Loss of CSL unlocks a hypoxic response and enhanced tumor growth potential in breast cancer cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Urban Lendahl
PROVIDER: E-GEOD-77308 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Notch signaling is frequently hyperactivated in breast cancer, but how the enhanced signaling contributes to the tumor process is less well understood. In this report, we identify the proinflammatory cytokine interleukin-6 (IL-6) as a novel Notch target in breast tumor cells. Enhanced Notch signaling upregulated IL-6 expression at the transcriptional level, leading to activation of autocrine and paracrine JAK/STAT signaling. IL-6 upregulation was mediated by non-canonical Notch signaling, as it could be effectuated by a cytoplasmically localized Notch intracellular domain and was independent on the DNA-binding protein CSL. Instead, Notch-mediated IL-6 upregulation was controlled by two other factors: IKKβ, a protein in the NF-kB signaling cascade, and p53. Activation of IL-6 by Notch required IKKβ function, but interestingly, did not engage canonical NF-κB signaling, in contrast to IL-6 activation by inflammatory agents such as tumor necrosis factor, which requires canonical NF-κB signaling. With regard to p53 status, IL-6 expression was upregulated by Notch when p53 was mutated or lost, but restoring wildtype 53 into p53-mutated or -deficient cells abrogated the IL-6 upregulation. Furthermore, Notch-induced genome-wide transcriptomes from p53 wildtype and -mutated breast tumor cell lines differed extensively, and in a subset of genes upregulated by Notch in a p53-mutant cell line, upregulation was reduced by wildtype p53. In conclusion, we identify IL-6 as a novel non-canonical Notch target gene, and reveal roles for p53 and IKKβ in non-canonical Notch signaling in breast cancer and in the generation of cell context-dependent diversity in the Notch signaling output. 30 microarray samples consisting of MCF7 (ER+, wild-type p53, luminal type B breast cancer) and MDA-MB-231 (ER-, mutated p53, basal breast cancer) cells cultured on immobilized 1 μg/ml JAGGED1-Fc or 1 μg/ml DLL4-Fc or 1 μg/ml Fc control with or without 5 μM DAPT for 6 hours in 3 biological replicates.
2012-06-29 | E-GEOD-36051 | biostudies-arrayexpress
Project description:Notch signaling is an important regulator of stem cell differentiation. All canonical Notch signaling is transmitted through the DNA-binding protein CSL and hyperactivated Notch signaling is associated with tumor development; thus it may be anticipated that CSL deficiency should reduce tumor growth. In contrast, we report that genetic removal of CSL in breast tumor cells caused accelerated growth of xenografted tumors. Loss of CSL unleashed a hypoxic response during normoxic conditions, manifested by stabilization of the HIF1± protein and acquisition of a polyploid giant-cell, cancer stem cell-like, phenotype. At the transcriptome level, loss of CSL upregulated more than 1750 genes and less than 3% of those genes were part of the Notch transcriptional signature. Collectively, this suggests that CSL exerts functions beyond serving as the central node in the Notch signaling cascade and reveals a novel role for CSL in tumorigenesis and regulation of the cellular hypoxic response.
2016-02-28 | GSE77308 | GEO
Project description:The Notch signaling pathway functions in a number of processes during embryologic development, especially the maintenance or aquisition of cell fate. We purturb the Notch signalling pathway in embryonic Xenopus laevis in order to 1) better characterize the downstream targets of Notch signalling, and 2) determine the extent to which early embryos can recover from transient purturbations to critical signalling pathways, if at all. Xenopus laevis embryos were unilaterally injected at the two cell stage with either GFP, GFP and ICD (Notch intracellular domain, an up-regulator of the Notch pathway), or GFP and DBM (domain-binding mutant, a downregulator of the Notch pathway). At stages 18, 28, and 38, for each injection, pooled total RNA from 10 embryos was extracted. Extraction was performed for three biological replicates for each time/injection condition. cDNA from total RNA was hybridized on Affymetrix Xenopus laevis Genome 2.0 arrays.
2014-08-06 | E-GEOD-60127 | biostudies-arrayexpress
Project description:This experiment aims at investigating the effect of AT2 overexpression and/or activation in breast cancer. Human breast cancer cells MDA-MB-231 were transfected with lentiviral construct containing flagged human AT2 receptor sequence (or empty vector) and were subcutaneously injected into immuno-deficient mice (2. 10e6 cells). When the tumors reached 5 mm3 volume, mice were treated daily with compound C21 (0.3 mg/kg) or vehicle for 43 days. Total RNA was extracted from tumors and submitted to DNA array analysis.
2020-11-07 | E-MTAB-8532 | biostudies-arrayexpress
Project description:The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target-selection and gene activation in each context. To investigate, we partitioned Drosophila chromatin into different states, based on histone modifications, establishing the preferred chromatin conditions for binding of CSL, the Notch pathway transcription factor. While most histone modifications were unchanged by CSL binding or Notch activation, rapid changes in H3K56 acetylation occurred at Notch regulated-enhancers. This modification required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation are a conserved indicator of enhancer activation, also occurring at mammalian Notch-regulated Hey1 and at Drosophila ecdysone-regulated genes. This core histone modification may therefore underpin the changes in chromatin accessibility needed to promote transcription following signaling activation. H3K56ac profile of control cells (KP) and of NICD overexpression cells (KN). In total 6 samples, with 2 input files (from 2 different conditions) and 2 replicates of H3K56ac ChIP samples of each condition.
2015-07-01 | E-GEOD-66221 | biostudies-arrayexpress
Project description:Gene expression profiling on IL-10-secreting and non-secreting murine Th1 cells, stimulated in the presence or absence of the Notch ligand Delta-like 4 (Dll4), was performed to identify transcription factors co-expressed with IL-10. Primary naM-CM-/ve T helper cells were isolated from lymph nodes and spleens of C57BL/6 wildtype mice. Cells were enriched using the Multisort Kit from Miltenyi Biotec for CD25-CD4+CD62L+, and afterwards cultured under Th1 polarizing conditions. For activation, 0.25E06 naM-CM-/ve T cells were co-cultured with 0.75E06 MACSi Beads in 96-well flat bottom plates. MACSi Beads were coated with anti-CD3 and anti-CD28 (30 M-BM-5g of total primary IgG antibody per 1.0E08 beads) prior to seeding. Notch activation via Dll4 was induced by additional co-culture with MACSi Beads covalently coated with recombinant mouse Dll4. After 5 days in culture, the cells were restimulated with PMA/Ionomycin and subjected to an IL-10-secretion assay (Miltenyi Biotec) to separate IL-10-secreting and non-secreting cells. Using a BD Aria or DIVA cell sorter (Becton Dickinson), living CD4+ IL-10-secreting and non-secreting cells, with (co-culture with Dll4; 'TH1Notch') or without ('TH1Control') activation of the Notch signaling pathway, were isolated. Total RNA was extracted using the RNeasy Mini kit (Qiagen). The integrity and amount of isolated RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The preparation for the hybridization to the chip was done using the GeneChip 3' IVT Express Kit. Fifteen micrograms of fragmented cRNA of each sample were hybridized to a total of 4 mouse genome 430 2.0 GeneChips (Affymetrix). Hybridization was performed in a Hybridization Oven 640, and chips were washed and stained in the Fluidics Station 400 (both Affymetrix). Finally, the arrays were scanned with a GeneChip Scanner 3000 using the GCOS software, version 1.1.1., both Affymetrix. The data was analyzed using the original GCOS CHP-file Signals, Excel and AmiGO website.
2014-05-08 | E-GEOD-57417 | biostudies-arrayexpress
Project description:Aortic valve calcification is the most common form of valvular heart disease, but the mechanisms of calcific aortic valve disease (CAVD) are unknown. NOTCH1 mutations are associated with aortic valve malformations and adult-onset calcification in families with inherited disease. The Notch signaling pathway is critical for multiple cell differentiation processes, but its role in the development of CAVD is not well understood. The aim of this study was to investigate the molecular changes that occur with inhibition of Notch signaling in the aortic valve. Notch signaling pathway members are expressed in adult aortic valve cusps, and examination of diseased human aortic valves revealed decreased expression of NOTCH1 in areas of calcium deposition. To identify downstream mediators of Notch1, we examined gene expression changes that occur with chemical inhibition of Notch signaling in rat aortic valve interstitial cells (AVICs). We found significant downregulation of Sox9 along with several cartilage-specific genes that were direct targets of the transcription factor, Sox9. Loss of expression Sox9 has been published to be associated with aortic valve calcification. Utilizing an in vitro porcine aortic valve calcification model system, inhibition of Notch activity resulted in accelerated calcification while stimulation of Notch signaling attenuated the calcific process. Finally, the addition of Sox9 was able to prevent the calcification of porcine AVICs that occurs with Notch inhibition. In conclusion, loss of Notch signaling contributes to aortic valve calcification via a Sox9-dependent mechanism. 3 samples of aortic valve interstitial cells treated with DAPT were compared with 3 samples of aortic valve interstitial cells treated with DMSO
2011-08-25 | E-GEOD-31668 | biostudies-arrayexpress
Project description:Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. Here we have analyzed the temporal changes in transcription following a short Notch activation treatment and have related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were cultured in Schneiders medium (Invitrogen) supplemented with 10% FBS (Sigma), 5ug/ml insulin (Sigma) and 5% penicillin / streptomycin (Sigma) according to standard protocols. Notch signalling was initiated by replacing cell media with 2mM EDTA in PBS. The cells were stimulated for 5 minutes before washing out EDTA using normal culture media. The cells were then collected at 18 time points at 0M-bM-^@M-^Y, 5M-bM-^@M-^Y, 10M-bM-^@M-^Y, 15M-bM-^@M-^Y, 20M-bM-^@M-^Y, 25M-bM-^@M-^Y, 30M-bM-^@M-^Y, 35M-bM-^@M-^Y, 40M-bM-^@M-^Y, 50M-bM-^@M-^Y, 60M-bM-^@M-^Y, 70M-bM-^@M-^Y, 80M-bM-^@M-^Y, 90M-bM-^@M-^Y, 100M-bM-^@M-^Y, 110M-bM-^@M-^Y, 120M-bM-^@M-^Y and 150M-bM-^@M-^Y after stimulation in 4 independent time trials. For control samples, for each time trial, media was replaced with fresh media to mimic the addition and removal of EDTA in corresponding experimental samples and then collected at equivalent times. For each trial 50 M-BM-5g of the untreated control RNA sample of each time point was pooled to create a trial specific reference sample. Samples from trials #1 and #3 were labelled with Cy5 and trails #2 and #4 as dye-swap with Cy3.
2012-12-31 | E-GEOD-35557 | biostudies-arrayexpress
Project description:Cellular responses to signalling pathways are often highly dynamic, however most analyses of developmental signalling pathways focus on a single endpoint. We have analyzed the temporal changes in transcription following a short Notch activation treatment and related these to the recruitment of the Notch pathway transcription factor, CSL [Suppressor of Hairless, Su(H), in Drosophila], and to the state of RNA Polymerase II (Pol II) binding. A total of 154 genes showed significant differential expression over time and their expression profiles stratified into 14 clusters based on temporal and quantitative differences in their responses. These differences were partially reflected in the profiles of Pol II and Su(H) binding. However, neither could fully account for the different response profiles. Furthermore, the timing of the different responses was unaffected by more prolonged Notch activation. Instead our data suggest that regulatory relationships between genes that segregate into different response clusters can partially account for the stratification. Thus, feed-forward repression, where products of early responding Enhancer of split bHLH genes (E(spl)bHLH) inhibit expression of endogenous repressors, is one mechanism that explains the profile of genes that exhibit delayed up-regulation. E(spl)bHLH genes may therefore be responsible for co-ordinating the Notch response of a wide spectrum of other targets, explaining their critical functions in many developmental and disease contexts. DmD8 cells were collected at 7 time points (0M-bM-^@M-^Y, 10M-bM-^@M-^Y, 20M-bM-^@M-^Y, 30M-bM-^@M-^Y, 40M-bM-^@M-^Y, 60M-bM-^@M-^Y and 100M-bM-^@M-^Y) after a 5 minute Notch stimulation as 3 independent replicates. Immunoprecipitation was performed with Pol II (Ser 2 and Ser 5 phosphorylated) antibody and compared to the total input DNA. Samples from replicates #1 and #3 were labelled with Cy5, and replicate #2 was labelled with Cy3 as a dye-swap.
2012-12-31 | E-GEOD-35715 | biostudies-arrayexpress
Project description:Notch signaling regulates a variety of developmental cell fates decisions in a cell-context dependent manner. Although Notch signaling directly regulates transcription via the RBP-J/CSL DNA binding protein, little is known about the genes in the respective tissues that are directly activated by Notch. To analyze how Notch signaling mediates its context dependent functions, we utilized a Tamoxifen(OHT)-inducible system (NERT) to activate Notch1 in embryonic stem cells (ESC) at different stages of mesodermal differentiation combined with global transcriptional analyses. Notch1 signaling pathway was analysed in mouse embryonic stem cells (mESCs) that were kept under self-renewal conditions enabling ectodermal, or mesodermal gene expression (ESCe or ESCm respectively), or were differentiated into mesodermal progenitors. NotchIC were induced or not induced by hydroxytamoxifen (OHT) and addititionally in some experiments the cells were treated with protein synthesis inhibitor cycloheximide (CHX) for 4 h.
2010-07-13 | E-GEOD-15268 | biostudies-arrayexpress