RelB Proteins Research Articles

BRD4/nuclear PD-L1/RelB circuit is involved in the stemness of breast cancer cells

BackgroundBreast cancer (BC) is the most common cancer diagnosed in women worldwide. BC stem cells (BCSCs) have been known to be involved in the carcinogenesis of the breast and contribute to therapeutic resistance. The programmed death-ligand 1 (PD-L1) expression of BC correlated with a poor prognosis. Immunotherapies that target PD-L1 have great potential and have been successful when applied to cancer treatment. However, whether PD-L1 regulates BCSC formation is unknown.MethodsBCSCs were enriched by serum-free suspension culture. The properties of BCSCs were examined by mammosphere formation assay, CD44+/Cd24−, aldehyde dehydrogenase (ALDH) assay, CSC marker analysis, and mammosphere growth assay. To elucidate the functions of bromodomain-containing protein 4 (BRD4), nuclear PD-L1, and RelB proteins in the stemness of BCSCs, mammosphere formation was examined using BRD4 inhibitor and degrader, PD-L1 degrader, and RelB inhibitor. The antitumor function of 3',4',7,8-tetrahydroxyflavone (THF), a specific BRD4 inhibitor, was studied through in vivo tumor model and mouse studies, and the protein levels of c-Myc, PD-L1, and RelB were examined in tumor model under THF treatment.ResultsBRD4 was upregulated in breast CSCs and regulates the stemness of BCs. The downregulation of BRD4 using BRD4 PROTAC, ARV-825, and BRD4 inhibitor, (+)-JQ1, inhibits mammosphere formation and reduces the levels of breast CSC markers (CD44+/CD24− and ALDH1), stem cell marker genes, and mammosphere growth. BRD4 inhibitor (JQ1) and degrader (ARV825) downregulate membrane and nuclear fractions of PD-L1 through the inhibition of PD-L1 transcript levels. The knockdown of PD-L1 inhibits mammosphere formation. Verteporfin, a PD-L1 degrader, inhibits the transcripts and protein levels of PD-L1 and downregulates the transcript and protein levels of RelB. Calcitriol, a RelB inhibitor, and the knockdown of RelB using si-RelB regulate mammosphere formation through interleukin-6 (IL-6) expression. THF is a natural product and a potent selective BRD4 inhibitor, inhibits mammosphere formation, and reduces the levels of CD44+/CD24− and mammosphere growth by downregulating c-Myc, PD-L1, and RelB. 3',4',7,8-THF shows tumoricidal activity and increased levels of CD3+CD4+ and CD3+CD8+ T-cells in the tumor and tumor-draining lymph nodes (TDLNs) in the murine tumor model using 4T1 and MC38 cells.ConclusionsThe results show the first evidence of the essential role of the BRD4/nuclear PD-L1/RelB axis in breast CSC formation. The nuclear PD-L1 regulates RelB, and the RelB/p65 complex induces IL6 and breast CSC formation. Targeting nuclear PD-L1 represents a potential and novel tool for immunotherapies of intractable BC.9Yv5TUoezTtpQRCQc9Q8K4Video

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

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.

Ginsenoside Rg3 alleviates inflammation in a rat model of myocardial infarction via the SIRT1/NF-κB pathway.

Inflammation serves an important role in myocardial infarction (MI). Ginsenoside Rg3 (Rg3), an activator of sirtuin 1 (SIRT1), has been identified to elicit anti-inflammatory effects via the NF-κB pathway. However, the function of Rg3 in MI remains unknown. In the present study, a MI rat model was established by coronary artery ligation and treated with Rg3 to explore whether Rg3 could inhibit inflammation in MI rats by inhibiting the SIRT1/NF-κB pathway. At 28 days post-MI, it was identified that Rg3 not only decreased the ST-segment ECG values in MI rats, but also significantly decreased serum LDH, CK-MB and cTnI levels in MI rats. In addition, Rg3 also significantly decreased serum tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6 levels and increased serum IL-10 levels in MI rats. In the heart tissues of the MI rats, Rg3 attenuated myocardial pathological changes and cell apoptosis caused by MI, decreased the gene expression levels of TNF-α, IL-1β and IL-6, but increased the gene expression level of IL-10. In addition, the expression levels of the SIRT1 and transcription factor RelB proteins were significantly increased following Rg3 treatment, and the expression level of p-p65/p65 protein was significantly decreased in the heart tissues of MI rats with Rg3 treatment compared with that in heart tissues of MI rats without Rg3 treatment. In conclusion, Rg3 alleviates inflammation in a rat model of MI via the SIRT1/NF-κB pathway.

Loss of BAP-1 influences the activation of p52 and RelB proteins in the Inflammatory microenvironment of uveal melanoma

Abstract Background In recent years, research has focussed on targeted immunotherapeutic therapies in UM are disappointing and questions remain regarding the mechanisms leading to metastases and the tumor’s resistance to treatment. Genetic predictors for metastatic tumor behavior is the loss of BRCA1-associated protein 1 (BAP1) expression. NF-κB is a principal coordinator of innate immunity and inflammation and has emerged as an essential endogenous tumor promoter. We hypothesize that genetic changes not only influence the immunological microenvironment but also drive metastasis in UM and that NC-NFκB proteins (p52 & RelB) are the consequence of a highly-inflammatory profile. Methods In our study, based on the expression of CD3 (infiltrating lymphocytes) and CD68 (infiltrating macrophages), we divided our study cohort into two categories: UM with inflammation and UM without inflammation. Expression of BAP-1 and NC-NFκB proteins (RelB & p52/NFκB2) was evaluated using immunohistochemistry. Real-time PCR was performed on 60 frozen tumor samples. The presence of p52/RelB heterodimer detected by Co-immunoprecipitation in UM with inflammation. Results In the inflammation group, activation of NC-NFκB proteins found in 82% and 64% of cases while the loss of BAP-1 was observed in 82% of cases. Loss of BAP-1 protein along with activation of NC-NFκB proteins was seen in 70% of cases of the inflammation group. Loss of BAP-1 along with activation of C-NFκB proteins was statically significant with inflammatory factors such as CD34 + (p = 0.036), IL-6 (p = 0.012), LBD>15mm (p = 0.031) and epithelioid cell type (p = 0.027). In the inflammation group fold-change value of RelB (5.21) & NFκB2 (4.65) genes was reduced to 2.85 (RelB) & 2.34 (NFκB2) gene in the non-inflammation group. Mutation of BAP-1 was more frequently seen in the inflammation group than the non-inflammation group. Loss of BAP-1, along with the activation of NC-NFκB proteins, was associated with reduced metastasis-free survival and overall survival (p Conclusion Our preliminary data reveal that in an inflammation group loss of BAP-1 showed the synergistic role with the activation of NC-NFκB proteins and are the poor prognostic indicators of overall survival. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Differential expression of p52 and RelB proteins in the metastatic and non-metastatic groups of uveal melanoma with patient outcome.

Non-canonical NFκB (NC-NFκB) pathway plays an influential role in metastasis, which promotes cancer proliferation and progression. The aim of the study was to examine the expression of NC-NFκB proteins and their correlation with clinicopathological factors associated with metastatic cases of uveal melanoma (UM) and with the patient outcome. Expression of NC-NFκB proteins (p52, RelB, and co-expression of p52/RelB) was evaluated in 75 formalin-fixed cases of uveal melanoma by immunohistochemistry. Validation of nuclear immunoreactivity was done by western blotting. Transcriptional status of NC-NFκB genes was assessed in 60 fresh tumor tissues by quantitative real-time PCR. Co-immunoprecipitation was performed to determine the presence of native p52/RelB heterodimer in UM. Prognostic relevance was determined using Cox proportional hazard and Kaplan-Meier methods. Immunohistochemical expression of p52, RelB, and their co-expression was observed in 81%, 68.7%, 56.2% of metastatic cases, respectively, while their expression was seen only in 38%, 33% and 30% of non-metastatic cases. Loss of BAP-1 was correlated with expression of p52 and RelB proteins. Co-immunoprecipitation assay confirmed the putative interaction of p52 with RelB protein in metastatic cases of uveal melanoma. Co-expression of p52/RelB and expression of p52 protein was significantly correlated with decreased metastasis-free survival (MFS) (p = 0.004; p = 0.002) and overall survival (OS) (p = 0.004; p = 0.032), while the RelB expression only correlated with reduced MFS (p = 0.003). Our data showed that non-canonical NFκB proteins were significantly higher in metastatic cases and associated with poor outcome of the patients. Furthermore, the p52 protein could be used as a potential therapeutic biomarker for metastatic cases in uveal melanoma.

ClpP is required for proteolytic regulation of type II toxin-antitoxin systems and persister cell formation in Streptococcus mutans.

The type II toxin–antitoxin (TA) modules, mazEF and relBE, in Streptococcus mutans have been implicated in stress response, antibiotic tolerance and persister cell formation. However, how S. mutans regulates these systems to prevent unwanted toxin activation and persister cell formation is unclear. In this study, we provide evidence that ClpP is required for the proteolytic regulation of these TA systems and persister cell formation in S. mutans following antibiotic challenge. A persister viability assay showed that S. mutans UA159 (WT) formed a larger quantity of persister cells than its isogenic mutant ΔclpP following antibiotic challenge. However, the lux reporter assay revealed that clpP deletion did not affect the transcriptional levels of mazEF and relBE, since no significant differences (P>0.05) in the reporter activities were detected between the wild-type and ΔclpP background. Instead, all antibiotics tested at a sub-minimum inhibitory concentration (sub-MIC) induced transcriptional levels of mazEF and relBE operons. We then examined the protein profiles of His-tagged MazE and RelB proteins in the UA159 and ΔclpP backgrounds by Western blotting analysis. The results showed that S. mutans strains grown under non-stress conditions expressed very low but detectable levels of MazE and RelB antitoxin proteins. Antibiotics at sub-MICs induced the levels of the MazE and RelB proteins, but the protein levels decreased rapidly in the wild-type background. In contrast, a stable level of MazE and RelB proteins could be detected in the ΔclpP mutant background, suggesting that both proteins accumulated in the ΔclpP mutant. We conclude that ClpP is required for the proteolytic regulation of cellular levels of the MazE and RelB antitoxins in S. mutans , which may play a critical role in modulating the TA activities and persister cell formation of this organism following antibiotic challenge.

Lymphotoxin‐β receptor‐NIK signaling induces alternative RELB/NF‐κB2 activation to promote metastatic gene expression and cell migration in head and neck cancer

Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-β (LTβ), receptor LTβR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTβR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTβ, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTβR, NIK, and RELB proteins. Recombinant LTβ, and siRNA depletion of endogenous LTβR and NIK, modulated expression of LTβR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTβ induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTβ-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTβ is expressed. Our findings show that LTβ/LTβR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.

Mechanisms of NF-κB p65 and strategies for therapeutic manipulation.

The transcription factor NF-κB is a critical regulator of immune and inflammatory responses. In mammals, the NF-κB/Rel family comprises five members: p50, p52, p65 (Rel-A), c-Rel, and Rel-B proteins, which form homo- or heterodimers and remain as an inactive complex with the inhibitory molecules called IκB proteins in resting cells. Two distinct NF-κB signaling pathways have been described: 1) the canonical pathway primarily activated by pathogens and inflammatory mediators, and 2) the noncanonical pathway mostly activated by developmental cues. The most abundant form of NF-κB activated by pathologic stimuli via the canonical pathway is the p65:p50 heterodimer. Disproportionate increase in activated p65 and subsequent transactivation of effector molecules is integral to the pathogenesis of many chronic diseases such as the rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and even neurodegenerative pathologies. Hence, the NF-κB p65 signaling pathway has been a pivotal point for intense drug discovery and development. This review begins with an overview of p65-mediated signaling followed by discussion of strategies that directly target NF-κB p65 in the context of chronic inflammation.

Abstract 08: Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies

Abstract The Bruton's tyrosine kinase (BTK) inhibitor ibrutinib (IBR) is FDA approved for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). However, IBR responses are incomplete in majority of cases, and not durable in MCL and high-risk CLL. We hypothesize that de novo resistance, incomplete responses, and rapid recurrence can be due to adaptive signaling that should be cotargeted with BTK to achieve deeper and durable responses. We previously showed that venetoclax (VEN), an inhibitor of Bcl-2, generates synergistic cytotoxicity with IBR in MCL lines (Axelrod et al. Leukemia 2014;28:407-10). Recently, we extended these findings to circulating leukemic B cells in CLL, MCL (Jayappa et al. ASH 2016), and follicular lymphoma (FL) patient PBMCs treated ex vivo. Based on these results we initiated a phase I/Ib trial for IBR+VEN combination in MCL. Our ex vivo cytotoxicity studies showed a surprisingly high frequency of de novo resistance to IBR+VEN. Resistance did not correlate with previous therapy or clinical characteristics of patients. However, FACS analysis of primary CLL cells showed that cells displaying an “activation” phenotype (CD5+/CD19+/CD69+) were less sensitive to cytotoxicity of IBR+VEN, suggesting that cellular activation by extrinsic factors could generate drug resistance. To test this, we cocultured or preincubated CLL patient PBMCs with various stromal cells, cytokines, and other agonists and found that soluble CD40L, IL10, and CpG oligodeoxynucleotides (ODN) generated variable resistance to IBR+VEN in CLL samples, with CpG ODN, a specific TLR9 agonist, being the most effective. The combination of CpG ODN, sCD40L, and IL10 (“agonist mix”) generated near-complete resistance to IBR+VEN in most CLL(N=22)/MCL(N=7)/FL(N=4) patient PBMCs. The agonist mix treatment significantly upregulated nuclear localization of RelA and RelB proteins in CLL/MCL/FL cells analyzed by ImageStream, suggesting activation of NF-kB signaling. Agonist mix treatment also induced the NF-kB dependent overexpression of anti-apoptotic proteins Mcl-1, Bcl-xL, and survivin, and inhibitors of NF-kB or of upregulated anti-apoptotic proteins overcame this drug resistance (Jayappa et al. ASH 2016). To further explore the role of TLR9 and other innate immune receptors in resistance, we performed IBR+VEN cytotoxicity analysis in CLL/MCL patient PBMCs cocultured with agonists of various TLRs and NOD1/2 receptors. Preliminary results showed that TLR9 agonist CpG ODN generated significant resistance to IBR+VEN in most CLL/MCL samples, and agonists of TLR1/2, TLR7, and NOD1/2 induced a moderate level of resistance in a MCL sample. The CpG ODN treatment induced a significant increase in NF-kB signaling measured by increased nuclear localization of RelB in CLL patient samples. To determine if soluble agonists in the extranodal macroenvironment of patients could generate NF-kB dependent resistance to IBR+VEN, we analyzed drug cytotoxicity in CLL(N=6) and MCL(N=1) patient PBMCs cultured with autologous plasma. We found that preincubation with autologous plasma induced variable levels of resistance to IBR+VEN in CLL/MCL samples, but did not induce resistance to a NF-kB inhibitor BMS-345541. In conclusion, soluble factors in the patient nodal/extranodal macroenvironment, particularly a TLR9 agonist, generate resistance to IBR+VEN by activating NF-kB signaling and overexpression of multiple anti-apoptotic proteins. The same resistance mechanism, based on innate immune signaling, occurs in three mature B-cell malignancies, CLL, MCL and FL, that differ in etiology and clinical course. The signaling pathway responsible for resistance contains new targets for therapeutic intervention with potential to prevent or reverse resistance to IBR and VEN in these and perhaps other cancers as well. Citation Format: Kallesh D. Jayappa, Craig A. Portell, Vicki L. Gordon, Timothy P. Bender, Michael E. Williams, Michael J. Weber. Soluble agonists in the in vivo macroenvironment generate phenotypic de novo resistance to BTK/Bcl-2 targeted therapies in diverse B-cell malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr 08.

Cooperation of Rel family members in regulating A\u03b21-40-mediated pro-inflammatory cytokine secretion by retinal pigment epithelial cells

Amyloid-beta (Aβ) is a hallmark component of age-related macular degeneration (AMD), which induces secretion of pro-inflammatory cytokines from retinal pigment epithelium (RPE). Previous studies have shown that p50/RelA (p65), a member of NF-κB family, is an essential pro-inflammatory transcription factor responding to Aβ1-40 stimulation, but few focused on the other two Rel transcription factor members – RelB and c-Rel – and their role in Aβ1-40-mediated inflammation. It was reported that RelA, RelB and c-Rel are also implicated in various NF-κB-mediated inflammatory diseases. Therefore, we infer that Aβ1-40-mediated inflammation targets not only the classical inflammation regulator, RelA, but also RelB and c-Rel. In this study, we demonstrate that intravitreally injected Aβ1-40 mice develop AMD-like pathologic changes, coupled with Rel protein (RelA, RelB and c-Rel) synthesis and nuclear translocation. To focus on the interaction mechanism of Rel proteins, we found that RelB and c-Rel formed a heterodimer with RelA in mice model. We also found that c-Rel silencing decreased the levels of Aβ1-40-dependent RelA expression, indicating that RelB and c-Rel may interact with RelA as coactivator and c-Rel is required to activate the expression of RelA. Moreover, Rel protein silencing decreased the expression of distinct pro-inflammatory cytokines. Together, we demonstrate that besides RelA, RelB and c-Rel can also be activated by Aβ1-40, all of which mediate pro-inflammatory cytokine transcription and RPE damage. Our findings imply that RPE-mediated inflammation under the stimulation of Aβ1-40 is multi-targeted and RelA, RelB and c-Rel proteins may be the new targets of anti-inflammatory agents.

Abstract 358: LTB and LTBR mediates alternative Nf-kB activation through NIk and RELB/NF-kB2 to promote cell migration of HNSCC

Abstract Nuclear factor-κB (NF-κB) includes a family of NF-κB/REL transcription factors that regulate key inflammatory and cancer genes, and which are aberrantly activated in head and neck squamous cell carcinomas (HNSCC). Lymphotoxin beta/Lymphotoxin beta receptor (LTβ/LTβR) can mediate activation of the “alternative” NF-κB pathway that includes NF-κB-inducing kinase (NIK), Inhibitor-κB kinase α (IKKα), and transcription factors RELB/NF-κB2. While LTβ is expressed in lymph nodes where HNSCC spread, the role of this pathway in pathogenesis of HNSCC is not well studied. Analysis of The Cancer Genome Atlas (TCGA) data reveals that LTβR is amplified or overexpressed in larynx or oral cavity HNSCC, while LTα/β, NIK, (MAP3K14) and RELB are overexpressed in oropharynx and tonsil cancers. Similarly, we observed overexpression of LTβR, NIK, and RELB proteins in subsets of HNSCC cell lines. In UM-SCC46, LTβ enhanced nuclear translocation of RELB/NF-κB2 (p52) subunit proteins, while LTβR siRNA knockdown decreased the expression of its target kinase NIK and RELB/NF-κB2 (p52). NIK knockdown also decreased RELB/NF-κB2 (p52) protein expression, whereas LTβ treatment stabilized NIK, and RELB/NF-κB2 (p52). Functional LTβ-mediated NF-κB activation was examined using a stably transfected NF-κB reporter cell line for β-lactamase/Fret activity. Knockdown of LTβR and NIK decreased NF-κB activity, whereas treatment with LTβ partially restored it. Treatment with a NIK inhibitor (1, 3[2H, 4H]-Iso-Quinoline Dione) reduced the protein expression of NIK, IKKα, and RELB/NF-κB2 (p52) in the cytoplasm and/or in the nucleus, and blocked LTβ induced translocation of RELB to nucleus. NIK and RELB knockdown by siRNA or using NIK inhibitor slowed HNSCC migration. Knockdown of NIK inhibited the LTβ-induced expression of MET, and SERPINE1 genes involved in migration, and knockdown of MET by siRNA inhibited the migration of HNSCC cell lines. Our findings show that LTβ/LTβ receptor promotes activation of the alternative NIK-IKKa-RELB/NF-κB2 pathway, to enhance MET-mediated cell migration in HNSCC. This may help explain why HNSCC exhibit migration and metastasis to local lymph nodes, where LTβ is expressed. The constitutive and LTβ induced activation of NIK and downstream NF-κB alternate pathway proteins, RELB and NF-κB2/p52 could be therapeutic targets in HNSCC. (Supported by NIDCD intramural projects ZIA-DC-000016, 74) Citation Format: Rita Das, Jamie Coupar, Anthony saleh, Paul E. Clavijo, Zhong Chen, Carter VanWaes. LTB and LTBR mediates alternative Nf-kB activation through NIk and RELB/NF-kB2 to promote cell migration of HNSCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 358. doi:10.1158/1538-7445.AM2017-358

Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins.

ObjectivesUbiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown.MethodsWe examined the expression levels of E3 ligases and NF-κB members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay.ResultsThe expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-κB members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-κB RelA and RelB proteins were found to bind to the NF-κB binding site in the mouse Itch promoter.ConclusionsOur findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing.Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154–161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.

Sequential Actions of SIRT1-RELB-SIRT3 Coordinate Nuclear-Mitochondrial Communication during Immunometabolic Adaptation to Acute Inflammation and Sepsis

We reported that NAD(+)-dependent SIRT1, RELB, and SIRT6 nuclear proteins in monocytes regulate a switch from the glycolysis-dependent acute inflammatory response to fatty acid oxidation-dependent sepsis adaptation. We also found that disrupting SIRT1 activity during adaptation restores immunometabolic homeostasis and rescues septic mice from death. Here, we show that nuclear SIRT1 guides RELB to differentially induce SIRT3 expression and also increases mitochondrial biogenesis, which alters bioenergetics during sepsis adaptation. We constructed this concept using TLR4-stimulated THP1 human promonocytes, a model that mimics the initiation and adaptation stages of sepsis. Following increased expression, mitochondrial SIRT3 deacetylase activates the rate-limiting tricarboxylic acid cycle (TCA) isocitrate dehydrogenase 2 and superoxide dismutase 2, concomitant with increases in citrate synthase activity. Mitochondrial oxygen consumption rate increases early and decreases during adaptation, parallel with modifications to membrane depolarization, ATP generation, and production of mitochondrial superoxide and whole cell hydrogen peroxide. Evidence of SIRT1-RELB induction of mitochondrial biogenesis included increases in mitochondrial mass, mitochondrial-to-nuclear DNA ratios, and both nuclear and mitochondrial encoded proteins. We confirmed the SIRT-RELB-SIRT3 adaptation link to mitochondrial bioenergetics in both TLR4-stimulated normal and sepsis-adapted human blood monocytes and mouse splenocytes. We also found that SIRT1 inhibition ex vivo reversed the sepsis-induced changes in bioenergetics.

RelB regulates Bcl-xl expression and the irradiation-induced apoptosis of murine prostate cancer cells.

Apoptosis in prostate cancer (PCa) induced by ionizing radiation (IR) is believed to play a critical role in radioresistance. Bcl-xl, an important member of the anti-apoptotic Bcl-2 family, has critical roles in tumor progression and development. The aim of the present study was to investigate the association of Bcl-xl expression and radiosensitivity from murine PCa RM-1 cells. An adenovirus-mediated RNA interference technique was employed to inhibit the expression of the RelB gene. RelB proteins were detected upon irradiation following transfection with small interfering (si)RelB, as shown by western blot analysis. The radiosensitivity of the RM-1 cells was determined by clonogenic assays. The apoptosis of the RM-1 cells were detected by flow cytometry assay, then quantitative polymerase chain reaction assays were performed to determine the expression level of Bcl-xl mRNA in the RM-1 cells. Radiation treatment increased the RelB protein levels from the cytosol and nucleus in the RM-1 cells. The protein expression levels of RelB in the pLentilox-sh-RelB-transfected RM-1 cells were significantly lower than in the negative interference group following radiation treatment. The percentage of cells undergoing apoptosis in the siRelB-RM-1 group was significantly higher than that in the control group following radiation treatment. Finally, a positive link between Bcl-xl expression and RelB activity was established in the RM-1 cells. Inhibition of RelB correlates with a decrease in expression of Bcl-xl. In conclusion, adenovirus-mediated siRNA targeting RelB inhibits Bcl-xl expression, enhances radiosensitivity and regulates the irradiation-induced apoptosis of the murine PCa RM-1 cell line.

Nuclear protein 1 promotes pancreatic cancer development and protects cells from stress by inhibiting apoptosis

Pancreatic ductal adenocarcinoma (PDAC) has the lowest survival rate of all cancers and shows remarkable resistance to cell stress. Nuclear protein 1 (Nupr1), which mediates stress response in the pancreas, is frequently upregulated in pancreatic cancer. Here, we report that Nupr1 plays an essential role in pancreatic tumorigenesis. In a mouse model of pancreatic cancer with constitutively expressed oncogenic Kras(G12D), we found that loss of Nupr1 protected from the development of pancreatic intraepithelial neoplasias (PanINs). Further, in cultured pancreatic cells, nutrient deprivation activated Nupr1 expression, which we found to be required for cell survival. We found that Nupr1 protected cells from stress-induced death by inhibiting apoptosis through a pathway dependent on transcription factor RelB and immediate early response 3 (IER3). NUPR1, RELB, and IER3 proteins were coexpressed in mouse PanINs from Kras(G12D)-expressing pancreas. Moreover, pancreas-specific deletion of Relb in a Kras(G12D) background resulted in delayed in PanIN development associated with a lack of IER3 expression. Thus, efficient PanIN formation was dependent on the expression of Nupr1 and Relb, with likely involvement of IER3. Finally, in patients with PDAC, expression of NUPR1, RELB, and IER3 was significantly correlated with a poor prognosis. Cumulatively, these results reveal a NUPR1/RELB/IER3 stress-related pathway that is required for oncogenic Kras(G12D)-dependent transformation of the pancreas.

Retroviral Vectors Induce Epigenetic Chromatin Modifications and IL-10 Production in Transduced B Cells via Activation of Toll-like Receptor 2

The immune response toward viral vectors used for gene therapy and genetic vaccination appears to be critically important in determining the therapeutic outcome. However, the mechanisms that control the immune response following gene transfer are poorly understood. Unexpectedly, we found that integrating retroviral vector particles induce stable interleukin-10 (IL-10) production in murine (BALB/c H-2(d)) transduced B cells. This requires a novel mechanism whereby the interaction of retroviral vector particle with its cognate cellular receptor activates intracellular signaling pathways resulting in stable epigenetic modifications. Murine B cells exposed to retroviral vector particles triggered the colocalization of the retroviral cellular receptor [mouse cationic amino acid transporter 1 (mCAT1)] and Toll-like receptor 2 (TLR2) into lipid microrafts, which in turn activated TLR2 signaling pathways. TLR2 activation induced STAT3 phosphorylation and increased phosphorylated histone 3 (H3) at the STAT3-binding site of the IL-10 promoter. In addition, TLR2 activation during transduction activates nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor, α (NFKBIA), thereby preventing the translocation of the nuclear factor-κB (NF-κB) complex to the nucleus and the transcription of proinflammatory cytokines. These findings open new perspectives for controlling immune responses following gene therapy and genetic vaccination.

MiR-301a as an NF-κB activator in pancreatic cancer cells

NF-κB is constitutively activated in most human pancreatic adenocarcinoma, which is a deadly malignancy with a 5-year survival rate of about 5%. In this work, we investigate whether microRNAs (miRNAs) contribute to NF-κB activation in pancreatic cancer. We demonstrate that miR-301a down-regulates NF-κB-repressing factor (Nkrf) and elevates NF-κB activation. As NF-κB promotes the transcription of miR-301a, our results support a positive feedback loop as a mechanism for persistent NF-κB activation, in which miR-301a represses Nkrf to elevate NF-κB activity, which in turn promotes miR-301a transcription. Nkrf was found down-regulated and miR-301a up-regulated in human pancreatic adenocarcinoma tissues. Moreover, miR-301a inhibition or Nkrf up-regulation in pancreatic cancer cells led to reduced NF-κB target gene expression and attenuated xenograft tumour growth, indicating that miR-301a overexpression contributes to NF-κB activation. Revealing this novel mechanism of NF-κB activation by an miRNA offers new avenues for therapeutic interventions against pancreatic cancer.

Interaction specificity, toxicity and regulation of a paralogous set of ParE/RelE‐family toxin–antitoxin systems

Toxin-antitoxin (TA) gene cassettes are widely distributed across bacteria, archaea and bacteriophage. The chromosome of the alpha-proteobacterium, Caulobacter crescentus, encodes eight ParE/RelE-superfamily toxins that are organized into operons with their cognate antitoxins. A systematic genetic analysis of these parDE and relBE TA operons demonstrates that seven encode functional toxins. The one exception highlights an example of a non-functional toxin pseudogene. Chromosomally encoded ParD and RelB proteins function as antitoxins, inhibiting their adjacently encoded ParE and RelE toxins. However, these antitoxins do not functionally complement each other, even when overexpressed. Transcription of these paralogous TA systems is differentially regulated under distinct environmental conditions. These data support a model in which multiple TA paralogs encoded by a single bacterial chromosome form independent functional units with insulated protein-protein interactions. Further characterization of the parDE(1) system at the single-cell level reveals that ParE(1) toxin functions to inhibit cell division but not cell growth; residues at the C-terminus of ParE(1) are critical for its stability and toxicity. While continuous ParE(1) overexpression results in a substantial loss in cell viability at the population level, a fraction of cells escape toxicity, providing evidence that ParE(1) toxicity is not uniform within clonal cell populations.

Single Nucleotide Changes in the Human Iγ1 and Ιγ4 Promoters Underlie Different Transcriptional Responses to CD40

Analysis of subclass-specific germline transcription in activated peripheral B cells revealed a highly biased expression pattern of the four Igamma transcripts to signals through CD40 and IL-4. This difference was most pronounced when comparing the profile of Igamma1 and Igamma4 transcripts and was not expected given the very high degree of sequence conservation between promoters. In this report, the influence of sequence differences on the regulation of the Igamma1 and Igamma4 promoters has been investigated given the highly muted transcriptional activity of the Igamma4 promoter. Two regions were analyzed where single nucleotide differences corresponded to major changes in transcriptional activity. These regions were the previously defined CD40 response region containing three putative NF-kappaB-binding sites and the downstream 36-bp region containing CREB/activating transcription factor and kappaB6 sites. Mutation of a single nucleotide at position 6 within the Igamma4 kappaB6 site increased promoter activity to approximately 50% of the activity of the Igamma1 promoter. Furthermore, elevated promoter strength corresponded with increased binding of p50, p65, c-Rel, RelB, and p300 proteins to a level comparable with that of Igamma1. Minor nucleotide changes to both the Igamma4 CD40 response region and the 36-bp element resulted in a response undistinguishable from an Igamma1 response, suggesting cooperation between the two regulatory regions for optimal transcriptional activity. Collectively, these mutational analyses suggest that minor sequence differences contribute to the composition and affinity of transcriptional protein complexes regulating subclass-specific germline transcription, which in part impacts the overall level of class switch recombination to targeted C(H) regions.