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.

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 . Keywords: cell type comparison

Project description:PD-L1 Inhibitor Regulates the miR-33a-5p/PTEN Signaling Pathway and Can Be Targeted to Sensitize Glioblastomas to Radiation. Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Ionizing radiation (IR) is a standard treatment for GBM patients and results in DNA damage. However, the clinical efficacy of IR is limited due to therapeutic resistance. The programmed death ligand 1 (PD-L1) blockade has a shown the potential to increase the efficacy of radiotherapy by inhibiting DNA damage and repair responses. The miR-33a-5p is an essential microRNA that promotes GBM growth and self-renewal. In this study, we investigated whether a PD-L1 inhibitor (a small molecule inhibitor) exerted radio-sensitive effects to impart an anti-tumor function in GBM cells by modulating miR-33a-5p. U87 MG cells and U251 cells were pretreated with PD-L1 inhibitor. The PD-L1 inhibitor-induced radio-sensitivity in these cells was assessed by assaying cellular apoptosis, clonogenic survival assays, and migration. TargetScan and luciferase assay showed that miR-33a-5p targeted the phosphatase and tensin homolog (PTEN) 3' untranslated region. The expression level of PTEN was measured by western blotting, and was also silenced using small interfering RNAs. The levels of DNA damage following radiation was measured by the presence of γ-H2AX foci, cell cycle, and the mRNA of the DNA damage-related genes, BRCA1, NBS1, RAD50, and MRE11. Our results demonstrated that the PD-L1 inhibitor significantly decreased the expression of the target gene, miR-33a-5p. In addition, pretreatment of U87 MG and U251 cells with the PD-L1 inhibitor increased radio-sensitivity, as indicated by increased apoptosis, while decreased survival and migration of GBM cells. Mir-33a-5p overexpression or silencing PTEN in U87 MG and U251 cells significantly attenuated PD-L1 radiosensitive effect. Additionally, PD-L1 inhibitor treatment suppressed the expression of the DNA damage response-related genes, BRCA1, NBS1, RAD50, and MRE11. Our results demonstrated a novel role for the PD-L1 inhibitor in inducing radio- sensitivity in GBM cells, where inhibiting miR-33a-5p, leading to PTEN activated, and inducing DNA damage was crucial for antitumor immunotherapies to treat GBM.

Project description:Background: Signaling by receptor tyrosine kinases (RTK) is frequently dysregulated in gliomas. Inter-individual variability in the causes for dysregulated RTK signaling may have hampered the efficacy of targeted therapies. Using gene expression modules around key regulators in the RAS-RAF-MEK-MAPK cascade and in the phosphatidylinositol 3-kinase-AKT pathways, we developed a âRMPAâ clustering scheme to distinguish gliomas with varying extents of RTK signaling. Results: We identified gene modules consistently co-expressed with NF1 (NF1-M), Sprouty (SPRY-M) and PTEN (PTEN-M) in gliomas. Their signatures enabled robust clustering of adult diffuse gliomas of WHO grades II-IV into RMPAhigh and RMPAlow phenotypes in a morphology-independent manner. In five independent data sets from three continents containing more than 1500 adult diffuse gliomas, RMPAhigh gliomas were associated with poor prognosis while RMPAlow gliomas were not. The RMPAhigh and RMPAlow glioma subtypes showed distinct levels of the activities of RAS-RAF-MEK-MAPK cascade and PI3K-AKT pathway and harbored unique sets of genomic alterations in the RTK signaling-related genes. The RMPAhigh gliomas contained large numbers of immature vessel cells and tumor associated macrophages and both cell types expressed high levels of pro-angiogenic RTKs including MET, VEGFR1, KDR, EPHB4 and NRP1. Conclusion: Inter-glioma variability in RTK signaling activities can be defined using the RMPA clustering scheme. The combined signatures of NF1-M, SPRY-M and PTEN-M reflect RTK signaling activity both in the glioma cells and in the glioma microenvironment. Our data show that RTK signaling in the glioma microenvironment may play a pivotal role in glioma progression. Transcriptome data from 22 fresh gliomas (2 astrocytoma II, 1 oligodendrocytoma II, 7 oligoastrocytoma II, 4 anaplastic oligoastrocytoma III and 8 GBM) were obtained using Affymetrix Human Gene 1.0 ST Array. Unsupervised hierarchical clustering of the expression data for the SPRY-M, NF1-M and PTEN-M was performed on these transcriptome data to identify samples with RMPAhigh or RMPAlow signature.

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Combined inhibition of TOP1 and PARP: a synergistic therapeutic strategy for PTEN-deficient glioblastoma

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