Human non-GCIMP gioblastoma subtypes evolve from a common proneural-like precursor glioma
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PROVIDER: PRJNA196865 | ENA |
REPOSITORIES: ENA
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Project description:In order to understand the relationships between the human non-GCIMP glioblastoma subgroups, we performed computational analysis of human genomic data to predict the temporal sequence in which the driver events arise during tumorigenesis. The order of evolutionary events for non-GCIMP GBM is 1) chr 7 gain and loss of chr 10, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes such as NF1 loss or focal amplification of PDGFRα or EGFR. We then developed a computational methodology to identify the drivers of broad copy number changes, identifying PDGF-A (chr 7) and PTEN (chr 10) as driving the initial non-disjunction events. These predictions were validated using mouse modeling, showing PDGF-A is sufficient to induce PN-like gliomas that are enhanced by loss of Ink4a-Arf, Tp53 or Pten. Additional Nf1 loss converts PN to the MES subtype. Our findings suggest non-GCIMP GBMs arise as, and evolve from, a common proneural-like precursor.
2014-04-03 | GSE45874 | GEO
Project description:In order to understand the relationships between the human non-GCIMP glioblastoma subgroups, we performed computational analysis of human genomic data to predict the temporal sequence in which the driver events arise during tumorigenesis. The order of evolutionary events for non-GCIMP GBM is 1) chr 7 gain and loss of chr 10, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes such as NF1 loss or focal amplification of PDGFRα or EGFR. We then developed a computational methodology to identify the drivers of broad copy number changes, identifying PDGF-A (chr 7) and PTEN (chr 10) as driving the initial non-disjunction events. These predictions were validated using mouse modeling, showing PDGF-A is sufficient to induce PN-like gliomas that are enhanced by loss of Ink4a-Arf, Tp53 or Pten. Additional Nf1 loss converts PN to the MES subtype. Our findings suggest non-GCIMP GBMs arise as, and evolve from, a common proneural-like precursor. hGBM_sphere: Three different human GBM sphere lines expressing two different NF1-shRNAs or empty shRNA-control were generated by a lentiviral infection with the relevant pLKO.1 vectors. The total RNAs were obtained from the GBM sphere lines and the gene expression profile of the NF1-shRNA cells was then compared to the control cells. hGBM_rapamycin: Two human GBM sphere lines (TS543 and TS667) expressing the NF1-shRNAs were generated by a lentiviral infection with two different pLKO.1-NF1-shRNA vectors (two different target sequence; #2 and #5). The total RNAs were obtained from the NF1-shRNA lines treated with a 1nM rapamycin or 0.1% DMSO control for 5 hours and the gene expression profile was then compared between the both groups. Murine_gliomas: Murine brain tumors were generated by an injection of DF1 cells producing the relevant RCAS virus into newborn pups or adult mice brain. When mice presented any symptoms of disease, the mice were sacrificed and the brain tumor tissues were macroscopically dissected. Then total RNAs were extracted from the murine brain tumors or normal brain tissues and the gene expression profile was compared between various tumor types and/or normal brain tissues. In this study, two different RCAS-shNf1 (GR249 and GF249) and shp53 (R696 and mR696) was used for the generation of the murine brain tumors. Each RCAS vector has different structure but the target sequence against the Nf1 and p53 is same and similar knockdown effect was observed. Tumor grade were determined with the residual sections after a piece of tumor tissue were excised for the RNA extraction. The detail information of the experiment was described in the associated-publication. Murine_shere: Murine neurosphere lines were generated by a retroviral infection with the relevant RCAS viruses. The total RNAs were obtained from the murine neurosphere lines expressing the RCAS-shGL2, shNf1, shp53 or shNf1+shp53 and the gene expression profiles of the shNf1, shp53 or shNf1/p53 cells were then compared to the control shGL2 cells.
2014-04-03 | E-GEOD-45874 | biostudies-arrayexpress
Project description:To understand the relationships between the non-GCIMP glioblastoma (GBM) subgroups, we performed mathematical modeling to predict the temporal sequence of driver events during tumorigenesis. The most common order of evolutionary events is 1) chromosome (chr) 7 gain and chr10 loss, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes. We then developed a computational methodology to identify drivers of broad copy number changes, identifying PDGFA (chr7) and PTEN (chr10) as driving initial nondisjunction events. These predictions were validated using mouse modeling, showing that PDGFA is sufficient to induce proneural-like gliomas and that additional NF1 loss converts proneural to the mesenchymal subtype. Our findings suggest that most non-GCIMP mesenchymal GBMs arise as, and evolve from, a proneural-like precursor.
| S-EPMC4143139 | biostudies-literature
Project description:In order to understand the relationships between the human non-GCIMP glioblastoma subgroups, we performed computational analysis of human genomic data to predict the temporal sequence in which the driver events arise during tumorigenesis. The order of evolutionary events for non-GCIMP GBM is 1) chr 7 gain and loss of chr 10, followed by 2) CDKN2A loss and/or TP53 mutation, and 3) alterations canonical for specific subtypes such as NF1 loss or focal amplification of PDGFRα or EGFR. We then developed a computational methodology to identify the drivers of broad copy number changes, identifying PDGF-A (chr 7) and PTEN (chr 10) as driving the initial non-disjunction events. These predictions were validated using mouse modeling, showing PDGF-A is sufficient to induce PN-like gliomas that are enhanced by loss of Ink4a-Arf, Tp53 or Pten. Additional Nf1 loss converts PN to the MES subtype. Our findings suggest non-GCIMP GBMs arise as, and evolve from, a common proneural-like precursor. hGBM_sphere: Three different human GBM sphere lines expressing two different NF1-shRNAs or empty shRNA-control were generated by a lentiviral infection with the relevant pLKO.1 vectors. The total RNAs were obtained from the GBM sphere lines and the gene expression profile of the NF1-shRNA cells was then compared to the control cells. hGBM_rapamycin: Two human GBM sphere lines (TS543 and TS667) expressing the NF1-shRNAs were generated by a lentiviral infection with two different pLKO.1-NF1-shRNA vectors (two different target sequence; #2 and #5). The total RNAs were obtained from the NF1-shRNA lines treated with a 1nM rapamycin or 0.1% DMSO control for 5 hours and the gene expression profile was then compared between the both groups. Murine_gliomas: Murine brain tumors were generated by an injection of DF1 cells producing the relevant RCAS virus into newborn pups or adult mice brain. When mice presented any symptoms of disease, the mice were sacrificed and the brain tumor tissues were macroscopically dissected. Then total RNAs were extracted from the murine brain tumors or normal brain tissues and the gene expression profile was compared between various tumor types and/or normal brain tissues. In this study, two different RCAS-shNf1 (GR249 and GF249) and shp53 (R696 and mR696) was used for the generation of the murine brain tumors. Each RCAS vector has different structure but the target sequence against the Nf1 and p53 is same and similar knockdown effect was observed. Tumor grade were determined with the residual sections after a piece of tumor tissue were excised for the RNA extraction. The detail information of the experiment was described in the associated-publication. Murine_shere: Murine neurosphere lines were generated by a retroviral infection with the relevant RCAS viruses. The total RNAs were obtained from the murine neurosphere lines expressing the RCAS-shGL2, shNf1, shp53 or shNf1+shp53 and the gene expression profiles of the shNf1, shp53 or shNf1/p53 cells were then compared to the control shGL2 cells.
| S-ECPF-GEOD-45874 | biostudies-other
Project description:Glioblastoma multiforme (GBM) is an aggressive, heterogeneous and highly vascularized brain tumor. GBM is thought to arise from glioblastoma stem-like cells (GSCs) which are characterized as being either proneural or mesenchymal. The former isolates of GSCs are tight sphere forming and slow growing while mesenchymal GSCs are lose sphere forming, fast growing, highly invasive and when dominant yield poorer patient prognosis. GSCs are known to be plastic in nature and can therefore evolve from a proneural to a mesenchymal state. Here, we observed that factors secreted by endothelial cells (which make up the brain vasculature) alter several properties of GSCs resulting in the acquisition of a more mesenchymal and invasive phenotype coupled with changes at the level of secretory and cellular proteome. Thus, using mass spectrometry, quantitative proteomic analysis and GO term filters, we identified several mesenchymal traits in proneural GSCs exposed to endothelial cell secretome. Specifically, proneural cells treated with the conditioned media derived from human umbilical vein endothelial cells (HUVEC) upregulated the expression of mesenchymal proteins such as CD44 and VIM, while downregulating the expression of the proneural proteins such as NOTCH1, activated NOTCH intracellular domain (NICD), SOX2 and NESTIN, which were validated using flow cytometry (FACS) and western blots (WB). Using DAVID analysis tool, we detected the features of cellular proteome indicative of the activation of NFkB, Wnt and several other pathways in the proneural cells treated with HUVEC conditioned media. Using conditioned media fractionation through several centrifugation steps we identified the extracellular vesicles (EV) sedimented at 100,000 x g using ultracentrifugation, as the source of activity in endothelial conditioned media capable of triggering mesenchymal shift in proneural GSCs. EVs are heterogeneous membrane structures containing multiple bioactive macromolecules, which have the ability to carry multiple bioactive proteins, transfer them to recipient cells and alter their function, signalling and biological programmes. We compared the effects of EVs, soluble fraction and unfractionated conditioned media in terms of their ability to trigger mesenchymal changes in the phenotype of proneural GSCs. Once the cultures were established, the culture medium was removed and replaced with HUVEC-derived material (conditioned media, supernatant or EV fraction) and responses evaluated over 7 days by microscopical analysis of sphere structures, and biochemically by following the aforementioned proneural or mesenchymal markers (WB, FACS). We observed an upregulation of mesenchymal proteins, as well as downregulation of the proneural proteins, mentioned above. These effects recapitulated those of unfractionated conditioned media and were absent from target cells exposed to EV-depleted conditioned media. The data analysis of EV proteome included canonical markers and pathways of cellular vesiculation as well as markers and pathways of interest with regards to the biological effects associated with treatment of GSC recipient cells. In this regard we observed several EV related tetraspanin markers, which were validated using western blot including CD9, CD63, CD81 and a purity control, BIP. Although we identified several potential effectors associated with endothelial cell EVs that could impact proneural cell phenotype, we focused on MMPs for at least three reasons: (i) evidence in the literature (see text) indicated that MMPs may induce differentiation programs in neural stem cells; (ii) MMPs in EV cargo were relatively abundant and have been implicated in various biological processes; (iii) MMPs released from endothelial cells could be functionally involved in disrupting proneural cell sphere structures that we observed in the presence of endothelial cell secretome. We noted the expressions of MMP1, MMP2, MMP11 and MP14 in our HUVEC-EV mass spectrometry dataset, the activity of which was validated using the MMP activity assay kit from abcam (ab112146). Using GO terms we also detected a signal for NFkB pathway activation in the proteome of endothelial (HUVEC) conditioned media-treated proneural GSCs. NFkB activation is regarded as hallmark of mesenchymal phenotype in GSCs and GBM cells. We validated that the upregulation of NFkB, was also true for the proneural cells treated with HUVEC derived EVs. Moreover, upon blocking MMP expression in proneural cells treated with endothelial cell EVs, we inhibited the activation of NFkB activity thereby documenting that the initial effects of MMPs trigger a shift in cellular phenotype toward NfkB activation and mesenchymal reprogramming. Briefly, we compared GO terms of GSC157 cells treated with their own or HUVEC-derived EVs. Validation of the NFkB pathway activation was analysed using WB and immunofluorescence for levels of NFkB and phosphor-NFkB.
2022-10-14 | PXD034819 | Pride
Project description:To identify potential targets of miR-34a, we performed transcriptional profiling on proneural TS543 GBM cells, focusing on mRNAs whose levels decreased in response to miR-34a transfection as compared to control oligonucleotide. Proneural TS543 GBM cells were transfected with 100 nM hsa-miR-34a or control oligonucleotide using Hiperfect transfection reagent (Qiagen). After 3 days, RNA was isolated and expression analyses were performed using Illumina HT-12 bead array. The microarray dataset was normalized using a variance stable normalization (VSN) procedure in the ‘lumi’ package from the Bioconductor framework.
2012-03-26 | E-GEOD-34242 | biostudies-arrayexpress
Project description:As many other tumors, a subset of gliobastoma is thought to be maintained by a restricted population of cancer cells, stem-like cells that express CD133 transmembrane protein. Expression levels of CD133 gene has been linked to a poor prognostic molecular subgroup and is not overexpressed by the PDGF-driven proneural group. Thus, the significance of CD133+ cells for gliomagenesis of the proneural group is undetermined. In addition, the role of the CD133 protein remains elusive and controversial, which results from the difficult isolation of CD133+ cells that has largely relied on the use of antibodies to ill-defined glycosylated epitopes of CD133. Here, we used a knockin lacZ reporter mouse, Prom1lacZ/+, to track Prom1+ cells in the brain and found that Prom1 (prominin1, murine CD133 homologue) is expressed by cells that co-express markers characteristic of neuronal, glial and vascular lineage phenotype. In proneural tumors derived from injection of RCAS-PDGF into the brain of tv-a;Ink4a-Arf-/- Prom1lacZ/+ mice, Prom1+ cells co-express markers for astrocytes and endothelial cells. Therefore, we characterize the tumor propagation in a murine model and found that the mice co-transplanted with Prom1 endothelium and proneural tumor spheres cells had significant tumor burden and vascular proliferation (angiogenesis). Specific genes in Prom1 endothelium are identified that code for endothelial signaling modulators that most likely support proneural tumor progression and can be potential targets for anti-angiogenic therapy. Cells were sorted via FACS to obtain a population of CD31+CD133- cells and a population of CD31+CD133+ cells. Total RNA was extracted from each population and gene expression was assayed on Affymetrix Mouse 430 2.0 arrays with one array per cell population.
2013-12-31 | E-GEOD-43931 | biostudies-arrayexpress
Project description:As many other tumors, a subset of gliobastoma is thought to be maintained by a restricted population of cancer cells, stem-like cells that express CD133 transmembrane protein. Expression levels of CD133 gene has been linked to a poor prognostic molecular subgroup and is not overexpressed by the PDGF-driven proneural group. Thus, the significance of CD133+ cells for gliomagenesis of the proneural group is undetermined. In addition, the role of the CD133 protein remains elusive and controversial, which results from the difficult isolation of CD133+ cells that has largely relied on the use of antibodies to ill-defined glycosylated epitopes of CD133. Here, we used a knockin lacZ reporter mouse, Prom1lacZ/+, to track Prom1+ cells in the brain and found that Prom1 (prominin1, murine CD133 homologue) is expressed by cells that co-express markers characteristic of neuronal, glial and vascular lineage phenotype. In proneural tumors derived from injection of RCAS-PDGF into the brain of tv-a;Ink4a-Arf-/- Prom1lacZ/+ mice, Prom1+ cells co-express markers for astrocytes and endothelial cells. Therefore, we characterize the tumor propagation in a murine model and found that the mice co-transplanted with Prom1 endothelium and proneural tumor spheres cells had significant tumor burden and vascular proliferation (angiogenesis). Specific genes in Prom1 endothelium are identified that code for endothelial signaling modulators that most likely support proneural tumor progression and can be potential targets for anti-angiogenic therapy.
2013-12-31 | GSE43931 | GEO
Project description:To identify potential targets of miR-34a, we performed transcriptional profiling on proneural TS543 GBM cells, focusing on mRNAs whose levels decreased in response to miR-34a transfection as compared to control oligonucleotide.
2012-03-27 | GSE34242 | GEO
Project description:Identification of critical survival determinants of PDGF-driven proneural glioma. Results provided information about the genes and pathways that are regulated by PDGF signaling in PDGF-driven proneural glioma and led to the assessment of the importance of the USP1-ID2 axis in proneural glioma.
2016-03-22 | GSE77423 | GEO