RESUMO
BACKGROUND: Chromosome instability (CIN) with recurrent copy number alterations is a feature of many solid tumors, including glioblastoma (GBM), yet the genes that regulate cell division are rarely mutated in cancers. Here, we show that the brain-abundant mitogen, platelet-derived growth factor-A (PDGFA) fails to induce the expression of kinetochore and spindle assembly checkpoint genes leading to defective mitosis in neural progenitor cells (NPCs). METHODS: Using a recently reported in vitro model of the initiation of high-grade gliomas from murine NPCs, we investigated the immediate effects of PDGFA exposure on the nuclear and mitotic phenotypes and patterns of gene and protein expression in NPCs, a putative GBM cell of origin. RESULTS: NPCs divided abnormally in defined media containing PDGFA with P53-dependent effects. In wild-type cells, defective mitosis was associated with P53 activation and cell death, but in some null cells, defective mitosis was tolerated. Surviving cells had unstable genomes and proliferated in the presence of PDGFA accumulating random and clonal chromosomal rearrangements. The outcome of this process was a population of tumorigenic NPCs with recurrent gains and losses of chromosomal regions that were syntenic to those recurrently gained and lost in human GBM. By stimulating proliferation without setting the stage for successful mitosis, PDGFA-transformed NPCs lacking P53 function. CONCLUSIONS: Our work describes a mechanism of transformation of NPCs by a brain-associated mitogen, raising the possibility that the unique genomic architecture of GBM is an adaptation to defective mitosis that ensures the survival of affected cells.
Assuntos
Glioblastoma , Células-Tronco Neurais , Humanos , Animais , Camundongos , Mitógenos/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Mitose , Células-Tronco Neurais/patologia , Glioblastoma/patologiaRESUMO
Glioblastomas (GBMs) are aggressive brain tumors characterized by extensive inter- and intratumor heterogeneity. Patient-derived models, such as organoids and explants, have recently emerged as useful models to study such heterogeneity, although the extent to which they can recapitulate GBM genomic features remains unclear. Here, we analyze bulk exome and single-cell genome and transcriptome profiles of 12 IDH wild-type GBMs, including two recurrent tumors, and of patient-derived explants (PDEs) and gliomasphere (GS) lines derived from these tumors. We find that PDEs are genetically similar to, and variably retain gene expression characteristics of, their parent tumors. Notably, PDEs appear to exhibit similar levels of transcriptional heterogeneity compared with their parent tumors, whereas GS lines tend to be enriched for cells in a more uniform transcriptional state. The approaches and datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived models, especially in the context of studying cellular heterogeneity.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular , Genômica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Recidiva Local de NeoplasiaRESUMO
Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Glioma/metabolismo , Glioma/patologia , Interleucina-33/metabolismo , Animais , Neoplasias Encefálicas/mortalidade , Carcinogênese , Núcleo Celular/metabolismo , Citocinas/metabolismo , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioma/mortalidade , Humanos , Inflamação , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos SCID , Microglia , Análise de Sobrevida , Linfócitos T/metabolismo , Linfócitos T/patologia , Microambiente Tumoral/imunologiaRESUMO
INTRODUCTION: Temozolomide (TMZ) is a life prolonging DNA alkylating agent active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately acquired TMZ resistance severely undermines its clinical efficacy. Using an in vitro model, we tested whether poly (ADP-ribose) polymerase-1 and -2 (PARP) inhibition could suppress the emergence of resistance to enhance the effectiveness of TMZ. METHODS: Using the MGMT-methylated GBM line U251N, in which TMZ resistance can be induced, we developed a method to rapidly recreate mechanisms of TMZ resistance seen in GBMs, including MMR mutations and MGMT re-expression. We then assessed whether TMZ resistant U251N sub-clones could be re-sensitized to TMZ by co-treatment with the PARP inhibitor ABT-888, and also whether the emergence of resistance could be suppressed by PARP inhibition. RESULTS: U251N cultures chronically exposed to TMZ developed discrete colonies that expanded during TMZ treatment. These colonies were isolated, expanded further as sub-clones, and assessed for mechanisms of TMZ resistance. Most resistant sub-clones had detectable mutations in one or more mismatch repair (MMR) genes, frequently MSH6, and displayed infrequent re-expression of MGMT. TMZ resistance was associated with isolated poly(ADP-ribose) (pADPr) up-regulation in one sub-clone and was unexplained in several others. TMZ resistant sub-clones regressed during co-treatment with TMZ and ABT-888, and early co-treatment of U251N parental cultures suppressed the emergence of TMZ resistant colonies. CONCLUSION: In a model of acquired resistance, co-treatment with TMZ and a PARP inhibitor had two important benefits: re-sensitization of TMZ resistant cells and suppression of TMZ resistance.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Temozolomida/farmacologia , Antineoplásicos Alquilantes/farmacologia , Apoptose , Proliferação de Células , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Células Tumorais CultivadasRESUMO
Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Microambiente Tumoral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose , Neoplasias Encefálicas/genética , Estudos de Casos e Controles , Proliferação de Células , Metilação de DNA , Resistencia a Medicamentos Antineoplásicos , Feminino , Perfilação da Expressão Gênica , Glioblastoma/genética , Humanos , Masculino , Camundongos , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , Transcriptoma , Células Tumorais Cultivadas , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic's function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity.
Assuntos
Neoplasias Encefálicas/patologia , Células-Tronco Neurais/patologia , Oligodendroglioma/patologia , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Animais , Astrócitos/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios/patologia , Oligodendroglia/citologia , Oligodendroglia/patologia , Cultura Primária de Células , Prosencéfalo/citologia , Prosencéfalo/patologia , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Temozolomide (TMZ) is active against glioblastomas (GBM) in which the O6-methylguanine-DNA methyltransferase (MGMT) gene is silenced. However, even in responsive cases, its beneficial effect is undermined by the emergence of drug resistance. Here, we tested whether inhibition of poly (ADP-ribose) polymerase-1 and -2 (PARP) enhanced the effectiveness of TMZ. METHODS: Using patient derived brain tumor initiating cells (BTICs) and orthotopic xenografts as models of newly diagnosed and recurrent high-grade glioma, we assessed the effects of TMZ, ABT-888, and the combination of TMZ and ABT-888 on the viability of BTICs and survival of tumor-bearing mice. We also studied DNA damage repair, checkpoint protein phosphorylation, and DNA replication in mismatch repair (MMR) deficient cells treated with TMZ and TMZ plus ABT-888. RESULTS: Cells and xenografts derived from newly diagnosed MGMT methylated high-grade gliomas were sensitive to TMZ while those derived from unmethylated and recurrent gliomas were typically resistant. ABT-888 had no effect on the viability of BTICs or tumor bearing mice, but co-treatment with TMZ restored sensitivity in resistant cells and xenografts from newly diagnosed unmethylated gliomas and recurrent gliomas with MSH6 mutations. In contrast, the addition of ABT-888 to TMZ had little sensitizing effect on cells and xenografts derived from newly diagnosed methylated gliomas. In a model of acquired TMZ resistance mediated by loss of MMR gene MSH6, re-sensitization to TMZ by ABT-888 was accompanied by persistent DNA strand breaks, re-engagement of checkpoint kinase signaling, and interruption of DNA synthesis. CONCLUSION: In laboratory models, the addition of ABT-888 to TMZ overcame resistance to TMZ.
Assuntos
Benzimidazóis/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioma/patologia , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Gradação de Tumores , RNA Interferente Pequeno/genéticaRESUMO
Glioblastoma (GBM) is the most lethal and aggressive adult brain tumor, requiring the development of efficacious therapeutics. Towards this goal, we screened five genetically distinct patient-derived brain-tumor initiating cell lines (BTIC) with a unique collection of small molecule epigenetic modulators from the Structural Genomics Consortium (SGC). We identified multiple hits that inhibited the growth of BTICs in vitro, and further evaluated the therapeutic potential of EZH2 and HDAC inhibitors due to the high relevance of these targets for GBM. We found that the novel SAM-competitive EZH2 inhibitor UNC1999 exhibited low micromolar cytotoxicity in vitro on a diverse collection of BTIC lines, synergized with dexamethasone (DEX) and suppressed tumor growth in vivo in combination with DEX. In addition, a unique brain-penetrant class I HDAC inhibitor exhibited cytotoxicity in vitro on a panel of BTIC lines and extended survival in combination with TMZ in an orthotopic BTIC model in vivo. Finally, a combination of EZH2 and HDAC inhibitors demonstrated synergy in vitro by augmenting apoptosis and increasing DNA damage. Our findings identify key epigenetic modulators in GBM that regulate BTIC growth and survival and highlight promising combination therapies.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Piridonas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dexametasona/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada , Epigênese Genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Piridonas/farmacologia , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Metabolic imaging of brain tumors using (13)C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-(13)C] pyruvate is a promising neuroimaging strategy which, after a decade of preclinical success in glioblastoma (GBM) models, is now entering clinical trials in multiple centers. Typically, the presence of GBM has been associated with elevated hyperpolarized [1-(13)C] lactate produced from [1-(13)C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-(13)C] lactate. However, to date, lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation, which, in addition to initiating tumor development, also induces metabolic reprogramming. In particular, mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1, MCT4), three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of (13)C MRS of hyperpolarized [1-(13)C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA, MCT1 and MCT4, and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-(13)C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-(13)C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors, in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas, which, when combined with other clinically available imaging methods, could be used to detect the presence of the IDH1 mutation in vivo.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13/métodos , Glioma/diagnóstico por imagem , Isocitrato Desidrogenase/metabolismo , Ácido Láctico/metabolismo , Ácido Pirúvico/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Glioma/genética , Glioma/metabolismo , Humanos , Células Tumorais CultivadasRESUMO
BACKGROUND: Glioblastoma (GBM) is a fatal cancer that has eluded major therapeutic advances. Failure to make progress may reflect the absence of a human GBM model that could be used to test compounds for anti-GBM activity. In this respect, the development of brain tumor-initiating cell (BTIC) cultures is a step forward because BTICs appear to capture the molecular diversity of GBM better than traditional glioma cell lines. Here, we perform a comparative genomic and genetic analysis of BTICs and their parent tumors as preliminary evaluation of the BTIC model. METHODS: We assessed single nucleotide polymorphisms (SNPs), genome-wide copy number variations (CNVs), gene expression patterns, and molecular subtypes of 11 established BTIC lines and matched parent tumors. RESULTS: Although CNV differences were noted, BTICs retained the major genomic alterations characteristic of GBM. SNP patterns were similar between BTICs and tumors. Importantly, recurring SNP or CNV alterations specific to BTICs were not seen. Comparative gene expression analysis and molecular subtyping revealed differences between BTICs and GBMs. These differences formed the basis of a 63-gene expression signature that distinguished cells from tumors; differentially expressed genes primarily involved metabolic processes. We also derived a set of 73 similarly expressed genes; these genes were not associated with specific biological functions. CONCLUSIONS: Although not identical, established BTIC lines preserve the core molecular alterations seen in their parent tumors, as well as the genomic hallmarks of GBM, without acquiring recurring BTIC-specific changes.
Assuntos
Neoplasias Encefálicas/genética , Variações do Número de Cópias de DNA/genética , Genoma Humano , Glioblastoma/genética , Células-Tronco Neoplásicas/patologia , Idoso , Autoanticorpos/uso terapêutico , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Feminino , Testes Genéticos , Glioblastoma/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism bof reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples.
Assuntos
Neoplasias Encefálicas/genética , Glutaratos/metabolismo , Isocitrato Desidrogenase/genética , Oligodendroglioma/genética , Proteínas Repressoras/genética , Western Blotting , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Humanos , Imunoprecipitação , Espectrometria de Massas , Mutação , Oligodendroglioma/metabolismoRESUMO
BACKGROUND: Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. METHODS: We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant ((mt)) and IDH wild-type ((wt)) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. RESULTS: We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH(mt) gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH(mt) derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH(wt)), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH(mt) glioblastomas. CONCLUSION: To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH(mt) gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.
Assuntos
Neoplasias do Tronco Encefálico/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glioma/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isocitrato Desidrogenase/genética , L-Lactato Desidrogenase/genética , Animais , Linhagem Celular Tumoral , Metilação de DNA , Glioblastoma/genética , Humanos , Isoenzimas/genética , Lactato Desidrogenase 5 , Camundongos , MutaçãoRESUMO
BACKGROUND: Embryonal tumor with multilayered rosettes (ETMR) is an aggressive central nervous system primitive neuroectodermal tumor (CNS-PNET) variant. ETMRs have distinctive histology, amplification of the chromosome 19 microRNA cluster (C19MC) at chr19q13.41-42, expression of the RNA binding protein Lin28, and dismal prognosis. Functional and therapeutic studies of ETMR have been limited by a lack of model systems. METHODS: We have established a first cell line, BT183, from a case of ETMR and characterized its molecular and cellular features. LIN28 knockdown was performed in BT183 to examine the potential role of Lin28 in regulating signaling pathway gene expression in ETMR. Cell line findings were corroborated with immunohistochemical studies in ETMR tissues. A drug screen of 73 compounds was performed to identify potential therapeutic targets. RESULTS: The BT183 line maintains C19MC amplification, expresses C19MC-encoded microRNAs, and is tumor initiating. ETMRs, including BT183, have high LIN28 expression and low let-7 miRNA expression, and show evidence of mTOR pathway activation. LIN28 knockdown increases let-7 expression and decreases expression of IGF/PI3K/mTOR pathway components. Pharmacologic inhibition of the mTOR pathway reduces BT183 cell viability. CONCLUSIONS: BT183 retains key genetic and histologic features of ETMR. In ETMR, Lin28 is not only a diagnostic marker but also a regulator of genes involved in growth and metabolism. Our findings indicate that inhibitors of the IGF/PI3K/mTOR pathway may be promising novel therapies for these fatal embryonal tumors. As the first patient-derived cell line of these rare tumors, BT183 is an important, unique reagent for investigating ETMR biology and therapeutics.
Assuntos
Cromossomos Humanos Par 19/genética , Amplificação de Genes , MicroRNAs/metabolismo , Neoplasias Embrionárias de Células Germinativas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Técnicas de Cultura de Células , Pré-Escolar , Variações do Número de Cópias de DNA , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Técnicas Imunoenzimáticas , Hibridização in Situ Fluorescente , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Glioblastoma multiforme (GBM) is characterized by an aggressive clinical course, therapeutic resistance, and striking molecular heterogeneity. GBM-derived brain tumor stem cells (BTSCs) closely model this molecular heterogeneity and likely have a key role in tumor recurrence and therapeutic resistance. Emerging evidence indicates that Janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 is an important mediator of tumor cell survival, growth, and invasion in a large group of GBM. Here, we used a large set of molecularly heterogeneous BTSCs to evaluate the translational potential of JAK2/STAT3 therapeutics. METHODS: BTSCs were cultured from GBM patients and MGMT promoter methylation, and the mutation statuses of EGFR, PTEN, and TP53 were determined. Endogenous JAK2/STAT3 activity was assessed in human GBM tissue, BTSCs, and orthotopic xenografts by immunohistochemistry and Western blotting. STAT3 short hairpin (sh)RNA, cucurbitacin-I, and WP1066 were used to inhibit JAK2/STAT3 activity in vitro and in vivo. RESULTS: The JAK2/STAT3 pathway was demonstrated to be highly activated in human GBM, molecularly heterogeneous BTSCs derived from these tumors, and BTSC xenografts. STAT3 shRNA knockdown or cucurbitacin-I and WP1066 administration resulted in on-target JAK2/STAT3 inhibition and dramatically reduced BTSC survival regardless of endogenous MGMT promoter methylation or EGFR, PTEN, and TP53 mutational status. BTSC orthotopic xenografts maintained the high levels of activated JAK2/STAT3 seen in their parent human tumors. Intraperitoneal WP1066 reduced intratumoral JAK2/STAT3 activity and prolonged animal survival. CONCLUSION: Our study demonstrates the in vitro and in vivo efficacy of on-target JAK2/STAT3 inhibition in heterogeneous BTSC lines that closely emulate the genomic and tumorigenic characteristics of human GBM.
Assuntos
Neoplasias Encefálicas/prevenção & controle , Glioblastoma/prevenção & controle , Janus Quinase 2/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT3/metabolismo , Idoso , Animais , Apoptose , Western Blotting , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células , Metilação de DNA , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Progressão da Doença , Receptores ErbB/metabolismo , Feminino , Citometria de Fluxo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Técnicas Imunoenzimáticas , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/genética , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Regiões Promotoras Genéticas/genética , Piridinas/farmacologia , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais , Triterpenos/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Tirfostinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oligodendroglioma is characterized by mutations of IDH and CIC, 1p/19q loss, and slow growth. We found that NHE-1 on 1p is silenced in oligodendrogliomas secondary to IDH-associated hypermethylation and 1p allelic loss. Silencing lowers intracellular pH and attenuates acid load recovery in oligodendroglioma cells. Others have shown that rapid tumor growth cannot occur without NHE-1-mediated neutralization of the acidosis generated by the Warburg glycolytic shift. Our findings show for the first time that the pH regulator NHE-1 can be silenced in a human cancer and also suggest that pH deregulation may contribute to the distinctive biology of human oligodendroglioma.
Assuntos
Neoplasias Encefálicas/genética , Deleção Cromossômica , Cromossomos Humanos Par 1/genética , Metilação de DNA , Mutação/genética , Oligodendroglioma/genética , Trocadores de Sódio-Hidrogênio/genética , Neoplasias Encefálicas/patologia , Humanos , Oligodendroglioma/patologia , Prótons , Células Tumorais CultivadasRESUMO
Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co-deletion to identify new molecular features at base-pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non-synonymous somatic mutations in 20 CIC-mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA-interacting domain and three were in exon 20 within an annotated protein-interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co-deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild-type tumours, in a background of 1p/19q co-deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co-deletion.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Isocitrato Desidrogenase/genética , Oligodendroglioma/genética , Proteínas Repressoras/genética , Biomarcadores Tumorais/análise , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 19/genética , Intervalo Livre de Doença , Humanos , Estimativa de Kaplan-Meier , Mutação , Gradação de Tumores , Oligodendroglioma/mortalidade , Oligodendroglioma/patologiaRESUMO
Somatic mutations in the catalytic domain of isocitrate dehydrogenase (IDH) 1/2 and accumulation of the oncometabolite 2-hydroxyglutarate (2-HG) appear to be among the earliest events in gliomagenesis and may contribute to malignant transformation. The lack of cell lines with endogenous mutations has been one of the major challenges in studying IDH1/2-mutant glioma and developing novel therapeutics for these tumors. Here, we describe the isolation of a glioma brain tumor stem cell line (BT142) with an endogenous R132H mutation in IDH1, aggressive tumor-initiating capacity, and 2-HG production. The neurosphere culture method was used to establish a brain tumor stem cell line from an IDH1-mutant anaplastic oligoastrocytoma sample, and an orthotopic xenograft system was developed to allow its rapid expansion. Production of 2-HG by glioma cells with endogenous IDH1 mutations was confirmed by mass spectrometry. BT142 retained an endogenous R132H IDH1 mutation in culture and possessed aggressive tumor-initiating capacity, allowing it to be readily propagated in orthotopic xenografts of nonobese diabetic/severe combined immune deficiency (NOD SCID) mice. Endogenous 2-HG production by BT142 was detectable in both cell culture medium and xenograft animal serum. BT142 is the first brain tumor cell line with an endogenous IDH1 mutation and detectable 2-HG production both in vitro and in vivo, which thus provides a unique model for studying the biology of IDH1-mutant glioma and in vivo validation of compounds targeting IDH1-mutant cells.
Assuntos
Neoplasias Encefálicas/genética , Glioma/genética , Isocitrato Desidrogenase/genética , Mutação Puntual/genética , Adulto , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Glioma/metabolismo , Glioma/patologia , Humanos , Isocitrato Desidrogenase/metabolismo , Masculino , Camundongos , Camundongos SCID , Modelos BiológicosRESUMO
The most effective chemotherapeutic for glioblastoma (GBM) is the DNA alkylating agent temozolomide (TMZ). In a recent study by Hegi et al. benefit from TMZ was significantly associated with methylation of the promoter of the O6-methylguanine-DNA methyltransferase (MGMT) gene; however, the correlation was imperfect. Some patients with methylated tumors were short survivors and others with unmethylated tumors were long survivors. These exceptions have raised the possibility that TMZ response might be influenced by non-MGMT mechanisms. The effect of p53 status on response to TMZ was explored in traditional glioma cell lines (U87MG, U251MG, U343MG, U373MG, SF767, LN443 and LNZ308) and brain tumor initiating cells (BTICs--BT012, BT025, BT042, BT048, BT060 and BT069) in two ways: (1) inhibition of p53 by RNAi and (2) sensitivity in relation to intrinsic p53 status, either wild-type or mutant. Traditional glioma cell lines that did not express a functional p53 were significantly more sensitive to TMZ than cell lines with functionally intact wild-type p53 expression. Altered p53 expression or function had only minor effects on TMZ sensitivity in BTICs and tended to decrease sensitivity to TMZ. RNAi specific for p53 had little effect on sensitivity in p53 null glioma cells. Absence of a functional p53 increases TMZ sensitivity in traditional glioma cell lines, an effect that is independent of MGMT status, and not seen in BTICs. P53 status may influence response to TMZ in differentiated cells in a GBM with a negligible affect on its initiating cells.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Glioma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Western Blotting , Neoplasias Encefálicas/metabolismo , Metilação de DNA , Dacarbazina/farmacologia , Glioma/metabolismo , Humanos , Mutação/genética , Células-Tronco Neoplásicas/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Temozolomida , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genéticaRESUMO
Investigating the biology of oligodendroglioma and its characteristic combined deletion of chromosomal arms 1p and 19q, mediated by an unbalanced translocation, t(1;19)(q10;p10), has been hampered by the lack of cell lines that harbor these traits. We grew cells from 2 anaplastic oligodendrogliomas in serum-free conditions. Serial propagation and expansion led to the establishment of permanent cell lines that maintained the genetic signature of the parent oligodendrogliomas and displayed features of brain tumor stem cells in vitro. One line was established from a treatment-naïve tumor and the other from a temozolomide resistant recurrent tumor. These lines may be important tools for understanding the biology of oligodendrogliomas and the function of their defining genetic traits.