Linking oncogenic signaling to transcriptional changes by epigenomic data in a cell line model of human glioblastoma
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE36900 | GEO | 2013/01/01
SECONDARY ACCESSION(S): PRJNA157207
REPOSITORIES: GEO
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Project description:We present a computational method for building a regulatory network from global phosphoproteomic and transcription profiling data. To recover the critical missing links between signaling events and transcriptional responses, we relate changes in chromatin accessibility to changes in expression and then uses these links to connect proteomic and transcriptome data. We applied our approach to integrate epigenomic, phosphoproteomic and transcriptome changes induced by the variant III mutation of the epidermal growth factor receptor (EGFRvIII) in a cell line model of glioblastoma multiforme (GBM). Genome-wide DNase I hypersensitivity followed by sequencing (DNase-Seq) to measure chromatin accessibility in a cell line derived from the U87MG glioblastoma cell line to express high level of EGFRvIII (U87H; 2 million copies of EGFRvIII per cell) and a control cell line expressing kinase dead EGFRvIII (U87DK; 2 million kinase dead EGFRvIII per cell). A prediction from the computational method, the transcriptional co-regulator p300, was experimentally validated by chromatin immunoprecipitation followed by sequencing (ChIP-Seq).
2013-01-01 | E-GEOD-36900 | biostudies-arrayexpress
Project description:EGFRvIII is the most common deletion mutant of EGFR in human cancer and its levels are highly correlated with poor prognosis in GBM. The deletion of exons 2-7 removes most of the extracellular ligand binding domain, so it is unable to bind EGF or other EGFR-binding ligands. Nevertheless, the mutant receptor is constitutively phosphorylated, and is capable of activating downstream signaling pathways at a low level. To comprehensively identify the downstream signaling consequences of the EGFRvIII, we incorporated phosphoproteomic, transcription profiling and DNase-Seq data from U87MG glioblastoma cells expressing titrated levels of this mutant receptor. Total RNA were extracted from U87MG cells engineered to expressed different levels of EGFRvIII: medium (U87M; 1.5 million copies of EGFRvIII receptor per cell), high (U87H; 2 million copies per cell), super-high (U87SH; 2.5 million copies per cell), and kinase-dead EGFRvIII (U87DK; 2 million copies of kinase dead EGFRvIII per cell). RNA was hybridized to Affymetrix microarrays.
2013-01-01 | E-GEOD-36901 | biostudies-arrayexpress
Project description:Analysis of human glioblastoma multiforme tumors revealed genes that are upregulated in tumors expressing EGFRvIII compared to those expressing wild-type EGFR We examined gene expression in 52 primary GBM tumors, which were stratified based on the presence or absence of EGFRvIII expression using EGFRvIII-specific RT-PCR (Tykocinski et al., 2012). The extracted RNA samples from these tumors were subjected to microarray analysis using HG-U133 2.0 Plus Affymetrix gene chips. Average expression of all transcripts were compared between EGFRvIII-positive and -negative GBMs.
2015-09-08 | E-GEOD-51062 | biostudies-arrayexpress
Project description:Analysis of human glioblastoma multiforme tumors revealed genes that are upregulated in tumors expressing EGFRvIII compared to those expressing wild-type EGFR
2015-09-08 | GSE51062 | GEO
Project description:EGFRvIII is the most common deletion mutant of EGFR in human cancer and its levels are highly correlated with poor prognosis in GBM. The deletion of exons 2-7 removes most of the extracellular ligand binding domain, so it is unable to bind EGF or other EGFR-binding ligands. Nevertheless, the mutant receptor is constitutively phosphorylated, and is capable of activating downstream signaling pathways at a low level. To comprehensively identify the downstream signaling consequences of the EGFRvIII, we incorporated phosphoproteomic, transcription profiling and DNase-Seq data from U87MG glioblastoma cells expressing titrated levels of this mutant receptor.
2013-01-01 | GSE36901 | GEO
Project description:Glioblastoma multiforme (GBM) is a highly aggressive and heterogeneous form of primary brain tumors, driven by a complex repertoire of oncogenic alterations, including the constitutively active epidermal growth factor receptor (EGFRvIII). EGFRvIII impacts both cell-intrinsic and non-cell autonomous aspects of GBM progression, including cell invasion, angiogenesis and modulation of the tumor microenvoiront, this is, at least in part, attributable to the release and intercellular trafficking of extracellular vesicles (EVs), heterogeneous membrane structures containing multiple bioactive macromolecules. Here we analyzed the impact of EGFRvIII on the profile of glioma EVs using a series of isogenic tumor cell lines, in which this oncogene exhibits a strong transforming activity. Thus, we observed that EGFRvIII expression alters several properties of glioma EVs, including their output and global protein composition. Using mass spectrometry, quantitative proteomic analysis and Gene Ontology terms filters, we observed that EVs released by EGFRVIII-transformed cells were enriched for extracellular exosome and focal adhesion related proteins. Among them, we validated the association of pro-invasive proteins (CD44, BSG, CD151) with EVs of EGFRvIII expressing cells, and down-regulation of exosomal markers (CD81 and CD82) in EVs of their indolent counterparts. Nano-flow cytometry revealed that the EV output from individual glioma cell lines was highly heterogeneous, such that only a fraction of EVs contained specific proteins such as EGFR, CD9, or others. Notably, cells expressing EGFRvIII released ample EVs double positive for CD44/BSG, and these proteins also co-localized in cellular filopodia. We also detected the expression of homophilic adhesion molecules and increased homologous EV uptake by EGFRvIII-positive glioma cells. These results suggest that oncogenic EGFRvIII reprograms the proteome of GBM-related EVs, a notion with considerable implications for their biological activity and properties relevant for development of EV-based cancer biomarkers.
2018-07-30 | PXD008311 | Pride
Project description:Next generation sequencing platforms were used to identify STAT3 targets in the background of EGFRvIII expresssion mRNA of 3 EGFRvIII positive brain tumor stem cell lines (BTSC #68, 73, and 90) were compared to an EGFRvIII negative line (#41) to identify EGFRvIII-regulated targets in human BTSC. EGFRvIII-dependent targets in mouse astrocytes were identified by mRNA-seq analyses of EGFRvIII- or MSCV-expressing astrocytes. STAT3-differentially regulated genes in EGFRvIII expressing mouse astrocytes were obtained by subjecting EGFRvIII,STAT3f/f and EGFRvIII, STAT3-/- astrocytes to mRNA-Seq analyses. Sites of STAT3 occupancy in EGFRvIII expressing astrocytes were identified by ChIP-Seq using a STAT3 antibody or IgG control. We identified OSMR as a direct target of STAT3 in both EGFRvIII-expressing human BTSC and mouse astrocytes. Therefore to identify OSMR-regulated genes, we used a lentiviral mediated RNAi system to knockdown OSMR in EGFRvIII-expressing astrocytes. OSMR-dependent differentially expressed genes were obtained by comparison of OSMR knockdown (KD1 and KD2) astrocytes to control groups (ShRNA control and Vector control)
2016-04-13 | E-GEOD-51281 | biostudies-arrayexpress
Project description:Cancer patients often experience changes in mental health, prompting an exploration into whether nerves infiltrating tumors contribute to these alterations by impacting brain functions. Using a mouse model for head and neck cancer and neuronal tracing we show that tumor-infiltrating nerves connect to distinct brain areas. The activation of this neuronal circuitry altered behaviors (decreased nest-building, increased latency to eat a cookie, and reduced wheel running). Tumor-infiltrating nociceptor neurons exhibited heightened calcium activity and brain regions receiving these neural projections showed elevated Fos as well as increased calcium responses compared to non-tumor-bearing counterparts.
2024-08-30 | GSE275481 | GEO
Project description:Background: EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2-7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation. Methods: We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis,quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis. Results: In the course of screening 30,000 substances, a reagent, “Ertredin,” was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis. Conclusion: We developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells.
2016-08-01 | GSE76959 | GEO