IKKβ/NF-κB) signalling pathways in the regulation of HNSCC EGFR inhibitor resistance.MethodsWe performed in vitro experiments in eight human HNSCC cell lines and a patient-derived HNSCC cell line as well as in vivo xenografts in a human HNSCC cell line.ResultsWe found that treatment of all HNSCC cells with Gefitinib and Erlotinib, two Food Drug Administration-approved EGFR inhibitors, blocked the activity of Akt/mammalian target of the rapamycin (mTOR) and extracellular signal-regulated kinase, two crucial downstream effectors of EGFR, but up-regulated IKKβ/NF-κB signalling. In addition, induction of IKKβ/NF-κB by EGFR inhibitors required HER2 and HER3 expression. In keeping with these, IKKβ inhibitor CmpdA synergistically enhanced the efficacy of EGFR inhibitors to further inhibit in vitro HNSCC cell growth. Importantly, we demonstrated that the combination of Gefitinib with CmpdA inhibited xenograft tumour formation.ConclusionOur data demonstrated that co-targeting EGFR and IKKβ with Gefitinib and IKKβ inhibitors could provide a potential novel therapy for head and neck squamous cell cancer.

USP14-mediated IκBα degradation exacerbates NF-κB activation and IL-1β-stimulated chondrocyte dedifferentiation

Osteoarthritis (OA) is an inflammatory joint disease. USP14, a deubiquitinating enzyme critical for ubiquitin-mediated proteasomal protein degradation, is implicated in inflammation regulation. However, its role and mechanism in OA are poorly understood. Here, we report that USP14 is upregulated in OA articular cartilage as well as in chondrocytes treated with IL-1β in vitro. USP14 upregulation depends on NF-κB pathway activation, since inhibition of this pathway by ACHP, a selective inhibitor of IKK-β, abolishes USP14 upregulation. We further show that USP14 in turn exacerbates NF-κB activation through promoting IκBα deubiquitination and degradation. Functionally, USP14 aggravates the dedifferentiation effect of IL-1β on chondrocytes, and NF-κB inhibition remarkably reverses this effect, highlighting an important role of NF-κB in mediating USP14 function. Collectively, our data reveal a previously unidentified feed-forward loop driven by USP14 and NF-κB pathway in promoting the dedifferentiation effect of IL-1β on chondrocytes. This mechanism might offer a useful hint for OA intervention.

Metformin inhibits pro-inflammatory responses via targeting nuclear factor-κB in HaCaT cells.

Psoriasis is a prevalent, chronic inflammatory skin disease that arises from rapid and excessive growth of keratinocytes induced by abnormal inflammatory responses. Metformin is the first-line drug in type 2 diabetes and has been proven to possess significant anti-inflammatory effects in various diseases. In the present study, we examined the role of metformin in nuclear factor kappa B (NF-κB)-mediated inflammatory responses in HaCaT cells, a cell line for the keratinocyte. Our results demonstrated that metformin significantly decreased the mRNA and protein levels of tumour necrosis factor-α (TNFα), interleukin (IL)-6, IL-8, and IL-1β induced by TNFα. Immunofluorescence staining and western blot analysis showed that metformin inhibited the nuclear localization of p65, a subunit of nuclear factor NF-κB. In addition, metformin suppressed the transcription activity of NF-κB by inhibiting the degradation of IκBα. The inhibitory effect of metformin on NF-κB signalling is comparable with a specific IKKβ inhibitor BI605906. Collectively, our data suggest that metformin may be a potential therapeutic agent in inflammatory skin diseases like psoriasis.

Paris Saponin II inhibits colorectal carcinogenesis by regulating mitochondrial fission and NF-κB pathway.

Colorectal cancer (CRC) is one of the leading causes of cancer-related morbidity and mortality worldwide. Accumulating evidence suggests that mitochondrial dynamics are closely implicated in carcinogenesis including CRC. Paris Saponin II (PSII), a major steroidal saponin extracted from Rhizoma Paris polyphylla, has emerged as a potential anticancer agent. However, the effects of PSII on CRC and its underlying mechanisms remain unknown. In the present study, we found PSII induced apoptosis and inhibited colony formation in HT 29 and HCT 116 cells, and cell cycle arrest in G1 phase. PSII inhibited the phosphorylation of ERK1/2 and mitochondrial translocation of dynamin-related protein 1 (Drp1) by dephosphorylating Drp1 at Ser616, leading to the suppression of mitochondrial fission. PSII also suppressed NF-κB activation as a result of the inhibition of IKKβ and p65 translocation. Drp1 knockdown remarkably downregulated the nuclear expression of p65 and its target genes cyclin D1 and c-Myc in HCT 116 cell, confirming the link between mitochondrial fission and NF-κB pathway. Silencing of Drp 1 enhanced the inhibitory effects of PSII on p65 phosphorylation and the expressions of cyclin D1 and c-Myc, revealing that the inhibitory effects of PSII on cyclin D1 and c-Myc were relevant in the suppression of Drp1 and NF-κB activation. An in vivo study demonstrated PSII remarkably decreased the xenograft tumor size and suppressed the phosphorylation of ERK1/2 and Drp1 at Ser616. Taken together, our results suggested that PSII could inhibit colorectal carcinogenesis, at least in part, by regulating mitochondrial fission and NF-κB pathway.

Inhibition of NF-κB improves sensitivity to irradiation and EGFR-TKIs and decreases irradiation-induced lung toxicity.

Resistance to radiotherapy and to EGFR tyrosine kinase inhibitors (EGFR-TKIs), as well as therapy-related lung toxicity, are serious problems in the treatment of lung cancer. NF-κB has been reported to be associated with radioresistance. Therefore, we evaluated its effects on sensitivity to irradiation and to EGFR-TKIs; irradiation-induced lung toxicity; and the effects of irradiation on sensitivity to EGFR-TKIs. We used IKKβ inhibitor IMD 0354 or p65 depletion to explore their effects on sensitivity to irradiation and to EGFR-TKIs in vitro and in vivo. We evaluated the efficacy of IMD 0354 in a radiation-induced pulmonary-fibrosis mouse model. Irradiation enhanced activation and expression of MET and therefore suppressed the sensitivity of lung cancer cells to irradiation or EGFR-TKIs. Inhibition of NF-κB by IMD 0354 or by p65 depletion reversed irradiation-induced MET activation and increased the sensitivity of lung cancer cells to irradiation, to EGFR-TKIs and to the combination thereof in vitro and in vivo. In addition, IMD 0354 significantly reduced lung toxicity in a murine model of irradiation-induced pneumonia and lung fibrosis. These findings indicated that NF-κB inhibition can improve sensitivity to irradiation and to EGFR-TKIs and can decrease irradiation-induced lung toxicity in lung cancer.

3,5-Disubstituted-indole-7-carboxamides as IKKβ Inhibitors: Optimization of Oral Activity via the C3 Substituent.

IκB kinase β (IKKβ or IKK2) is a key regulator of nuclear factor kappa B (NF-κB) and has received attention as a therapeutic target. Herein we report on the optimization of a series of 3,5-disubstituted-indole-7-carboxamides for oral activity. In doing so, we focused attention on potency, ligand efficiency (LE), and physicochemical properties and have identified compounds 24 and (R)-28 as having robust in vivo activity.

NF-κB-IKKβ Pathway as a Target for Drug Development: Realities, Challenges and Perspectives.

Nuclear factor κB (NF-κB) comprises a family of proteins that act as transcription factors promoting the expression of many genes. Activation of NF-κB biochemical cascades is associated with the regulation of innate and adaptive immune responses and inflammation, among other physiological responses. However, genetic abnormalities and continuous stimulation of the NF- κB-IKKβ pathway are directly related to many types of inflammatory and autoimmune diseases, as well as to the genesis and survival of tumor cells. Inhibition of the NF-κB-IKKβ cascade can be considered an attractive therapeutic method for the genesis of new prototypes to combat these chronic multifactorial diseases. This review describes some prototypes and drugs that act to inhibit the NF-κB-IKKβ pathway, highlighting the realities, challenges and perspectives for therapeutic use. Although only proteasome inhibitors, such as bortezomib and carfilzomib, are a reality as therapeutically useful drugs among the known modulators of possible targets in the NF-κB-IKKβ pathway, some other prototypes described as IKKβ inhibitors have entered clinical stages as drug candidates for the control of inflammatory diseases. It is important to note that some classical drugs available on the pharmaceutical market, such as acetylsalicylic acid, were also described more recently as NF-κB pathway modulators as IKKβ inhibitors.

GLK-IKKβ signaling induces dimerization and translocation of the AhR-RORγt complex in IL-17A induction and autoimmune disease.

Retinoic-acid-receptor-related orphan nuclear receptor γt (RORγt) controls the transcription of interleukin-17A (IL-17A), which plays critical roles in the pathogenesis of autoimmune diseases. Severity of several human autoimmune diseases is correlated with frequencies of germinal center kinase-like kinase (GLK) (also known as MAP4K3)-overexpressing T cells; however, the mechanism of GLK overexpression-induced autoimmunity remains unclear. We report the signal transduction converging on aryl hydrocarbon receptor (AhR)-RORγt interaction to activate transcription of the IL-17A gene in T cells. T cell-specific GLK transgenic mice spontaneously developed autoimmune diseases with selective induction of IL-17A in T cells. In GLK transgenic T cells, protein kinase Cθ (PKCθ) phosphorylated AhR at Ser36 and induced AhR nuclear translocation. AhR also interacted with RORγt and transported RORγt into the nucleus. IKKβ (inhibitor of nuclear factor κB kinase β)-mediated RORγt Ser489 phosphorylation induced the AhR-RORγt interaction. T cell receptor (TCR) signaling also induced the novel RORγt phosphorylation and subsequent AhR-RORγt interaction. Collectively, TCR signaling or GLK overexpression induces IL-17A transcription through the IKKβ-mediated RORγt phosphorylation and the AhR-RORγt interaction in T cells. Our findings suggest that inhibitors of GLK or the AhR-RORγt complex could be used as IL-17A-blocking agents for IL-17A-mediated autoimmune diseases.

Overexpression of miR-381-3p promotes the recovery of spinal cord injury.

To study the effects of miR-381-3p on spinal cord injury and its underlying mechanism. After the spinal cord injury rat model of was established, Sprague Dawley (SD) rats were randomly divided into the control group and the acute spinal cord injury (ASCI) group. Microglial BV2 cells were used as experimental cells, and the cells were divided into the control group and the lipopolysaccharide (LPS) group. The mRNA and protein expression level of miR-381-3p, IKKβ, inflammatory factors, and p-p65 were detected by quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. Dual-luciferase reporter gene assay and Western blot were used to detect the regulatory effect of IKKβ on miR-381-3p. Changes in grip ability and rotary performance of rats in the ASCI group were evaluated after miR-381-3p overexpression in vivo. The expression of miR-381-3p was downregulated in rats of the ASCI group, while the expression of IKKβ and p-p65 were upregulated. In vitro experiments demonstrated that LPS could inhibit the expression of miR-381-3p and promote the upregulation of IKKβ and p-p65. Overexpression of miR-381-3p could inhibit the mRNA and protein expression of IKKβ. The upregulated expression of IKKβ, p-p65, tumor necrosis factor-alpha (TNF-α), and interleukins-1β (IL-1β) induced by LPS in BV2 cells were reversed by miR-381-3p mimic transfection. Besides, upregulated TNF-α and IL-1β induced by miR-381-3p inhibitor in BV2 cells were reversed by IKKβ inhibitor (BMS-345541). Results of animal experiments indicated that miR-381-3p was overexpressed in rats of the ASCI group. The protein levels of IKKβ and p-p65, and the mRNA expression levels of inflammatory cytokines TNF-α and IL-1β were remarkably decreased in the ASCI group than those of the control group. The grip ability, coordination, and anti-fatigue performance of rats in the ASCI group recovered quicker than those of the control group. MiR-381-3p was downregulated in ASCI rats. The overexpression of miR-381-3p could recover the motor ability of rats in the ASCI group earlier and might inhibit injury aggravation by inhibiting inflammatory responses via the IKKβ-NF-κB pathway.

Targeting IKKβ in Cancer: Challenges and Opportunities for the Therapeutic Utilisation of IKKβ Inhibitors.

Deregulated NF-κB signalling is implicated in the pathogenesis of numerous human inflammatory disorders and malignancies. Consequently, the NF-κB pathway has attracted attention as an attractive therapeutic target for drug discovery. As the primary, druggable mediator of canonical NF-κB signalling the IKKβ protein kinase has been the historical focus of drug development pipelines. Thousands of compounds with activity against IKKβ have been characterised, with many demonstrating promising efficacy in pre-clinical models of cancer and inflammatory disease. However, severe on-target toxicities and other safety concerns associated with systemic IKKβ inhibition have thus far prevented the clinical approval of any IKKβ inhibitors. This review will discuss the potential reasons for the lack of clinical success of IKKβ inhibitors to date, the challenges associated with their therapeutic use, realistic opportunities for their future utilisation, and the alternative strategies to inhibit NF-κB signalling that may overcome some of the limitations associated with IKKβ inhibition.

Repurposing Thioridazine (TDZ) as an anti-inflammatory agent

Nuclear factor-kB (NF-kB) is a crucial transcription factor in the signal transduction cascade of the inflammatory signaling. Activation of NF-κB depends on the phosphorylation of IκBα by IκB kinase (IKKβ) followed by subsequent ubiquitination and degradation. This leads to the nuclear translocation of the p50- p65 subunits of NF-κB, and further triggers pro-inflammatory cytokine gene expression. Thus, in the need of a more effective therapy for the treatment of inflammatory diseases, specific inhibition of IKKβ represents a rational alternative strategy to the current therapies. A computer-aided drug identification protocol was followed to identify novel IKKβ inhibitors from a database of over 1500 Food and Drug Administration (FDA) drugs. The best scoring compounds were compared with the already known high-potency IKKβ inhibitors for their ability to bind and inhibit IKKβ by evaluating their docking energy. Finally, Thioridazinehydrochloride (TDZ), a potent antipsychotic drug against Schizophrenia was selected and its efficiency in inhibiting IκBα protein degradation and NF-κB activation was experimentally validated. Our study has demonstrated that TDZ blocks IκBα protein degradation and subsequent NF-κB activation to inhibit inflammation. Thus, it is a potential repurposed drug against inflammation.

MMB triazole analogs are potent NF-κB inhibitors and anti-cancer agents against both hematological and solid tumor cells

Triazole derivatives of melampomagnolide B (MMB) have been synthesized via click chemistry methodologies and screened against a panel of 60 human cancer cell lines. Several derivatives showed promising anti-cancer activity, affording growth inhibition (GI50) values in the nanomolar range (GI50 = 0.02–0.99 μM). Lead compound 7h exhibited EC50 values of 400 nM and 700 nM, respectively, against two AML clinical specimens. Compound 7h was significantly more potent than parthenolide as an inhibitor of p65 phosphorylation in both hematological and solid tumor cell lines, indicating its ability to inhibit the NF-κB pathway. In TMD-231 breast cancer cells, treatment with 7h reduced DNA binding activity of NF-κB through inhibition of IKK-β mediated p65 phosphorylation and caused elevation of basal IκBα levels through inhibition of constitutive IκBα turnover and NF-κB activation. Molecular docking and dynamic modeling studies indicated that 7h interacts with the kinase domain of the monomeric IKKβ subunit, leading to inhibition of IKKβ activation, and compromising phosphorylation of downstream targets of the NF-κB pathway; dynamic modeling studies show that this interaction also causes unwinding of the α-helix of the NEMO binding site on IKKβ. Molecular docking studies with 10, a water-soluble analog of 7h, demonstrate that this analog interacts with the dimerization/oligomerization domain of monomeric IKKβ and may inhibit oligomer formation and subsequent autophosphorylation. Sesquiterpene lactones 7h and 10 are considered ideal candidates for potential clinical development.

Fusicoccane-Derived Diterpenoids from Alternaria brassicicola: Investigation of the Structure-Stability Relationship and Discovery of an IKKβ Inhibitor.

The structure-stability relationships of 1-6 were investigated to show that 1 converted to 5 via a kinetic, solution-mediated autoxidation. In addition, alterbrassicene A (7), a fusicoccane-derived diterpenoid bearing an unprecedented 5/9/4-fused carbocyclic skeleton with a rare fused 2-cyclobuten-1-one motif, was characterized from Alternaria brassicicola. Its absolute structure was elucidated by spectroscopic analyses and quantum chemical calculations. Compound 7 was the first fusicoccane derivative acting as a potent IKKβ inhibitor in the NF-κB signaling pathway.

BMS‑345541 inhibits airway inflammation and epithelial‑mesenchymal transition in airway remodeling of asthmatic mice.

The present study aimed to investigate the possible effects and regulatory mechanism of the inhibitor of nuclear factor-κB kinase complex β subunit (IKKβ) inhibitor BMS-345541 on airway inflammation, airway remodeling and epithelial-mesenchymal transition (EMT) in an ovalbumin (OVA) exposure asthma model in mice. The asthma mouse model was generated by sensitization and challenge with OVA. BMS-345541/dimethyl sulfoxide (DMSO) was administered perorally dairy in two therapeutic groups throughout the entire OVA challenge process. At 24 h following the last challenge, airway hyperresponsiveness (AHR) and airway inflammation were examined, and serum, bronchoalveolar lavage fluid (BALF) and lung samples were collected. Lung tissue was stained and assessed for pathological changes. The total number and classification of inflammatory cells in the BALF were examined. Levels of transforming growth factor β1 (TGFβ1) in the serum and BALF were measured using an enzyme-linked immunosorbent assay. The differential expression of EMT regulators E-cadherin and vimentin was detected by immunohistochemical staining, reverse transcription-quantitative polymerase chain reaction analysis and western blot analysis. The results showed that OVA successfully induced allergic asthma. The asthmatic mice had AHR, airway inflammation, airway remodeling, a high expression of TGFβ1, and evidence of EMT. Following BMS-345541 treatment, there was significant inhibition of pathophysiological signs, including increased pulmonary eosinophilia infiltration, mucus hypersecretion and AHR. Treatment with BMS-345541 significantly reduced levels of TGFβ1. In addition, BMS-345541 notably downregulated the expression of vimentin and increased the expression of E-cadherin. These data suggested that the increased secretion of TGFβ1 induced by asthmatic inflammation can lead to EMT, and the IKKβ inhibitor BMS-345541 may alter airway remodeling by preventing EMT in an OVA asthma model. Therefore, IKKβ inhibitors require investigation as potential asthma therapies.

Anti-survivin effect of the small molecule inhibitor YM155 in RCC cells is mediated by time-dependent inhibition of the NF-\u03baB pathway

Constitutive activation of the NF-κB signaling cascade is associated with tumourigenesis and poor prognosis in many human cancers including RCC. YM155, a small molecule inhibitor of survivin, was previously shown to potently inhibit the viability of immortalized and patient derived renal cell carcinoma (RCC) cell lines. Here we investigated the role of NF-κB signaling to the anti-cancer properties of YM155 in RCC786.0 cells. YM155 diminished nuclear levels of p65 and phosphorylated p65 and attenuated the transcriptional competencies of the p65/p50 heterodimers. Accordingly, we found that YM155 diminished the transcription of NF-κB target gene survivin. Events that led to the interception of the nuclear translocation of p65/p50 were the activation of the deubiquinating enzyme CYLD by YM155, which led to the inhibition of IKKβ, stabilization of IκBα and retention of NF-κB heterodimers in the cytosol. Importantly, the suppressive effects of YM155 were time-dependent and observed at the 24 h time point, and not earlier. TNF-α, a stimulator of NF-κB signaling did not affect its inhibitory properties. The ability of YM155 to intercept a major transcriptional pathway like NF-κB, would have important ramifications on the pharmacodynamics effects elicited by this unusual molecule.

A dual role of miR-22 modulated by RelA/p65 in resensitizing fulvestrant-resistant breast cancer cells to fulvestrant by targeting FOXP1 and HDAC4 and constitutive acetylation of p53 at Lys382

Antiestrogen resistance is a major challenge encountered during the treatment of estrogen receptor alpha positive (ERα+) breast cancer. A better understanding of signaling pathways and downstream transcription factors and their targets may identify key molecules that can overcome antiestrogen resistance in breast cancer. An aberrant expression of miR-22 has been demonstrated in breast cancer; however, its contribution to breast cancer resistance to fulvestrant, an antiestrogen drug, remains unknown. In this study, we demonstrated a moderate elevation in miR-22 expression in the 182R-6 fulvestrant-resistant breast cancer line we used as a model system, and this elevation was positively correlated with the expression of the miRNA biogenesis enzymes AGO2 and Dicer. The level of phosphorylated HER2/neu at Tyr877 was also upregulated in these cells, whereas the level of RelA/p65 phosphorylated at Ser536 (p-p65) was downregulated. Knockdown of HER2/neu led to an induction of p-p65 and a reduction in miR-22 levels. Luciferase assays identified two NF-κB binding motifs in the miR-22 promoter that contributed to transcriptional repression of miR-22. Activation of RelA/p65, triggered by LPS, attenuated miR-22 expression, but this expression was restored by sc-514, a selective IKKβ inhibitor. Inhibition of miR-22 suppressed cell proliferation, induced apoptosis and caused cell cycle S-phase arrest, whereas enhancing expression of p21Cip1/Waf1 and p27Kip1. Surprisingly, ectopic expression of miR-22 also suppressed cell proliferation, induced apoptosis, caused S-phase arrest, and promoted the expression of p21Cip1/Waf1 and p27Kip1. Ectopic overexpression of miR-22 repressed the expression of FOXP1 and HDAC4, leading to a marked induction of acetylation of HDAC4 target histones. Conversely, inhibition of miR-22 promoted the expression of both FOXP1 and HDAC4, without the expected attenuation of histone acetylation. Instead, p53 acetylation at lysine 382 was unexpectedly upregulated. Taken together, our findings demonstrated, for the first time, that HER2 activation dephosphorylates RelA/p65 at Ser536. This dephosphoryalted p65 may be pivotal in transactivation of miR-22. Both increased and decreased miR-22 expression cause resensitization of fulvestrant-resistant breast cancer cells to fulvestrant. HER2/NF-κB (p65)/miR-22/HDAC4/p21 and HER2/NF-κB (p65)/miR-22/Ac-p53/p21 signaling circuits may therefore confer this dual role on miR-22 through constitutive transactivation of p21.

Progestin and AdipoQ Receptor 3 Upregulates Fibronectin and Intercellular Adhesion Molecule-1 in Glomerular Mesangial Cells via Activating NF-κB Signaling Pathway Under High Glucose Conditions.

Progestin and adipoQ receptor 3 (PAQR3), is a Golgi-anchored membrane protein containing seven transmembrane helices. It has been demonstrated that PAQR3 mediates insulin resistance, glucose and lipid metabolism, and inflammation. In addition, kidney inflammatory fibrosis is an important pathological feature of diabetic nephropathy (DN). Therefore, we aimed to investigate the role of PAQR3 in diabetic kidney fibrosis as well as inflammation in DN. The effect of PAQR3 on NF-κB signaling pathway, expressions of fibronectin (FN) and intercellular adhesion molecule-1 (ICAM-1) in glomerular mesangial cells (GMCs) cultured by high glucose (HG) were examined. Diabetic mouse and rat models were induced by streptozotocin (STZ). GMCs were treated with HG and transfected with PAQR3 plasmids or small-interfering RNA targeting PAQR3 or NF-κB. The protein levels of FN and ICAM-1 were examined by Western blotting, and the transcriptional activity and DNA binding activity of NF-κB were measured by dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). The interaction between PAQR3 and IKKβ (inhibitor of nuclear factor κB kinase β) was analyzed by co-immunoprecipitation. PAQR3 was increased in both STZ-induced diabetic models and HG-treated GMCs. PAQR3 overexpression further increased HG-induced FN and ICAM-1 upregulation. In contrast, silencing of PAQR3 suppressed the expressions of FN and ICAM-1. PAQR3 overexpression promoted the nuclear accumulation, DNA binding activity, and transcriptional activity of NF-κB. Mechanically, PAQR3 directly interacted with IKKβ. The upregulation effect of PAQR3 overexpression on the expressions of FN and ICAM-1 was abolished by the treatment of NF-κB siRNA or PDTC (ammonium pyrrolidinedithiocarbamate) in HG-treated GMCs. PAQR3 promotes the expressions of FN and ICAM-1 via activating NF-κB signaling pathway. Mechanistically, PAQR3 activates NF-κB signaling pathway to mediate kidney inflammatory fibrosis through direct interaction with IKKβ in DN.

IκB Kinase α Is Required for Development and Progression of KRAS-Mutant Lung Adenocarcinoma.

Although oncogenic activation of NFκB has been identified in various tumors, the NFκB-activating kinases (inhibitor of NFκB kinases, IKK) responsible for this are elusive. In this study, we determined the role of IKKα and IKKβ in KRAS-mutant lung adenocarcinomas induced by the carcinogen urethane and by respiratory epithelial expression of oncogenic KRASG12D Using NFκB reporter mice and conditional deletions of IKKα and IKKβ, we identified two distinct early and late activation phases of NFκB during chemical and genetic lung adenocarcinoma development, which were characterized by nuclear translocation of RelB, IκBβ, and IKKα in tumor-initiated cells. IKKα was a cardinal tumor promoter in chemical and genetic KRAS-mutant lung adenocarcinoma, and respiratory epithelial IKKα-deficient mice were markedly protected from the disease. IKKα specifically cooperated with mutant KRAS for tumor induction in a cell-autonomous fashion, providing mutant cells with a survival advantage in vitro and in vivo IKKα was highly expressed in human lung adenocarcinoma, and a heat shock protein 90 inhibitor that blocks IKK function delivered superior effects against KRAS-mutant lung adenocarcinoma compared with a specific IKKβ inhibitor. These results demonstrate an actionable requirement for IKKα in KRAS-mutant lung adenocarcinoma, marking the kinase as a therapeutic target against this disease.Significance: These findings report a novel requirement for IKKα in mutant KRAS lung tumor formation, with potential therapeutic applications. Cancer Res; 78(11); 2939-51. ©2018 AACR.