RelA Expression Research Articles

674 dclk1+ Tuft Cells Display Quiescent Stem Cell-Like Properties in the Small Intestine

Background: To date no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. Here we present a discrete candidate gene signature of Dclk1+ cells obtained from Dclk1-CreER;Rosa26-YFP mice, which will be a valuable animal model for future work seeking to uncover the latent abilities and functional role of Dclk1 expressing tuft cells and their subsets. Methods: The Dclk1-CreER;Rosa26-YFP compound mice were injected with tamoxifen to induce the expression of YFP driven by Dclk1 promoter. We FACS-sorted Dclk1+ cells from the small intestinal epithelium of Dclk1-CreER;Rosa26-YFP mice. We determined the mRNA expression levels of Dclk1 and other stem cell associated markers (Bmi1 and Lgr5), and pluripotency factors in the isolated Dclk1+ and Dclk1cells. To analyze the expression of key markers of quiescence survival and longevity, mRNA expression of cell cycle regulators and survival factors were analyzed. To explore the clonogenic capacity of the otherwise quiescent cells, we investigated self-renewal capacity in the Dclk1+ and Dclk1population. Results: Following tamoxifen administration, intestines were collected for YFP and Dclk1 staining, and we found that YFP co-localized with Dclk1 expression. Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFPcells, confirming successful isolation of Dclk1+ cells. The Dclk1+ population was enriched (~6 fold) for the quiescent putative stem cell marker Bmi1, whereas the Dclk1fraction was enriched ~174 fold for Lgr5. We observed a relatively higher expression of pluripotency genes (~5-80 fold) as well as a significant increase in pro-survival genes (~4-9 fold) and quiescence regulatory markers (~8-24 fold) in the Dclk1+ cells compared to Dclk1cells. A 14-fold increase in self-renewal capability was observed in in vitro isolated Dclk1+ cells by clonogenic assay. The unique genetic profile presented in this study suggests that Dclk1+ cells may be relatively quiescent under normal homeostatic conditions but maintain their pluripotency and metabolic machinery required for survival/longevity through the expression of Cdkn1A, Cdkn1B, Oct4, Sox2, Nanog, and Klf4, Wif1, RelA, Akt and AMPK, and vital regulation of Raptor, Rictor, p53 and Survivin. Conclusion: Taken as a whole, these results provide additional support for the hypothesis that while the majority of Dclk1+ cells are committed tuft cells, they also maintain the molecular capability for stemness in reserve, and could play a role in the maintenance of the stem cell niche in an injury repair environment.

675 Depletion of CDC42 Leads to Disrupted Villus-Crypt Architecture, Increasing the Sensitivity to Intestinal Mucosal Injury

Background: To date no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. Here we present a discrete candidate gene signature of Dclk1+ cells obtained from Dclk1-CreER;Rosa26-YFP mice, which will be a valuable animal model for future work seeking to uncover the latent abilities and functional role of Dclk1 expressing tuft cells and their subsets. Methods: The Dclk1-CreER;Rosa26-YFP compound mice were injected with tamoxifen to induce the expression of YFP driven by Dclk1 promoter. We FACS-sorted Dclk1+ cells from the small intestinal epithelium of Dclk1-CreER;Rosa26-YFP mice. We determined the mRNA expression levels of Dclk1 and other stem cell associated markers (Bmi1 and Lgr5), and pluripotency factors in the isolated Dclk1+ and Dclk1cells. To analyze the expression of key markers of quiescence survival and longevity, mRNA expression of cell cycle regulators and survival factors were analyzed. To explore the clonogenic capacity of the otherwise quiescent cells, we investigated self-renewal capacity in the Dclk1+ and Dclk1population. Results: Following tamoxifen administration, intestines were collected for YFP and Dclk1 staining, and we found that YFP co-localized with Dclk1 expression. Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFPcells, confirming successful isolation of Dclk1+ cells. The Dclk1+ population was enriched (~6 fold) for the quiescent putative stem cell marker Bmi1, whereas the Dclk1fraction was enriched ~174 fold for Lgr5. We observed a relatively higher expression of pluripotency genes (~5-80 fold) as well as a significant increase in pro-survival genes (~4-9 fold) and quiescence regulatory markers (~8-24 fold) in the Dclk1+ cells compared to Dclk1cells. A 14-fold increase in self-renewal capability was observed in in vitro isolated Dclk1+ cells by clonogenic assay. The unique genetic profile presented in this study suggests that Dclk1+ cells may be relatively quiescent under normal homeostatic conditions but maintain their pluripotency and metabolic machinery required for survival/longevity through the expression of Cdkn1A, Cdkn1B, Oct4, Sox2, Nanog, and Klf4, Wif1, RelA, Akt and AMPK, and vital regulation of Raptor, Rictor, p53 and Survivin. Conclusion: Taken as a whole, these results provide additional support for the hypothesis that while the majority of Dclk1+ cells are committed tuft cells, they also maintain the molecular capability for stemness in reserve, and could play a role in the maintenance of the stem cell niche in an injury repair environment.

677 IBS Patients Have SCN5A Mutations That Result in Decreased NaV1.5 Current and Mechanosensitivity

Background: To date no discrete genetic signature has been defined for isolated Dclk1+ tuft cells within the small intestine. Furthermore, recent reports on the functional significance of Dclk1+ cells in the small intestine have been inconsistent. These cells have been proposed to be fully differentiated cells, reserve stem cells, and tumor stem cells. Here we present a discrete "candidate" gene signature of Dclk1+ cells obtained from Dclk1-CreER;Rosa26-YFP mice, which will be a valuable animal model for future work seeking to uncover the latent abilities and functional role of Dclk1 expressing tuft cells and their subsets. Methods: The Dclk1-CreER;Rosa26-YFP compound mice were injected with tamoxifen to induce the expression of YFP driven by Dclk1 promoter. We FACS-sorted Dclk1+ cells from the small intestinal epithelium of Dclk1-CreER;Rosa26-YFP mice. We determined the mRNA expression levels of Dclk1 and other stem cell associated markers (Bmi1 and Lgr5), and pluripotency factors in the isolated Dclk1+ and Dclk1cells. To analyze the expression of key markers of quiescence survival and longevity, mRNA expression of cell cycle regulators and survival factors were analyzed. To explore the clonogenic capacity of the otherwise quiescent cells, we investigated self-renewal capacity in the Dclk1+ and Dclk1population. Results: Following tamoxifen administration, intestines were collected for YFP and Dclk1 staining, and we found that YFP co-localized with Dclk1 expression. Analysis of sorted YFP+ cells demonstrated marked enrichment (~6000 fold) for Dclk1 mRNA compared with YFPcells, confirming successful isolation of Dclk1+ cells. The Dclk1+ population was enriched (~6 fold) for the quiescent putative stem cell marker Bmi1, whereas the Dclk1fraction was enriched ~174 fold for Lgr5. We observed a relatively higher expression of pluripotency genes (~5-80 fold) as well as a significant increase in pro-survival genes (~4-9 fold) and quiescence regulatory markers (~8-24 fold) in the Dclk1+ cells compared to Dclk1cells. A 14-fold increase in self-renewal capability was observed in in vitro isolated Dclk1+ cells by clonogenic assay. The unique genetic profile presented in this study suggests that Dclk1+ cells may be relatively quiescent under normal homeostatic conditions but maintain their pluripotency and metabolic machinery required for survival/longevity through the expression of Cdkn1A, Cdkn1B, Oct4, Sox2, Nanog, and Klf4, Wif1, RelA, Akt and AMPK, and vital regulation of Raptor, Rictor, p53 and Survivin. Conclusion: Taken as a whole, these results provide additional support for the hypothesis that while the majority of Dclk1+ cells are committed tuft cells, they also maintain the molecular capability for stemness in reserve, and could play a role in the maintenance of the stem cell niche in an injury repair environment.

Effects of Pholiota nameko polysaccharide on NF-κB pathway of murine bone marrow-derived dendritic cells

This study investigated the effect of a polysaccharide purified from Pholiota nameko (PNPS-1) on the NF-κB signaling pathway of murine bone marrow-derived dendritic cells (BMDCs) and relevant mechanisms. The results showed that PNPS-1 could decrease the expression of maturation markers CD40 and CD80 on BMDCs. PNPS-1 also could decrease the mRNA expression of Myd88, TRAF6, TIRAP, IRAKI, IKBKB, NFKB1, NFKB2 and RelA in immature BMDCs determined by RT-PCR, and decreased the IKKβ and P65 production in BMDCs determined by Western blot, and decreased the NF-кB P65 production determined by ELISA. In addition, the effects of PNPS-1 on BMDCs were significantly impaired by treating the cells with anti-TLR2 antibody prior to PNPS-1 treatment, implying direct interaction between PNPS-1 and TLR2 on cell surface. These results indicate that PNPS-1 regulates BMDCs through TLR2 and downstream NF-кB signalings.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) promotes glioma cell invasion through induction of NF-κB-inducing kinase (NIK) and noncanonical NF-κB signaling.

BackgroundHigh-grade gliomas are one of the most invasive and therapy-resistant cancers. We have recently shown that noncanonical NF-κB/RelB signaling is a potent driver of tumorigenesis and invasion in the aggressive, mesenchymal subtype of glioma. However, the relevant signals that induce activation of noncanonical NF-κB signaling in glioma and its function relative to the canonical NF-κB pathway remain elusive.MethodsThe ability of tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) to regulate NF-κB signaling and promote tumor progression was investigated in both established and primary high-grade glioma tumor lines using a three-dimensional (3-D) collagen invasion assay. The roles of specific NF-κB proteins in regulating glioma cell invasion and expression of Matrix Metalloproteinase 9 (MMP9) in response to TWEAK were evaluated using shRNA-mediated loss-of-function studies. The ability of NF-κB-inducing kinase (NIK) to promote glioma growth in vivo was investigated using an orthotopic xenograft mouse model.ResultsIn glioma cells that display elevated noncanonical NF-κB signaling, loss of RelB attenuates invasion without affecting RelA expression or phosphorylation and RelB is sufficient to promote invasion in the absence of RelA. The cytokine TWEAK preferentially activates the noncanonical NF-κB pathway through induction of p100 processing to p52 and nuclear accumulation of both RelB and p52 without activating the canonical NF-κB pathway. Moreover, TWEAK, but not TNFα, significantly increases NIK mRNA levels. TWEAK also promotes noncanonical NFκB-dependent MMP9 expression and glioma cell invasion. Finally, expression of NIK is sufficient to increase gliomagenesis in vivo.ConclusionsOur data establish a key role for NIK and noncanonical NF-κB in mediating TWEAK-induced, MMP-dependent glioma cell invasion. The findings also demonstrate that TWEAK induces noncanonical NF-κB signaling and signal-specific regulation of NIK mRNA expression. Together, these studies reveal the important role of noncanonical NF-κB signaling in regulating glioma invasiveness and highlight the therapeutic potential of targeting activation of NIK in this deadly disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-014-0273-1) contains supplementary material, which is available to authorized users.

The restrained expression of NF-kB in renal tissue ameliorates folic acid induced acute kidney injury in mice.

The Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) represent family of structurally-related eukaryotic transcription factors which regulate diverse array of cellular processes including immunological responses, inflammation, apoptosis, growth & development. Increased expression of NF-kB has often been seen in many diverse diseases, suggesting the importance of genomic deregulation to disease pathophysiology. In the present study we focused on acute kidney injury (AKI), which remains one of the major risk factor showing a high rate of mortality and morbidity. The pathology associated with it, however, remains incompletely known though inflammation has been reported to be one of the major risk factor in the disease pathophysiology. The role of NF-kB thus seemed pertinent. In the present study we show that high dose of folic acid (FA) induced acute kidney injury (AKI) characterized by elevation in levels of blood urea nitrogen & serum creatinine together with extensive tubular necrosis, loss of brush border and marked reduction in mitochondria. One of the salient observations of this study was a coupled increase in the expression of renal, relA, NF-kB2, and p53 genes and proteins during folic acid induced AKI (FA AKI). Treatment of mice with NF-kB inhibitor, pyrrolidine dithio-carbamate ammonium (PDTC) lowered the expression of these transcription factors and ameliorated the aberrant renal function by decreasing serum creatinine levels. In conclusion, our results suggested that NF-kB plays a pivotal role in maintaining renal function that also involved regulating p53 levels during FA AKI.

Annexin-A1 regulates microRNA-26b* and microRNA-562 to directly target NF-κB and angiogenesis in breast cancer cells.

Annexin 1 (ANXA1) is an endogenous anti-inflammatory protein implicated in cancer. ANXA1 was previously shown to be regulated by hsa-miR-196a. However, whether ANXA1 itself regulates microRNA (miR) expression is unknown. Therefore, we investigated the regulation of miR by ANXA1 in MCF7 breast cancer cells. MCF7-EV (Empty vector) and MCF7-V5 (ANXA1-V5 expressing cells) were subjected to a miR microarray. Microarray analysis revealed a number of miRNAs which were dysregulated in MCF7-V5 cells. 2 novel miRNAs (miR562 and miR26b*) were validated, cloned and functionally characterized. As ANXA1 constitutively activates NF-κB activity to modulate breast cancer metastasis, we found that miR26b* and miR562 directly targeted the canonical NF-κB pathway by targeting the 3′ UTR and inhibiting expression of Rel A (p65) and NF-κB1 (p105) respectively. MiR562 inhibited wound healing, which was reversed when ANXA1 was overexpressed. Overexpression of either miR562 or miR26b* in MCF-7 cells enhanced endothelial tube formation when cocultured with human umbilical cord endothelial cells while conversely, treatment of MCF7 cells with either anti-miR562 or anti-miR26b* inhibited endothelial tube formation after co-culture. Further analysis of miR562 revealed that miR562-transfected cell conditioned media enhances endothelial cell tube formation, indicating that miR562 increased angiogenic secreted factors from MCF-7 breast tumor cells. TNFα was increased upon overexpression of miR562, which was reversed when ANXA1 was co-transfected In conclusion, this data suggests that ANXA1-regulated miR26b* and miR562 may play a role in wound healing and tumor-induced endothelial cell tube formation by targeting NF-κB expression and point towards a potential therapeutic target for breast cancer.

NF-κB Subunit c-Rel Cooperates with Myc and Mutated p53 to Confer Significantly Worse Survival in Patients with Diffuse Large B-Cell Lymphoma: A Report from the International DLBCL Rituximab-CHOP Consortium Program

Objective:To evaluate the prognostic significance of RELamplification, expression and c-Rel activation in DLBCL patients and to identify potential mechanisms for impact of c-Rel activation on patient survival.Patients and Design: The study cohort consists 460 de novo DLBCL patients (median follow-up, 46.8 moths) treated with R-CHOP. We assessed the nuclear expression/activation of c-Rel and other NF-κB subunits by immunohistochemistry, REL gene amplification by fluorescence in situ hybridization, and gene expression profiling using Affymetrix GeneChips array. Correlations between expression of nuclear c-Rel, REL mRNA, and expression of TP53, MDM2, MDM4, MYC, BCL2, AKT1, NFKB1, RELA, NFKB2, and RELB, both at the mRNA and protein levels were analyzed using t-tests. The prognostic significance of c-Rel activation, REL mRNA expression, and REL amplification was evaluated in the overall cohort, and different subgroups stratified by COO, status of TP53 mutation (wide type/WT, or mutated/MUT) and expression, Myc, Bcl-2 overexpression, and nuclear expression of other NF-κB subunits.Results:Nuclear c-Rel expression was observed in 29.6% of DLBCL patients and did not correlate with REL mRNA levels (P=0.95) and COO (P=0.77). In contrast, REL mRNA was significantly higher in germinal center B-like (GCB) subtype (P<0.0001). In GCB-DLBCL, nuclear c-Rel expression was associated with significantly lower Myc, p53, MDM4, and pAKT protein levels but not at the transcriptional level. In contrast, in ABC-DLBCL, c-Rel activation was associated with significantly higher MUT-TP53mRNA level and reduced pAKT expression.Correspondingly to the lack of associations with reduced Myc, pAKT, and p53 in ABC-DLBCL, nuclear c-Rel expression showed prognostic impact only in ABC- but not in GCB- DLBCL, especially when it was concurrent with Myc overexpression (P<0.0001 for OS and P=0.0012 for PFS). Importantly, patients with c-Rel activation and p53 mutations had significantly worse survival (for OS, hazard ratio, 3.56; P=0.0011; median survival, 16.2 vs 87.3 months. For PFS, hazard ratio, 3.976; P=0.0004; median survival, 10.4 vs 55.5 months) compared with other MUT-p53 DLBCL patients. The additive impact of c-Rel activation to TP53 mutations was more apparent in the ABC-DLBCL subtype, in which c-Rel activation was associated with significantly upregulated MUT-TP53 transcription (P=0.0087). Conversely, MUT-p53 expression was associated significantly with upregulated REL mRNA expression (P=0.0021), predominantly in the ABC-DLBCL subtype (P=0.0047). Only in ABC-DLBCL patients, TP53 mutations were associated with elevated nuclear c-Rel levels with a borderline P value (P=0.05).In addition to the association of nuclear p65 in GCB-DLBCL (P=0.003), and p50, NFKB1 and RELA mRNA in ABC-DLBCL (P=0.0023), the prognostic significance of c-Rel appears to depend on p50 (P=0.08) and p65 expression (P=0.12). Also, supporting that c-Rel transactivates anti-apoptotic BCL2L1/BCLXL by previous studies, there was no significant difference in survival of ABC-DLBCL patients with isolated BCL2 overexpression and with nuclear c-Rel expression. Comprehensive gene-expression profiling analysis and cell line study are undergoing in order to identify pathways to activate c-Rel and oncogenic mechanisms for c-Rel–mediated chemoresistance.REL amplification was predominantly observed in GCB-DLBCL (frequency, 7%) and correlated with significantly higher mRNA level (P<0.0001). However, REL amplification did not correlation with nuclear c-Rel expression or patient survival, illustrating the importance of posttranslational regulations in c-Rel activation and function. Consistent with the adverse impact of c-Rel activation at the protein level, a strong trend toward poor survival was observed for elevated RELmRNA in ABC- but not in GCB-DLBCL.Conclusions: Nuclear c-Rel activation was associated with reduced level of proteins whose degradation involves with ubiquitin-proteasome in GCB-DLBCL, and upregulated TP53 transcription in ABC-DLBCL. Reciprocal regulation of c-Rel and MUT-p53 at the transcriptional level may underlie the synergetic adverse effect of c-Rel activation and TP53 mutations. c-Rel cooperated with Myc and conferred significantly worse survival in ABC-DLBCL patients. These subsets of c-Rel+ DLBCL patients likely will benefit from c-Rel targeted therapies. DisclosuresNo relevant conflicts of interest to declare.

HPV16 E2 enhances the expression of NF-κB and STAT3 target genes and potentiates NF-κB activation by inflammatory mediators

HPV-transformed cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but very little is known about their regulation under inflammatory conditions before HPV integration. This study reports that cervical tissues with stromal inflammation and intact HPV16 E2 gene show increased expression of target genes of NF-κB and/or STAT3 which can regulate cell survival (cyclin D1, c-Myc, survivin and Bcl2) and inflammatory responses (TNF-α, IL-1β, IL-6, IL-8 and CCR2). Increased expression of RelA, p-IκBα, STAT3, p-STAT3 (Ser727), Pin1 (peptidyl-prolyl cis/trans isomerase) and MCM2 in the squamous epithelia of cervices with stromal inflammation supports early activation of NF-κB-STAT3. Furthermore, HPV16 E2 potentiated NF-κB activation induced by inflammatory mediators, IL-1β and SDF-1α, in HEK293 cells. These results reveal a novel role for E2 in regulating the activities of NF-κB and STAT3 that may have implications in carcinogenic progression of HPV16-infected cells under conditions of stromal inflammation.

Tinospora cordifolia Induces Differentiation and Senescence Pathways in Neuroblastoma Cells.

Children diagnosed with neuroblastomas often suffer from severe side as well as late effects of conventional treatments like chemotherapy and radiotherapy. Recent advances in understanding of molecular pathways involved in cellular differentiation and apoptosis have helped in the development of new therapeutic approach based on differentiation-based therapy of malignant tumours. Natural medicines with their holistic therapeutic approach are known to selectively eliminate cancer cells thus provide a better substitute for the conventional treatment modes. The current study was aimed to investigate the anti-cancer potential of aqueous ethanolic extract of Tinospora cordifolia (TCE) using IMR-32 human neuroblastoma cell line as a model system. TCE is highly recommended in Ayurveda for its general body and metal health-promoting properties. TCE treatment was seen to arrest the majority of cells in G0/G1 phase and modulated the expression of DNA clamp sliding protein (PCNA) and cyclin D1. Further, TCE-treated cells showed differentiation as revealed by their morphology and the expression of neuronal cell specific differentiation markers NF200, MAP-2 and NeuN in neuroblastoma cells. The differentiated phenotype was associated with induction of senescence and pro-apoptosis pathways by enhancing expression of senescence marker mortalin and Rel A subunit of nuclear factor kappa beta (NFkB) along with decreased expression of anti-apoptotic marker, Bcl-xl. TCE exhibited anti-metastatic activity and significantly reduced cell migration in the scratched area along with downregulation of neural cell adhesion molecule (NCAM) polysialylation and secretion of matrix metalloproteinases (MMPs). Our data suggest that crude extract or active phytochemicals from this plant may be a potential candidate for differentiation-based therapy of malignant neuroblastoma cells.

Abstract 134: HNF4α inhibits liver cancer metastasis via suppression of NF-кB activity

Abstract BACKGROUND &amp; AIMS: Hepatocyte nuclear factor 4α (HNF4α) is a liver enriched transcription factor and is indispensable for liver development. However, the role of HNF4α in hepatocellular carcinoma (HCC) metastasis remains largely unknown. METHODS: 429 HCC tissues were involved in the correlation analysis of HNF4α levels with tumor features and patient outcomes. Effect of HNF4α on HCC metastasis was monitored through in vivo imaging system. Expression of HNF4α, RelA(p65) or microRNAs in human HCCs was determined by Real-time PCR, western blot or immunohistochemistry. Point mutation and ChIP assay were performed to detect the interaction of HNF4α with the promoter of target microRNA. 3’UTR reporter assay was conducted to determine the regulation of RelA by microRNAs. RESULTS: Reduced HNF4α expression correlated well with the aggressive clinicopathological characteristics of HCC and predicted poor prognosis of patients. HNF4α levels were even lower in metastatic HCCs, and ectopic HNF4α expression suppressed the metastasis of hepatoma cells both in vitro and in vivo. HNF4α suppressed HCC metastasis through inhibiting RelA expression and NFκB activity. miR-7 and miR-124, which were transcriptionally up-regulated by HNF4α, repressed RelA expression via interaction with RelA-3’UTR. Herein, HNF4α suppresses NF-κB activity and RelA expression via a miR-7 and miR-124-dependent manner. Moreover, combination of HNF4α and NF-κB exhibited more powerful predictive efficiency of patient prognosis. CONCLUSIONS: Considering the importance of HNF4α and NF-κB in chronic liver disease, the suppression of NF-κB by HNF4α is not only essential in connecting hepatic inflammation and tumorigenesis, but also indicates novel strategy in HCC prevention and therapy. Citation Format: Bei-Fang Ning, Jiao Liu, Wen-Ping Xu, Chuan Yin, Xin Zhang, Wei-Fen Xie. HNF4α inhibits liver cancer metastasis via suppression of NF-кB activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 134. doi:10.1158/1538-7445.AM2014-134

Abstract 2463: Suppression of hepatocyte nuclear factor 4α by nuclear factor κB in hepatoma cells

Abstract BACKGROUND &amp; AIMS: HCC predominantly arises from cirrhotic liver where there usually has been repeated wound-healing response upon chronic inflammation. However, the mechanistic link between hepatic inflammation and carcinogenesis remains largely unknown. METHODS: Expression of HNF4α, RelA(p65) or microRNAs in human HCCs was determined by Real-time PCR, western blot or immunohistochemistry. 3′UTR reporter assay was conducted to determine the regulation of HNF4α by microRNAs. The recombinant adenoviruses carrying HNF4α gene were injected into diethylinitrosamine(DEN)-administrated Wistar rats through tail vein. Tissue microarray was utilized to assess the expression of HNF4α and RelA HCC tissues. RESULTS: Overexpression or interference of RelA remarkably decreased or increased HNF4α expression in hepatoma cells respectively, suggesting a negative regulation of HNF4α by NF-κB. Interestingly, ecotopic RelA expression dramatically reduced the activation of HNF4α-3′UTR indicating microRNA could be involved in the feedback regulation. MiR-21 was predicted to be regulated by NF-κB using Targetscan analysis, and overexpression of RelA evidently enhanced the miR-21 expression. Delivery of miR-21 mimic notably suppressed HNF4α-3′UTR activity and HNF4α expression. Moreover, treatment of miR-21 inhibitor increased HNF4α levels and attenuated the down-regulation of HNF4α by RelA. Consistently, downregulation of HNF4α and upregulation of RelA was detected during DEN-induced rat hepatocarcingenesis, negative correlation of HNF4α and RelA was also observed in human HCCs. CONCLUSIONS: These findings not only broaden the knowledge on hepatic inflammation and cancer initiation, but also indicates novel strategy in HCC prevention. Note: This abstract was not presented at the meeting. Citation Format: Bei-Fang Ning, Xin Zhang, Wei-Fen Xie. Suppression of hepatocyte nuclear factor 4α by nuclear factor κB in hepatoma cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2463. doi:10.1158/1538-7445.AM2014-2463

Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer.

BackgroundAbout 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients.MethodsCTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn’s post-test was used for comparisons between groups. Statistical significance was set at p < 0.05.ResultsEpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis.ConclusionsOur results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-223) contains supplementary material, which is available to authorized users.

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway.

MicroRNA-7 protects against 1-methyl-4-phenylpyridinium-induced cell death by targeting RelA.

Parkinson's disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Mitochondrial complex I impairment in PD is modeled in vitro by the susceptibility of dopaminergic neurons to the complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+). In the present study, we demonstrate that microRNA-7 (miR-7), which is expressed in tyrosine hydroxylase-positive nigral neurons in mice and humans, protects cells from MPP+-induced toxicity in dopaminergic SH-SY5Y cells, differentiated human neural progenitor ReNcell VM cells, and primary mouse neurons. RelA, a component of nuclear factor-κB (NF-κB), was identified to be downregulated by miR-7 using quantitative proteomic analysis. Through a series of validation experiments, it was confirmed that RelA mRNA is a target of miR-7 and is required for cell death following MPP+ exposure. Further, RelA mediates MPP+-induced suppression of NF-κB activity, which is essential for MPP+-induced cell death. Accordingly, the protective effect of miR-7 is exerted through relieving NF-κB suppression by reducing RelA expression. These findings provide a novel mechanism by which NF-κB suppression, rather than activation, underlies the cell death mechanism following MPP+ toxicity, have implications for the pathogenesis of PD, and suggest miR-7 as a therapeutic target for this disease.

123. Response and habituation of pro- and anti-inflammatory gene expression to repeated psychosocial stress

Rationale: Acute stress is accompanied by an inflammatory response and some individuals show sensitization to repeated stress. Increased peripheral inflammation is a risk factor for many age-related illnesses, highlighting the need to understand factors controlling inflammation. No studies have examined changes in pro- and anti-inflammatory gene expression in response to repeated acute stress in humans. Methods: We recruited n = 31 healthy individuals and examined gene expression of IL-6, IL-1beta, RelA and IkappaB before, 30 and 120 min after exposure to the Trier Social Stress Test (TSST) on two days. We assessed the inflammatory response by measuring plasma IL-6, and cortisol as the major anti-inflammatory stress hormone. Results: Stress exposure induced inflammatory and cortisol responses (IL-6: F = 49.3, p F = 13.7, p ∗ time interaction F = 4.2, p = 0.011) but IL-6 responses did not (n.s.). IL-6 and IL-1beta gene expression increased on Day 1, but not on Day 2 (day ∗ time interaction: IL-6: F = 106, p = 0.001; IL-1beta: F = 7.9, p = 0.006). RelA expression increased on both days (time effect F = 5.0, p = 0.013). IkB expression increased then decreased below baseline on Day 1, and decreased post-TSST on Day 2 (day ∗ time interaction: F (2,54) = 5.4, p = 0.08). There was no relationship between IL-1Beta, IkB, and RelA with plasma IL-6 or cortisol, or between cortisol and plasma IL-6. Conclusions: Taken together, results show a response pattern of pro- and anti-inflammatory gene expression characterized by differential stress responses and habituation patterns that are in disjunction with patterns of cortisol and plasma IL-6 responses to repeated stress. This indicates that increases in plasma concentrations of inflammatory cytokines are unlikely to originate solely from de novo transcription within PBMCs, but that alternative or additional sources of plasma inflammatory cytokines need to be explored.

Hepatocyte nuclear factor 4α-nuclear factor-κB feedback circuit modulates liver cancer progression.

Hepatocyte nuclear factor 4α (HNF4α) is a liver enriched transcription factor and is indispensable for liver development. However, the role of HNF4α in hepatocellular carcinoma (HCC) progression remains to be elucidated. We report that reduced HNF4α expression correlated well with the aggressive clinicopathological characteristics of HCC and predicted poor prognosis of patients. HNF4α levels were even lower in metastatic HCCs, and ectopic HNF4α expression suppressed the metastasis of hepatoma cells both in vitro and in vivo. Forced HNF4α expression attenuated the expression and nuclear translocation of RelA (p65) and impaired NF-κB activation through an IKK-independent mechanism. Blockage of RelA robustly attenuated the suppressive effect of HNF4α on hepatoma cell metastasis. MicroRNA (miR)-7 and miR-124 were transcriptionally up-regulated by HNF4α, which repressed RelA expression by way of interaction with RelA-3' untranslated region (UTR). In addition, nuclear factor kappa B (NF-κB) up-regulated the expression of miR-21 in hepatoma cells, resulting in decreased HNF4α levels through down-regulating HNF4α-3'UTR activity. Collectively, an HNF4α-NF-κB feedback circuit including miR-124, miR-7, and miR-21 was identified in HCC, and the combination of HNF4α and NF-κB exhibited more powerful predictive efficiency of patient prognosis. These findings broaden the knowledge of hepatic inflammation and cancer initiation/progression, and also provide novel prognostic biomarkers and therapeutic targets for HCC.

HPV16 E2-mediated potentiation of NF-κB activation induced by TNF-α involves parallel activation of STAT3 with a reduction in E2-induced apoptosis

Human papilloma virus is associated with cervical and other tumors, and several cellular conditions also play an important role in carcinogenesis. Human papilloma virus (HPV)-infected cells exhibit activation of NF-κB and STAT3 (mediators of inflammation), but little is known about their regulation by HPV. This study attempts to understand the role of HPV16 E2, an important early protein of HPV16, in the regulation of NF-κB and STAT3 by reporter assays, quantitative reverse transcriptase-polymerase chain reaction, and immunoblotting. We demonstrate that E2 enhances NF-κB activation induced by TNF-α, a proinflammatory cytokine, in both non-tumor- and tumor-derived epithelial cell lines besides potentiating STAT3 transcriptional activity induced by TNF-α in HEK293 cells. E2 increases the expression of RelA and its transcriptional activation, and retention of E2 was observed in the nucleus with significant interaction with RelA (immunoprecipitation) upon TNF-α treatment. Transfection with shRNA-RelA or pretreatment with a STAT3 inhibitor had a negative effect on the ability of E2 to enhance TNF-α-induced NF-κB activation. Experiments with co-expression of a mutant of STAT3 with E2 also suggested that the activation of STAT3 is indispensible for TNF-α-induced NF-κB activation. Inhibition of STAT3 activation enhanced E2-induced apoptosis, whereas parallel activation of NF-κB and STAT3 by the combined action of E2 and TNF-α increased the expression of their common targets, cyclin-D1, c-Myc, survivin, and Bcl-2, leading to a decrease in E2-induced apoptosis (viability and cell cycle). Our results reveal novel mechanisms by which E2 may regulate NF-κB and STAT3 activation in the presence of TNF-α with implications on the survival of HPV-infected cells.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Forkhead transcription factor FOXO1 inhibits nuclear factor‐κB in gastric cancer

FOXO1, a forkhead box O (FOXO) transcription factor, and nuclear factor-κB (NF-κB) are prognostically significant transcription factors in gastric cancer. As their relationship has been inconsistent depending on the cell type, we aimed to investigate whether FOXO1 is associated with NF-κB p65 (RelA) in gastric cancer. Immunohistochemistry was performed on tissue array slides containing 298 gastric carcinoma specimens. We found that the cytoplasmic expression of pFOXO1, the inactive form of FOXO1, was positively correlated with nuclear RelA expression (p=0.024). In addition, the expressions of pFOXO1 and RelA were positively related with cyclin D1 expression (p=0.014 and p=0.001, respectively) and Ki-67 labeling index (p=0.025 and p=0.017, respectively). However, they did not show association with the expressions of cyclin E, p53 and pRb. Cell culture experiments showed that FOXO1 overexpression by transfection of FOXO1 AAA mutant gene suppressed NF-κB activation in SNU-484 gastric cancer cells. These results suggest that FOXO1 and NF-κB are negatively associated and that FOXO1 is a negative upstream regulator of NF-κB in gastric cancer.