Aberrant non-canonical NF-κB signalling reprograms the epigenome landscape to drive oncogenic transcriptomes in multiple myeloma.

In multiple myeloma, abnormal plasma cells establish oncogenic niches within the bone marrow by engaging the NF-κB pathway to nurture their survival while they accumulate pro-proliferative mutations. Under these conditions, many cases eventually develop genetic abnormalities endowing them with constitutive NF-κB activation. Here, we find that sustained NF-κB/p52 levels resulting from such mutations favours the recruitment of enhancers beyond the normal B-cell repertoire. Furthermore, through targeted disruption of p52, we characterise how such enhancers are complicit in the formation of super-enhancers and the establishment of cis-regulatory interactions with myeloma dependencies during constitutive activation of p52. Finally, we functionally validate the pathological impact of these cis-regulatory modules on cell and tumour phenotypes using in vitro and in vivo models, confirming RGS1 as a p52-dependent myeloma driver. We conclude that the divergent epigenomic reprogramming enforced by aberrant non-canonical NF-κB signalling potentiates transcriptional programs beneficial for multiple myeloma progression.

LRRC8A as a central mediator promotes colon cancer metastasis by regulating PIP5K1B/PIP2 pathway.

Colorectal cancer (CRC) has been the third most common malignancy and the second cause of cancer-related mortality. As the core of volume-sensitive chloride currents, leucine-rich repeat-containing 8A (LRRC8A) contributes to tumor progression but is not consistent, especially for whom the roles in colon carcinoma metastasis were not fully elucidated. Herein, LRRC8A proteins were found highly expressed in hematogenous metastasis from human colorectal cancer samples. The oxaliplatin-resistant HCT116 cells highly expressed LRRC8A, which was related to impaired proliferation and enhanced migration. The over-expressed LRRC8A slowed proliferation and increased migration ex vivo and in vivo. The elevated LRRC8A upregulated the focal adhesion, MAPK, AMPK, and chemokine signaling pathways via phosphorylation and dephosphorylation. Inhibition of LRRC8A impeded the TNF-α signaling cascade and TNF-α-induced migration. LRRC8A binding to PIP5K1B regulated the PIP2 formation, providing a platform for LRRC8A to mediate cell signaling transduction. Importantly, LRRC8A self-regulated its transcription via NF-κB1 and NF-κB2 pathways and the upregulation of NIK/NF-κB2/LRRC8A transcriptional axis was unfavorable for colon cancer patients. Collectively, our findings reveal that LRRC8A is a central mediator in mediating multiple signaling pathways to promote metastasis and targeting LRRC8A proteins could become a potential clinical biomarker-driven treatment strategy for colon cancer patients.

The preliminary study suggests an association between NF-ĸB pathway activation and increased plasma 20S proteasome activity in intracranial aneurysm patients.

The significant role of increased activation of 20S proteasomes in the development of abdominal aortic aneurysms has been well-established in a mouse model. The available literature lacks similar studies concerning brain aneurysms. The aim of the study was to verify the hypothesis that patients with unruptured intracranial aneurysms (UIA) have increased 20S proteasome ChT-L activity compared to the control group of individuals without vascular lesions in the brain. In the next step, the relationship between the activity of 20S proteasomes ChT-L and precursor proteins from the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) family, namely NF-κB1 (p105), NF-κB2 (p100), NF-κB p65, and the inflammatory chemokine MCP-1, was examined. Patients with UIA had significantly higher 20S ChT-L proteasome activity compared to the control group. Patients with multiple aneurysms had significantly higher 20S proteasome ChT-L activity compared to those with single aneurysms. In patients with UIA, the activity of the 20S proteasome ChT-L negatively correlated with the concentration of NF-κB1 (p105) and NF-κB p65 precursor proteins and positively correlated with the concentration of the cerebrospinal fluid chemokine MCP-1. Our results may suggest that increased 20S proteasome ChT-L activity in UIA patients modulates inflammation in the cerebral arterial vessel via the MCP-1 chemokine as a result of activation of the canonical NF-κB pathway.

TRIM55 promotes noncanonical NF-κB signaling and B cell-mediated immune responses by coordinating p100 ubiquitination and processing.

The ubiquitination-dependent processing of NF-κB2 (also known as p100) is a critical step in the activation of the noncanonical NF-κB pathway. We investigated the molecular mechanisms regulating this process and showed that TRIM55 was the E3 ubiquitin ligase that mediated the ubiquitination of p100 and coordinated its processing. TRIM55 deficiency impaired noncanonical NF-κB activation and B cell function. Mice with a B cell-specific Trim55 deficiency exhibited reduced germinal center formation and antibody production. These mice showed less severe symptoms than those of control mice upon the induction of a systemic lupus-like disease, suggesting B cell-intrinsic functions of TRIM55 in humoral immune responses and autoimmunity. Mechanistically, the ubiquitination of p100 mediated by TRIM55 was crucial for p100 processing by VCP, an ATPase that mediates ubiquitin-dependent protein degradation by the proteasome. Furthermore, we found that TRIM55 facilitated the interaction between TRIM21 and VCP as well as TRIM21-mediated K63-ubiquitination of VCP, both of which were indispensable for the formation of the VCP-UFD1-NPL4 complex and p100 processing. Together, our results reveal a mechanism by which TRIM55 fine-tunes p100 processing and regulates B cell-dependent immune responses in vivo, highlighting TRIM55 as a potential therapeutic target for lupus-like disease.

Integrated molecular modeling and dynamics approaches revealed potential natural inhibitors of NF-κB transcription factor as breast cancer therapeutics.

Breast cancer is a silent killer malady among women and a serious economic burden in health care management. A case of breast cancer is diagnosed among women every 19 s, and every 74 s, a woman dies of breast cancer somewhere in the world. Despite the pop-up of progressive research, advanced treatment approaches, and preventive measures, breast cancer remains amplifying ailment. The nuclear factor kappa B (NF-κB) is a key transcription factor that links inflammation with cancer and is demonstrated as being involved in the tumorigenesis of breast cancer. The NF-κB transcription factor family in mammals consists of five proteins; c-Rel, RelA(p65), RelB, NF-κB1(p50), and NF-κB2(p52). The antitumor effect of NF-κB has also been explored in breast cancer, however, the actual treatment for breast cancer is yet to be discovered. This study is attributed to the identification of novel drug targets against breast cancer by targeting c-Rel, RelA(p65), RelB, NF-κB1(p50), and NF-κB2(p52) proteins. To identify the putative active compounds, a structure-based 3D pharmacophore model to the protein active site cavity was generated followed by virtual screening, molecular docking, and molecular dynamics (MD) simulation. Initially, a library of 45000 compounds were docked against the target protein and five compounds namely Z56811101, Z653426226, Z1097341967, Z92743432, and Z464101066 were selected for further analysis. The relative binding affinity of Z56811101, Z653426226, Z1097341967, Z92743432, and Z464101066 with NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel proteins were -6.8, -8, -7.0, -6.9, and -7.2 kcal/mol, respectively which remained stable throughout the simulations of 200 ns. Furthermore, all of these compounds depict maximum drug-like properties. Therefore, the proposed compounds can be a potential candidate for patients with breast cancer, but, experimental validation is needed to ensure their safety.Communicated by Ramaswamy H. Sarma.

NEK6 Regulates Redox Balance and DNA Damage Response in DU-145 Prostate Cancer Cells.

NEK6 is a central kinase in developing castration-resistant prostate cancer (CRPC). However, the pathways regulated by NEK6 in CRPC are still unclear. Cancer cells have high reactive oxygen species (ROS) levels and easily adapt to this circumstance and avoid cell death by increasing antioxidant defenses. We knocked out the NEK6 gene and evaluated the redox state and DNA damage response in DU-145 cells. The knockout of NEK6 decreases the clonogenic capacity, proliferation, cell viability, and mitochondrial activity. Targeting the NEK6 gene increases the level of intracellular ROS; decreases the expression of antioxidant defenses (SOD1, SOD2, and PRDX3); increases JNK phosphorylation, a stress-responsive kinase; and increases DNA damage markers (p-ATM and γH2AX). The exogenous overexpression of NEK6 also increases the expression of these same antioxidant defenses and decreases γH2AX. The depletion of NEK6 also induces cell death by apoptosis and reduces the antiapoptotic Bcl-2 protein. NEK6-lacking cells have more sensitivity to cisplatin. Additionally, NEK6 regulates the nuclear localization of NF-κB2, suggesting NEK6 may regulate NF-κB2 activity. Therefore, NEK6 alters the redox balance, regulates the expression of antioxidant proteins and DNA damage, and its absence induces the death of DU-145 cells. NEK6 inhibition may be a new strategy for CRPC therapy.

How do nuclear factor kappa B (NF-κB)1 and NF-κB2 defects lead to the incidence of clinical and immunological manifestations of inborn errors of immunity?

INTRODUCTION: Genetic defects affect the manner of the immune system's development, activation, and function. Nuclear factor-kappa B subunit 1 (NF-κB1) and NF-κB2 are involved in different biological processes, and deficiency in these transcription factors may reveal clinical and immunological difficulties. AREAS COVERED: This review article gathers the most frequent clinical and immunological remarkable characteristics of NF-κB1 and NF-κB2 deficiencies. Afterward, an effort is made to describe the biological mechanism, which is likely to be the cause of these clinical and immunological abnormalities. EXPERT OPINION: The present review article has explained the mechanism of contributions of the NF-κB1 and NF-κB2 deficiency in revealing immunodeficiency symptoms, specifically immunological and clinical manifestations. These mechanisms demonstrate the importance of NF-κB1 and NF-κB2 signaling pathways for B and T cell development, activation, antibody production, and immunotolerance. The manifestation of a mutation can range from no symptoms to severe complications in a family.

NF-κB1 and NF-κB2 are the transcription factors that have an essential role in the development and function of the immune system. Several mutations in the NF-κBs could lead to inborn errors of immune manifestations.There are different reports of NF-κB mutations with various clinical and immunological manifestations, whereas the mechanisms behind the appearance of these have been less discussed. We collected frequent clinical and immunological manifestations from the literature and discussed the likely biological cause of their occurrence. Here we clarify the potential mechanism that defects in NF-κB1 and NF-κB2 signaling lead to inadequate B and T cells function, specifically, insufficient switched memory B cells and class-switched antibodies and autoimmunity. We also focused on the straight correlation between deficiencies in immune system responses caused by defects in NF-κB and susceptibility to recurrent infections and other clinical problems.Since our understanding of the mechanisms of signaling pathways such as NF-κB deficiencies from genetic mutations to clinical manifestations in PIDs is limited, further in vitro and animal model studies are necessary to assess these pathways' comprehensive roles in immunodeficiency more accurately. [Figure: see text].

Changes in expression of nuclear factor kappa B subunits in the ovine thymus during early pregnancy.

There is a pregnant maternal immunological tolerance that protects the fetus and promotes its growth, and nuclear factor kappa B (NF-κB) family participates in the regulation of innate immune and adaptive immune responses. The thymus is related to establishing central tolerance, and early pregnancy has effects on expression of a good number of genes and proteins in the maternal thymus in sheep. However, it is unclear whether early pregnancy changes expression of NF-κB subunits in the ovine thymus. In this study, the thymic samples were collected from day 16 of non-pregnant ewes, and days 13, 16 and 25 of pregnant ewes, and the expression of NF-κB members (NF-κB1, NF-κB2, RelA, RelB and c-Rel) was analyzed through real-time quantitative PCR, Western blot and immunohistochemical analysis. The results showed that c-Rel mRNA and protein upregulated at day 25 of pregnancy, and NF-κB1 mRNA and proteins increased at days 16 and 25 of pregnancy, and RelB mRNA and proteins enhanced during early pregnancy. However, expression levels of NF-κB2 and RelA were decreased during early pregnancy, but upregulated from day 13 to 25 of pregnancy. In addition, the RelA protein was located in the epithelial reticular cells, capillaries and thymic corpuscles. This paper reported for the first time that early pregnancy induced expression of NF-κB1, RelB and c-Rel, but inhibited expression of NF-κB2 and RelA in the maternal thymus during early pregnancy, which is involved in the central immune tolerance, and helpful for successful pregnancy in sheep.

Nfkb2 deficiency and its impact on plasma cells and immunoglobulin expression in murine small intestinal mucosa.

The alternative (noncanonical) nuclear factor-κB (NF-κB) signaling pathway predominantly regulates the function of the p52/RelB heterodimer. Germline Nfkb2 deficiency in mice leads to loss of p100/p52 protein and offers protection against a variety of gastrointestinal conditions, including azoxymethane/dextran sulfate sodium (DSS)-induced colitis-associated cancer and lipopolysaccharide (LPS)-induced small intestinal epithelial apoptosis. However, the common underlying protective mechanisms have not yet been fully elucidated. We applied high-throughput RNA-Seq and proteomic analyses to characterize the transcriptional and protein signatures of the small intestinal mucosa of naïve adult Nfkb2-/- mice. Those data were validated by immunohistochemistry and quantitative ELISA using both small intestinal tissue lysates and serum. We identified a B-lymphocyte defect as a major transcriptional signature in the small intestinal mucosa and immunoglobulin A as the most downregulated protein by proteomic analysis in Nfkb2-/- mice. Small intestinal immunoglobulins were dramatically dysregulated, with undetectable levels of immunoglobulin A and greatly increased amounts of immunoglobulin M being detected. The numbers of IgA-producing, cluster of differentiation (CD)138-positive plasma cells were also reduced in the lamina propria of the small intestinal villi of Nfkb2-/- mice. This phenotype was even more striking in the small intestinal mucosa of RelB-/- mice, although these mice were equally sensitive to LPS-induced intestinal apoptosis as their RelB+/+ wild-type counterparts. NF-κB2/p52 deficiency confers resistance to LPS-induced small intestinal apoptosis and also appears to regulate the plasma cell population and immunoglobulin levels within the gut.NEW & NOTEWORTHY Novel transcriptomic analysis of murine proximal intestinal mucosa revealed an unexpected B cell signature in Nfkb2-/- mice. In-depth analysis revealed a defect in the CD38+ B cell population and a gut-specific dysregulation of immunoglobulin levels.

Expression of nuclear factor kappa B components in the ovine maternal liver in early pregnancy periods.

There is a systemic immunological adaptation to maintaining tolerance towards the allogeneic fetus, and the liver participates in the adaptive immune tolerance during normal pregnancy. Nuclear factor kappa B (NF-κB) signalings contribute to immune regulation and liver homoeostasis. The objective of this study is to explore the effects of early pregnancy on expression of NF-κB components in the maternal liver in sheep. The maternal livers were sampled on Day 16 of the estrous cycle, and Days 13, 16, and 25 of gestation, and the expression of NF-κB components, including NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel, was detected by quantitative real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistochemical analysis. Our data revealed that early pregnancy inhibited the expression of NF-κB1 and c-Rel, but the expression of NF-κB1 and c-Rel was increased during early pregnancy. However, early pregnancy enhanced the expression of NF-κB2, RelA, and RelB with the pregnancy progress. In conclusion, early pregnancy regulates the expression of NF-κB components in the maternal livers, which may contribute to maintaining maternal liver homeostasis and immune tolerance during early pregnancy in sheep.

Exogenous interleukin-1 beta promotes the proliferation and migration of HeLa cells via the MEK/ERK signaling pathway.

OBJECTIVE: Interleukin-1 beta (IL-1ß) is a crucial cytokine that has been implicated in cancer and metastasis development. However, its possible mechanistic role in cervical cancer remains unclear. This study aimed to investigate the functions of exogenous IL-1ß in cervical cancer cell proliferation and migration. METHODS: HeLa cell proliferation and migration were measured using MTT and Transwell assays. A lentivirus-mediated packaging system was used to construct an IL-1ß overexpressing cell line. MEK/ERK signal transduction was inhibited by pretreatment with the MEK inhibitor PD98059. qRT-PCR and Western blotting were used to test the expression of relevant genes. RESULTS: Exogenous IL-1ß promoted the proliferation and migration of HeLa cells. In addition, overexpression of IL-1ß in HeLa cells promoted cell proliferation. Mechanistically, exogenous IL-1ß increased the phosphorylated MEK and ERK levels in HeLa cells and the expression of JUN, RELB, and NF-κB2. Alternatively, blockade of MEK inhibited the promoting proliferation effects of IL-1ß and the expression of JUN, RELB, and NF-κB2. CONCLUSIONS: Our data suggest that exogenous IL-1ß regulates HeLa cell functions by regulating the MEK/ERK signaling pathway and by targeting JUN, RELB, and NF-κB2. Our study uncovered a potential association across IL-1ß, cervical tumor development, and cancer progression.

Trim14 promotes osteoclastogenesis and noncanonical NF-κB activation by targeting p100/p52 in chronic periodontitis.

BACKGROUND: Periodontitis disease infection initiates host immune response, and alveolar bone damage is a hallmark of periodontitis. Bone damage occurs due to changes in osteoclast activity in response to local inflammation. Nuclear factor κB (NF-κB) signaling is essential for inflammatory responses and plays a pivotal role in osteoclast formation and activation. Tripartite motif 14 (Trim14) is a crucial regulator of the noncanonical NF-κB signaling. Here, we investigated the role of Trim14 in chronic periodontitis. METHODS: The development of immune cells and osteoclast formation was evaluated with flow cytometry, qRT-PCR, and histochemical staining. Proinflammatory cytokines were checked by ELISA and qRT-PCR. Protein expression was determined by immunoblotting. Also, the cemento-enamel junction-alveolar bone crest distance was evaluated in the mouse model. RESULTS: Development of innate and adaptive cells was not impaired from the deletion of Trim14. However, the genetic loss of Trim14 remarkably suppressed RANKL-induced osteoclastogenesis, without affecting TLR-induced proinflammatory cytokines except for Il-23a expression. The Trim14 deletion also suppressed the activation of noncanonical NF-κB signaling by targeting p100/p52. Importantly, the deletion of NIK diminished the effects of Trim14 on the inflammatory responses in vivo on chronic periodontitis responses. CONCLUSION: TRIM14 may be a positive regulator to promote osteoclastogenesis and proinflammatory cytokine secretion.

Alpha-(1,6)-fucosyltransferase (FUT8) affects the survival strategy of osteosarcoma by remodeling TNF/NF-κB2 signaling.

Glycosylation is an important modification of membrane proteins that results in functional changes in many cellular activities, from cell-cell recognition to regulatory signaling. Fucosyltransferase 8 (FUT8) is the sole enzyme responsible for core fucosylation, and aberrant fucosylation by dysregulated expression of fucosyltransferases is responsible for the growth of various types of carcinomas. However, the function of FUT8 in the progress of osteosarcoma (OS) has not been reported. In this study, we found that FUT8 is expressed at lower levels in patients with OS and in human OS cell lines such as MNNG/HOS, U2OS, and 143B, suggesting that attenuated expression of FUT8 is involved in the growth and progression of OS. Mechanistically, FUT8 affects the survival strategy of OS by modifying core-fucosylation levels of TNF receptors (TNFRs). Lower fucosylation of TNFRs activates the non-canonical NF-κB signaling pathway, and in turn, decreases mitochondria-dependent apoptosis in OS cells. Together, our results point to FUT8 being a negative regulator of OS that enhances OS-cell apoptosis and suggests a novel therapeutic strategy for treating OS.

A Kinase Assay for Measuring the Activity of the NIK-IKK1 Complex Induced via the Noncanonical NF-κB Pathway.

Nuclear factor-kappa B (NF-κB) inducing kinase (NIK), a key component of the noncanonical NF-κB pathway, directs a range of physiological processes, such as lymphoid organogenesis, immune cell differentiation, and immune responses. Aberrant noncanonical NF-κΒ signaling also causes human ailments, including autoimmune and neoplastic diseases. As such, NIK is constitutively degraded in resting cells, and accumulates upon noncanonical NF-κB signaling. NIK then associates with and phosphorylates IkappaB kinase 1 (IKK1, alternately IKKα). Subsequently, the NIK-IKK1 complex mediates the phosphorylation of p100 that triggers partial proteolysis of p100 into p52. Typically, accumulation of NIK or processing of p100 is estimated by immunoblot analyses, and these indirect measurements are used as a surrogate for cellular NIK activity. However, studies involving knockout and cancerous cells indicated that the activity of NIK-IKK1 might not always correlate with the abundance of NIK or with the relative level of p52 and p100. In this report, we describe a specific and sensitive assay for direct evaluation of cellular NIK-IKK1 activity. Here, NIK immunoprecipitates are examined for the presence of IKK1-dependent kinase activity toward p100. The NIK-IKK1 assay captured selectively noncanonical NF-κB activation in the context of multiple cell activating stimuli and cell types, including patient-derived myeloma cells. We suggest that our assay may help advance our understanding of the role of NIK in health and diseases.

An epithelial Nfkb2 pathway exacerbates intestinal inflammation by supplementing latent RelA dimers to the canonical NF-κB module.

Aberrant inflammation, such as that associated with inflammatory bowel disease (IBD), is fueled by the inordinate activity of RelA/NF-κB factors. As such, the canonical NF-κB module mediates controlled nuclear activation of RelA dimers from the latent cytoplasmic complexes. What provokes pathological RelA activity in the colitogenic gut remains unclear. The noncanonical NF-κB pathway typically promotes immune organogenesis involving Nfkb2 gene products. Because NF-κB pathways are intertwined, we asked whether noncanonical signaling aggravated inflammatory RelA activity. Our investigation revealed frequent engagement of the noncanonical pathway in human IBD. In a mouse model of experimental colitis, we established that Nfkb2-mediated regulations escalated the RelA-driven proinflammatory gene response in intestinal epithelial cells, exacerbating the infiltration of inflammatory cells and colon pathologies. Our mechanistic studies clarified that cell-autonomous Nfkb2 signaling supplemented latent NF-κB dimers, leading to a hyperactive canonical RelA response in the inflamed colon. In sum, the regulation of latent NF-κB dimers appears to link noncanonical Nfkb2 signaling to RelA-driven inflammatory pathologies and may provide for therapeutic targets.

NFKB2 inhibits NRG1 transcription to affect nucleus pulposus cell degeneration and inflammation in intervertebral disc degeneration.

Extracellular matrix degradation, reactive oxygen species (ROS) generation, and inflammation in nucleus pulposus (NP) cells contribute to the progression of intervertebral disc degeneration (IDD). NRGs (Neuregulins) play a vital role in the development of the nervous system. In the present study, we found that NRG1 was downregulated within degenerative intervertebral disc and NP tissues, according to both bioinformatics and experimental analyses. Within IL-1ß-stimulated NP cells, we observed degenerative and inflammatory changes, including inhibited cell viability, promoted cell apoptosis and ROS accumulation, reduced collagen II and aggrecan proteins, elevated MMP-3/13 and ADAMTS-4/5 proteins, and upregulated IL-6 and TNF-α mRNA levels. Within IL-1ß-stimulated NP cells, NRG1 expression was also downregulated. NRG1 overexpression attenuated, whereas NRG1 silencing aggravated IL-1ß-induced degenerative and inflammatory changes. Moreover, NRG1 regulated ErbB2/3 activation, contributing to the NRG1 protective function in NP cells. NFKB2 directly targeted the promoter region of NRG1 and inhibited NRG1 expression. In IL-1ß-stimulated NP cells, silencing NFKB2 attenuated, whereas silencing NRG1 aggravated the degenerative changes and inflammation; the effects of NFKB2 silencing were significantly reversed by NRG1 silencing. In conclusion, NRG1 expression is downregulated within degenerative NP tissue samples and IL-1ß-stimulated NP cells. NRG1 might protect against IL-1ß-induced degenerative changes and inflammation. The upregulated NFKB2 might be the reason of NRG1 downregulation in degenerative NP tissues.

BET inhibitor suppresses melanoma progression via the noncanonical NF-κB/SPP1 pathway.

Background: Bromodomain and extra-terminal domain (BET) inhibitors have shown profound efficacy against hematologic malignancies and solid tumors in preclinical studies. However, the underlying molecular mechanism in melanoma is not well understood. Here we identified secreted phosphoprotein 1 (SPP1) as a melanoma driver and a crucial target of BET inhibitors in melanoma. Methods: Bioinformatics analysis and meta-analysis were used to evaluate the SPP1 expression in normal tissues, primary melanoma, and metastatic melanoma. Real-time PCR (RT-PCR) and Western blotting were employed to quantify SPP1 expression in melanoma cells and tissues. Cell proliferation, wound healing, and Transwell assays were carried out to evaluate the effects of SPP1 and BET inhibitors in melanoma cells in vitro. A xenograft mouse model was used to investigate the effect of SPP1 and BET inhibitors on melanoma in vivo. Chromatin immunoprecipitation (ChIP) assay was performed to evaluate the regulatory mechanism of BET inhibitors on SPP1. Results: SPP1 was identified as a melanoma driver by bioinformatics analysis, and meta-analysis determined it to be a diagnostic and prognostic biomarker for melanoma. SPP1 overexpression was associated with poor melanoma prognosis, and silencing SPP1 suppressed melanoma cell proliferation, migration, and invasion. Through a pilot drug screen, we identified BET inhibitors as ideal therapeutic agents that suppressed SPP1 expression. Also, SPP1 overexpression could partially reverse the suppressive effect of BET inhibitors on melanoma. We further demonstrated that bromodomain-containing 4 (BRD4) regulated SPP1 expression. Notably, BRD4 did not bind directly to the SPP1 promoter but regulated SPP1 expression through NFKB2. Silencing of NFKB2 resembled the phenotype of BET inhibitors treatment and SPP1 silencing in melanoma. Conclusion: Our findings highlight SPP1 as an essential target of BET inhibitors and provide a novel mechanism by which BET inhibitors suppress melanoma progression via the noncanonical NF-κB/SPP1 pathway.

Circulating Myeloid-derived Suppressor Cells Facilitate Invasion of Thyroid Cancer Cells by Repressing miR-486-3p.

BACKGROUND: Myeloid-derived suppressor cells (MDSCs) have become increasingly recognized as facilitators of tumor development. However, the role of MDSCs in papillary thyroid carcinoma (PTC) progression has not been clearly explored. OBJECTIVE: We aimed to evaluate the levels and function of circulating MDSCs in PTC. METHODS: The proportion of circulating polymorphonuclear (PMN)-MDSCs and mononuclear-MDSCs from patients with PTC or benign thyroid nodules and healthy controls was measured using flow cytometry. For immunosuppressive activity analysis, sorted circulating MDSCs were cocultured with CD3/CD28-costimulated T lymphocytes and the proliferation of T cells was determined. PTC cell lines (TPC-1 and BC-PAP) were cocultured with PMN-MDSCs, and the effects on cell migration, invasion, proliferation, and apoptosis were evaluated. The differential expressed microribonucleic acids (RNAs) and messenger RNAs and their function were also explored in TPC-1 cells cocultured with or without PMN-MDSCs. RESULTS: PMN-MDSCs were increased in peripheral blood mononuclear cells of patients with PTC. Circulating PMN-MDSCs displayed strong T cell suppressive activity. PTC cells demonstrated enhanced invasive capabilities in vitro and in vivo when cocultured with sorted PMN-MDSCs. PMN-MDSCs decreased expression of miR-486-3p and activated nuclear factor kappa B2 (NF-κB2), a direct target of miR-486-3p. Rescue of miR-486-3p diminished the cell migration and invasion induced by PMN-MDSCs. CONCLUSION: Collectively, our work indicates that circulating PMN-MDSCs promote PTC progression. By suppressing miR-486-3p, PMN-MDSCs promote the activity of the NF-κB2 signaling pathway, resulting in accelerated invasion of PTC cells, which may provide new therapeutic strategies for treatment of thyroid cancer.

Excessive supply of glucose elicits an NF-κB2-dependent glycolysis in lactating goat mammary glands.

During lactation, an improper glucose supply often threatens mammary gland (MG) health. However, information is limited on the metabolic trajectories and molecules that regulate lactating MGs with an excessive glucose supply. Based on the network analysis of transcriptome and microRNAs, we found that the oversupply of glucose-induced severe glucose metabolic disorders in MGs of lactating goats, shifting lactose synthesis to acute fermentative glycolysis which caused increased flux of glucose metabolism into lactate. Moreover, NF-κB2 played a key role in regulating glycolysis, exhibiting a metabolic shift when MGs had an excessive supply of glucose. In primary mammary epithelial cells, fermentative glycolysis, and intracellular concentration of reactive oxygen species (ROS) were reduced by ganoderic acid A through blocking NF-κB2, while activation of NF-κB2 with phorbol myristate acetate (PMA) upregulated fermentative glycolysis and increased cellular ROS accumulation under excessive glucose. Thus, we established an NF-κB2-targeting method to reform the metabolic shift toward glycolysis caused by glucose oversupply by integrating NF-κB2 blockade and intracellular ROS scavenging.