Unknown,Transcriptomics,Genomics,Proteomics

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Oncogenic alterations in multiple core signaling pathways are required for glioblastoma pathogenesis in vitro and in vivo

ABSTRACT: Here, we use a series of genetically-defined murine cortical astrocytes with conditional inactivation of Rb/Pten and activated Kras to systematically investigate the individual and combinatorial roles of these pathways during gliomagenesis. We show that genetic disruption of all three pathways, which frequently occurs in human GBM, leads to maximal in vitro growth, migration, and invasion and produces stem-like transcriptomal profiles similar to the proneural subtype of human GBM. Genetic alterations in all three pathways are also required for efficient tumorigenesis in an orthotopic syngeneic allograft model system in vivo. These findings show that cortical astrocytes can form GBM and identify a potential model for proneural GBM that can be used to test subtype-specific therapies. Transcriptional profiling of transformed murine cortical astrocytes shows correlation between mutated genes, invasive capability, neural lineage, and human astrocytoma/GBM signatures. 10 samples Cortical astrocytes were isolated from neonatal mice (C57BL/6 background strain) and cultured. Cells were infected with adeneoviral-Cre for 6 hours to induce recombination. This resulted in expression of an N-terminal 121 amino acid truncation mutant of SV40 large T antigen (T121, hereafter called T) from the human glial fibrillary acidic protein (GFAP) promoter, which inactivates all three members of the Rb family (Rb, p107, p130) and results in proliferation and defective G1/S ratios. Additionally, cells contained KrasG12D mutant (R) and/or contained either heterozygous or homozygous deletion of Pten (P+/- or P-/-) . Here are 23 cell lines comprising 6 different genotypes. 10 Samples (Sample Title: TR_2, T_1, TRP_null_3, TR_1, TRP_null_4, TRP_het_1, T_2, TRP_null_2, T_3, TRP_null_5) from one batch were combined with the remaining 13 samples to remove batch related effects (see data processing for method).

ORGANISM(S): Mus musculus

SUBMITTER: Mark Vitucci

PROVIDER: E-GEOD-40265 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:BACKGROUND: Glioma stem cells (GSCs) from human glioblastomas (GBMs) are resistant to radiation and chemotherapy and may drive recurrence. Treatment efficacy may depend on GSCs, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. METHODS: To model genetic alterations in human GBM core signaling pathways, we induced Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Neurosphere culture, differentiation, and orthotopic transplantation assays were used to assess whether these mutations induced de-differentiation into GSCs. Genome-wide chromatin landscape alterations and expression profiles were examined by formaldehyde-assisted isolation of regulatory elements (FAIRE) seq and RNA-seq. Radiation and temozolomide efficacy were examined in vitro and in an allograft model in vivo. Effects of radiation on transcriptome subtype were examined by microarray expression profiling. RESULTS: Cultured triple mutant astrocytes gained unlimited self-renewal and multilineage differentiation capacity. These cells harbored significantly altered chromatin landscapes that were associated with downregulation of astrocyte- and upregulation of stem cell-associated genes, particularly the Hoxa locus of embryonic transcription factors. Triple-mutant astrocytes formed serially transplantable glioblastoma allografts that were sensitive to radiation but expressed MGMT and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of GBM allografts from proneural to mesenchymal. CONCLUSION: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into GSCs with altered chromatin landscapes and transcriptomes. This non-germline genetically engineered mouse model mimics human proneural GBM on histopathological, molecular, and treatment response levels. It may be useful for dissecting the mechanisms of treatment resistance and developing more effective therapies.

Project description:Glioblastoma (GBM) is a highly lethal brain tumor presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as high-grade disease that typically harbors EGFR, PTEN and Ink4a/Arf mutations, and the secondary GBM subtype evolves from the slow progression of low-grade disease that classically possesses PDGF and p53 events1. Here, we show that concomitant CNS-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with striking clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted p53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of p53 as well the expected PTEN mutations. Integrated transcriptomic profling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives elevated c-Myc levels and its associated signature. Functional studies validated increased c-Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of p53-Pten null NSCs as well as tumor neurospheres (TNSs) derived from this model. c-Myc also serves to maintain robust tumorigenic potential of p53-Pten null TNSs. These murine modeling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumor suppressor mutation profile in human primary GBM and establish c-Myc as a key target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential. We used microarrays to detail the gene expression difference of the p53-null and p53/Pten-doubly null neural stem cell after differentiation . Experiment Overall Design: transcriptome comparisons of 2 independent p53-null with 3 p53/Pten double-null murine NSCs at 1 day post exposure to the differentiation inducer.

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Oncogenic alterations in multiple core signaling pathways are required for glioblastoma pathogenesis in vitro and in vivo

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