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Background<p>Metabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB) play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS) and osteosarcoma (OS), has not been characterized.</p>Methods<p>Classical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XF^e24 was used to analyze oxygen consumption rate as a measure of aerobic respiration.</p>Results<p>Inhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK) 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells.</p>Conclusion<p>These findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.</p

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Background<p>Metabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB) play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS) and osteosarcoma (OS), has not been characterized.</p>Methods<p>Classical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XF^e24 was used to analyze oxygen consumption rate as a measure of aerobic respiration.</p>Results<p>Inhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK) 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells.</p>Conclusion<p>These findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.</p

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Background<p>Metabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB) play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS) and osteosarcoma (OS), has not been characterized.</p>Methods<p>Classical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XF^e24 was used to analyze oxygen consumption rate as a measure of aerobic respiration.</p>Results<p>Inhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK) 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells.</p>Conclusion<p>These findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.</p

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Background<p>Metabolic reprogramming has emerged as a cancer hallmark, and one of the well-known cancer-associated metabolic alterations is the increase in the rate of glycolysis. Recent reports have shown that both the classical and alternative signaling pathways of nuclear factor κB (NF-κB) play important roles in controlling the metabolic profiles of normal cells and cancer cells. However, how these signaling pathways affect the metabolism of sarcomas, specifically rhabdomyosarcoma (RMS) and osteosarcoma (OS), has not been characterized.</p>Methods<p>Classical NF-κB activity was inhibited through overexpression of the IκBα super repressor of NF-κB in RMS and OS cells. Global gene expression analysis was performed using Affymetrix GeneChip Human Transcriptome Array 2.0, and data were interpreted using gene set enrichment analysis. Seahorse Bioscience XF^e24 was used to analyze oxygen consumption rate as a measure of aerobic respiration.</p>Results<p>Inhibition of classical NF-κB activity in sarcoma cell lines restored alternative signaling as well as an increased oxidative respiratory metabolic phenotype in vitro. In addition, microarray analysis indicated that inhibition of NF-κB in sarcoma cells reduced glycolysis. We showed that a glycolytic gene, hexokinase (HK) 2, is a direct NF-κB transcriptional target. Knockdown of HK2 shifted the metabolic profile in sarcoma cells away from aerobic glycolysis, and re-expression of HK2 rescued the metabolic shift induced by inhibition of NF-κB activity in OS cells.</p>Conclusion<p>These findings suggest that classical signaling of NF-κB plays a crucial role in the metabolic profile of pediatric sarcomas potentially through the regulation of HK2.</p

TNF regulates Notch-1 expression.

<p><b>A.</b> Mononuclear cell cultures were prepared from <i>mdx</i> muscles and cultured under proliferation conditions and either left untreated or treated with TNF. After 24 hrs cells were harvested and processed for total RNA, and subsequently semi-quantitative RT-PCR reactions were performed probing for satellite cell markers. GAPDH was used as a control. <b>B.</b> Quantitative real-time RT-PCR analysis of Notch-1 from satellite cell cultures untreated and treated with TNF. Values shown were normalized to GAPDH levels. Asterisk denotes statistical significance, p = 0.03708. <b>C.</b> Western blot analysis probing for total Notch-1 receptor from duplicate <i>mdx</i> satellite cell cultures treated with TNF. The blot was stripped and re-probed for α-tubulin used as a loading control <b>D.</b> Quantitative real-time RT-PCR was repeated as in (B) from FACS sorted mononuclear cells enriched for a CD34⁺, α7integrin⁺, Sca1⁻ population. <b>E.</b> C2C12 myoblasts were cultured under proliferating conditions and either not treated or treated with TNF. Total RNA was prepared and quantitative RT-PCR analysis was performed probing for Notch-1 and Notch-1 targets, Hes-1 and Hey-1. Asterisk denotes statistical significance; Notch-1, p = 0.04844; Hes-1, p = 0.001; Hey-1, p = 0.0149. <b>F.</b> C2C12 myoblasts were cultured in treated in duplicate with TNF and protein lysates were subsequently probed with the intercellular activated form of Notch-1 (NICD), or with Notch signaling components, Delta and Jagged-1.</p

TNF promotes Dnmt-3b recruitment and DNA methylation on the Notch-1 promoter.

<p><b>A.</b> Schematic illustration of the Notch-1 promoter indicating the CpG islands in the BS1 and BS2 regions proximal to the TSS. <b>B.</b> C2C12 myoblasts were treated with TNF for 48 hrs at which time cells were prepared for quantitative ChIP analysis for Dnmt-1, Dnmt-3a, and Dnmt-3b. <b>C.</b> C2C12 myoblasts were transfected with scrambled siRNA or siRNA against Ezh2 and the following day cells were treated for TNF for 48 hrs and ChIP analysis for Dnmt-3b was subsequently performed. <b>D.</b> C2C12 myoblasts were treated with TNF for up to 7 days and at indicated times, cells were processed for bisulfite sequencing of the BS1 region within the Notch-1 promoter. Note the increase in BS1 methylation at discrete CpG dinucleotides over time with TNF treatment.</p

TNF regulates Notch-1 expression in vivo.

<p><b>A.</b> Muscle homogenates were prepared from 7–8 week old <i>mdx</i> mice that either contained or lacked IKKβ and western analysis was performed probing for Notch-1. The blot was stripped and reprobed for α-tubulin used as a loading control. <b>B.</b> Nude mice were implanted with vector control CHO cells or CHO cells expressing TNF. Once tumors were established, muscle injury was induced to tibialis anterior muscles with cardiotoxin injections. At indicated days post-toxin injections, muscles were harvested and homogenates prepared for western analysis probing for Notch-1 and α-tubulin as a loading control.</p

TNF repression of Notch-1 occurs through the recruitment of Ezh2.

<p><b>A.</b> Time course analysis of Notch-1 expression following TNF treatment of C2C12 myoblasts. <b>B.</b> Schematic illustration of the Notch-1 gene indicating conserved regions of CpG content (blue bars) that are located in positions proximal to the transcriptional start site (TSS), and where probes were designed for ChIP analysis. Also indicated is a CpG island immediately surrounding the TSS (red bar). <b>C, D.</b> C2C12 myoblasts were treated with TNF and at indicated times cells were harvested and subsequently prepared for ChIP analysis probing for Ezh2 by both semi-quantitative (<b>C</b>) and quantitative (<b>D</b>) RT-PCR. <b>E.</b> Using cell extracts prepared in C, additional ChIPs were performed for methylation of H3K27. F. C2C12 myoblasts were transfected with scrambled siRNA (siControl) or siRNA targeting Ezh2 (siEzh2) and following 48 hrs, western blot analyze was performed probing for Ezh2 and α-tubulin used as a loading control. G. C2C12 myoblasts transfections were performed with siControl and siEzh2 oligonucleotides and next day cells were treated for TNF for 48 hrs, and subsequently processed for Notch-1 expression by real-time RT-PCR. Asterisks denotes p = 0.00314).</p