Core pathway mutations induce de-differentiation of murine astrocytes into glioblastoma stem cells that are sensitive to radiation, but resistant to temozolomide
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE75592 | GEO | 2016/02/01
SECONDARY ACCESSION(S): PRJNA304640
REPOSITORIES: GEO
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Project description:BackgroundGlioma 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.MethodsTo 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.ResultsCultured 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.ConclusionA 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.
| S-EPMC4896545 | biostudies-literature
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.
2016-01-31 | GSE59116 | GEO
Project description:Introduction: Glioma stem cells isolated from human glioblastomas are resistant to radiation and cytotoxic chemotherapy and may drive tumor recurrence. Treatment efficacy may depend on the presence of glioma stem cells, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. Methods: To model genetic alterations in the core signaling pathways of human glioblastoma, we induced conditional Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Serial neurosphere culture, multi-lineage differentiation, and orthotopic transplantation were used to assess whether these mutations induced de-differentiation of cortical astrocytes into glioma stem cells. Efficacy of radiation and temozolomide was examined in vitro and in an allograft model in vivo. The effects of radiation on transcriptome subtype was examined by expression profiling. Results: G1/S-defective, Rb knockout astrocytes gained unlimited self-renewal and multi-lineage differentiation capacity, in both the presence and absence of Kras and Pten mutations. Only triple mutant astrocytes formed serially-transplantable glioblastoma allografts. Triple mutant astrocytes and allografts were sensitive to radiation, but expressed Mgmt and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of glioblastoma allografts from proneural to mesenchymal. Conclusion: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into glioma stem cells. This non-germline genetically engineered mouse model mimics human proneural glioblastoma on histopathological, molecular, and treatment response levels. It may be useful in dissecting the genetic and cellular mechanisms of treatment resistance and developing more effective therapies.
2016-02-01 | GSE75589 | GEO
Project description:Introduction: Glioma stem cells isolated from human glioblastomas are resistant to radiation and cytotoxic chemotherapy and may drive tumor recurrence. Treatment efficacy may depend on the presence of glioma stem cells, expression of DNA repair enzymes such as methylguanine methyltransferase (MGMT), or transcriptome subtype. Methods: To model genetic alterations in the core signaling pathways of human glioblastoma, we induced conditional Rb knockout, Kras activation, and Pten deletion mutations in cortical murine astrocytes. Serial neurosphere culture, multi-lineage differentiation, and orthotopic transplantation were used to assess whether these mutations induced de-differentiation of cortical astrocytes into glioma stem cells. Efficacy of radiation and temozolomide was examined in vitro and in an allograft model in vivo. The effects of radiation on transcriptome subtype was examined by expression profiling. Results: G1/S-defective, Rb knockout astrocytes gained unlimited self-renewal and multi-lineage differentiation capacity, in both the presence and absence of Kras and Pten mutations. Only triple mutant astrocytes formed serially-transplantable glioblastoma allografts. Triple mutant astrocytes and allografts were sensitive to radiation, but expressed Mgmt and were resistant to temozolomide. Radiation induced a shift in transcriptome subtype of glioblastoma allografts from proneural to mesenchymal. Conclusion: A defined set of core signaling pathway mutations induces de-differentiation of cortical murine astrocytes into glioma stem cells. This non-germline genetically engineered mouse model mimics human proneural glioblastoma on histopathological, molecular, and treatment response levels. It may be useful in dissecting the genetic and cellular mechanisms of treatment resistance and developing more effective therapies.
2016-02-01 | GSE73262 | GEO
Project description:Glioblastoma (GBM) is the most frequent malignant primary tumor of the CNS in adults, with a median survival of 14.6 months after diagnosis. The effectiveness of GBM therapies remains poor, highlighting the need for new therapeutic alternatives. In this work, we evaluated the effect of 4-methylumbelliferone (4MU), a coumarin derivative without adverse effects reported, in combination with temozolomide (TMZ) or vincristine (VCR) on U251, LN229, U251-TMZ resistant (U251-R) and LN229-TMZ resistant (LN229-R) human GBM cells. We determined cell proliferation by BrdU incorporation, migration through wound healing assay, metabolic and MMP activity by XTT and zymography assays, respectively, and cell death by PI staining and flow cytometry. 4MU sensitizes GBM cell lines to the effect of TMZ and VCR and inhibits metabolic activity and cell proliferation on U251-R cells. Interestingly, the lowest doses of TMZ enhance U251-R and LN229-R cell proliferation, while 4MU reverts this and even sensitizes both cell lines to TMZ and VCR effects. We showed a marked antitumor effect of 4MU on GBM cells alone and in combination with chemotherapy and proved, for the first time, the effect of 4MU on TMZ-resistant models, demonstrating that 4MU would be a potential therapeutic alternative for improving GBM therapy even on TMZ-refractory patients.
| S-EPMC10249561 | biostudies-literature
Project description:Adaptive plasticity to the standard chemotherapeutic temozolomide (TMZ) leads to glioblastoma progression. Here, we examine early stages of this process in patient-derived cellular models, exposing the human lysine-specific demethylase 5B (KDM5B) as a prospective indicator for subclonal expansion. By integration of a reporter, we show its preferential activity in rare, stem-like ALDH1A1+ cells, immediately increasing expression upon TMZ exposure. Naive, genetically unmodified KDM5Bhigh cells phosphorylate AKT (pAKT) and act as slow-cycling persisters under TMZ. Knockdown of KDM5B reverses pAKT levels, simultaneously increasing PTEN expression and TMZ sensitivity. Pharmacological inhibition of PTEN rescues the effect. Interference with KDM5B subsequent to TMZ decreases cellular vitality, and clonal tracing with DNA barcoding demonstrates high individual levels of KDM5B to predict subclonal expansion already before TMZ exposure. Thus, KDM5Bhigh treatment-naive cells preferentially contribute to the dynamics of drug resistance under TMZ. These findings may serve as a cornerstone for future biomarker-assisted clinical trials.
| S-EPMC10762356 | biostudies-literature
Project description:Temozolomide is the current first-line treatment for glioblastoma patients but, because many patients are resistant to it, there is an urgent need to develop antitumor agents to treat temozolomide-resistant glioblastoma. Gossypol, a natural polyphenolic compound, has been studied as a monotherapy or combination therapy for the treatment of glioblastoma. The combination of gossypol and temozolomide has been shown to inhibit glioblastoma, but it is not clear yet whether gossypol alone can suppress temozolomide-resistant glioblastoma. We find that gossypol suppresses the growth of temozolomide-resistant glioblastoma cells in both tumor sphere and adherent culture conditions, with tumor spheres showing the greatest sensitivity. Molecular docking and binding energy calculations show that gossypol has a similar affinity to the Bcl2 (B-cell lymphoma 2) family of proteins and several dehydrogenases. Gossypol reduces mitochondrial membrane potential and cellular ATP levels before cell death, which suggests that gossypol inhibits several dehydrogenases in the cell's metabolic pathway. Treatment with a Bcl2 inhibitor does not fully explain the effect of gossypol on glioblastoma. Overall, this study demonstrates that gossypol can suppress temozolomide-resistant glioblastoma and will be helpful for the refinement of gossypol treatments by elucidating some of the molecular mechanisms of gossypol in glioblastoma.
| S-EPMC6843396 | biostudies-literature
Project description:Background and purposeGlioblastoma multiforme (GBM) represents the most common and deadly primary brain malignancy, particularly due to temozolomide (TMZ) and radiation (RT) resistance. To better understand resistance mechanisms, we examined global kinase activity (kinomic profiling) in both treatment sensitive and resistant human GBM patient-derived xenografts (PDX or "xenolines").Materials and methodsThirteen orthotopically-implanted xenolines were examined including 8 with known RT sensitivity/resistance, while 5 TMZ resistant xenolines were generated through serial TMZ treatment in vivo. Tumors were harvested, prepared as total protein lysates, and kinomically analyzed on a PamStation®12 high-throughput microarray platform with subsequent upstream kinase prediction and network modeling.ResultsKinomic profiles indicated elevated tyrosine kinase activity associated with the radiation resistance phenotype, including FAK and FGFR1. Furthermore, network modeling showed VEGFR1/2 and c-Raf hubs could be involved. Analysis of acquired TMZ resistance revealed more kinomic variability among TMZ resistant tumors. Two of the five tumors displayed significantly altered kinase activity in the TMZ resistant xenolines and network modeling indicated PKC, JAK1, PI3K, CDK2, and VEGFR as potential mediators of this resistance.ConclusionGBM xenolines provide a phenotypic model for GBM drug response and resistance that when paired with kinomic profiling identified targetable pathways to inherent (radiation) or acquired (TMZ) resistance.
| S-EPMC4119546 | biostudies-literature
Project description:New strategies that improve median survivals of only ~15-20 months for glioblastoma (GBM) with the current standard of care (SOC) which is concurrent temozolomide (TMZ) and radiation (XRT) treatment are urgently needed. Inhibition of polo-like kinase 1 (PLK1), a multifunctional cell cycle regulator, overexpressed in GBM has shown therapeutic promise but has never been tested in the context of SOC. Therefore, we examined the mechanistic and therapeutic impact of PLK1 specific inhibitor (volasertib) alone and in combination with TMZ and/or XRT on GBM cells. We quantified the effects of volasertib alone and in combination with TMZ and/or XRT on GBM cell cytotoxicity/apoptosis, mitochondrial membrane potential (MtMP), reactive oxygen species (ROS), cell cycle, stemness, DNA damage, DNA repair genes, cellular signaling and in-vivo tumor growth. Volasertib alone and in combination with TMZ and/or XRT promoted apoptotic cell death, altered MtMP, increased ROS and G2/M cell cycle arrest. Combined volasertib and TMZ treatment reduced side population (SP) indicating activity against GBM stem-like cells. Volasertib combinatorial treatment also significantly increased DNA damage and reduced cell survival by inhibition of DNA repair gene expression and modulation of ERK/MAPK, AMPK and glucocorticoid receptor signaling. Finally, as observed in-vitro, combined volasertib and TMZ treatment resulted in synergistic inhibition of tumor growth in-vivo. Together these results identify new mechanisms of action for volasertib that provide a strong rationale for further investigation of PLK1 inhibition as an adjunct to current GBM SOC therapy.
| S-EPMC8533781 | biostudies-literature
Project description:Glioblastoma multiforme (GBM) is an aggressive cancer without currently effective therapies. Radiation and temozolomide (radio/TMZ) resistance are major contributors to cancer recurrence and failed GBM therapy. Heat shock proteins (HSPs), through regulation of extracellular matrix (ECM) remodeling and epithelial mesenchymal transition (EMT), provide mechanistic pathways contributing to the development of GBM and radio/TMZ-resistant GBM. The Friend leukemia integration 1 (Fli-1) signaling network has been implicated in oncogenesis in GBM, making it an appealing target for advancing novel therapeutics. Fli-1 is linked to oncogenic transformation with up-regulation in radio/TMZ-resistant GBM, transcriptionally regulating HSPB1. This link led us to search for targeted molecules that inhibit Fli-1. Expression screening for Fli-1 inhibitors identified lumefantrine, an antimalarial drug, as a probable Fli-1 inhibitor. Docking and isothermal calorimetry titration confirmed interaction between lumefantrine and Fli-1. Lumefantrine promoted growth suppression and apoptosis in vitro in parental and radio/TMZ-resistant GBM and inhibited tumor growth without toxicity in vivo in U87MG GBM and radio/TMZ-resistant GBM orthotopic tumor models. These data reveal that lumefantrine, an FDA-approved drug, represents a potential GBM therapeutic that functions through inhibition of the Fli-1/HSPB1/EMT/ECM remodeling protein networks.
| S-EPMC7275698 | biostudies-literature