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Glioblastoma is universally fatal and characterized by frequent chromosomal copy number alterations harboring oncogenes and tumor suppressors. In this study, we analyzed exome-wide human glioblastoma copy number data and found that cytoband 6q27 is an independent poor prognostic marker in multiple data sets. We then combined CRISPR-Cas9 data, human spatial transcriptomic data, and human and mouse RNA sequencing data to nominate PDE10A as a potential haploinsufficient tumor suppressor in the 6q27 region. Mouse glioblastoma modeling using the RCAS/tv-a system confirmed that Pde10a suppression induced an aggressive glioma phenotype in vivo and resistance to temozolomide and radiation therapy in vitro. Cell culture analysis showed that decreased Pde10a expression led to increased PI3K/AKT signaling in a Pten-independent manner, a response blocked by selective PI3K inhibitors. Single-nucleus RNA sequencing from our mouse gliomas in vivo, in combination with cell culture validation, further showed that Pde10a suppression was associated with a proneural-to-mesenchymal transition that exhibited increased cell adhesion and decreased cell migration. Our results indicate that glioblastoma patients harboring PDE10A loss have worse outcomes and potentially increased sensitivity to PI3K inhibition.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Humanos , Animais , Camundongos , Glioblastoma/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Haploinsuficiência , Glioma/genética , PTEN Fosfo-Hidrolase/genética , Diester Fosfórico Hidrolases/genética , Linhagem Celular Tumoral , Neoplasias Encefálicas/genéticaRESUMO
YAP1 is a transcriptional coactivator regulated by the Hippo signaling pathway, including NF2. Meningiomas are the most common primary brain tumors; a large percentage exhibit heterozygous loss of chromosome 22 (harboring the NF2 gene) and functional inactivation of the remaining NF2 copy, implicating oncogenic YAP activity in these tumors. Recently, fusions between YAP1 and MAML2 have been identified in a subset of pediatric NF2 wild-type meningiomas. Here, we show that human YAP1-MAML2-positive meningiomas resemble NF2 mutant meningiomas by global and YAP-related gene expression signatures. We then show that expression of YAP1-MAML2 in mice induces tumors that resemble human YAP1 fusion-positive and NF2 mutant meningiomas by gene expression. We demonstrate that YAP1-MAML2 primarily functions by exerting TEAD-dependent YAP activity that is resistant to Hippo signaling. Treatment with YAP-TEAD inhibitors is sufficient to inhibit the viability of YAP1-MAML2-driven mouse tumors ex vivo. Finally, we show that expression of constitutively active YAP1 (S127/397A-YAP1) is sufficient to induce similar tumors, suggesting that the YAP component of the gene fusion is the critical driver of these tumors. In summary, our results implicate YAP1-MAML2 as a causal oncogenic driver and highlight TEAD-dependent YAP activity as an oncogenic driver in YAP1-MAML2 fusion meningioma as well as NF2 mutant meningioma in general.
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YAP1 is a transcriptional coactivator and the principal effector of the Hippo signaling pathway, which is causally implicated in human cancer. Several YAP1 gene fusions have been identified in various human cancers and identifying the essential components of this family of gene fusions has significant therapeutic value. Here, we show that the YAP1 gene fusions YAP1-MAMLD1, YAP1-FAM118B, YAP1-TFE3, and YAP1-SS18 are oncogenic in mice. Using reporter assays, RNA-seq, ChIP-seq, and loss-of-function mutations, we can show that all of these YAP1 fusion proteins exert TEAD-dependent YAP activity, while some also exert activity of the C'-terminal fusion partner. The YAP activity of the different YAP1 fusions is resistant to negative Hippo pathway regulation due to constitutive nuclear localization and resistance to degradation of the YAP1 fusion proteins. Genetic disruption of the TEAD-binding domain of these oncogenic YAP1 fusions is sufficient to inhibit tumor formation in vivo, while pharmacological inhibition of the YAP1-TEAD interaction inhibits the growth of YAP1 fusion-expressing cell lines in vitro. These results highlight TEAD-dependent YAP activity found in these gene fusions as critical for oncogenesis and implicate these YAP functions as potential therapeutic targets in YAP1 fusion-positive tumors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Carcinogênese/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Sinais de Localização Nuclear , Motivos de Nucleotídeos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes.
Assuntos
Separação de Fases , Transcriptoma , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , OncogenesRESUMO
Glioblastoma is the most frequently occurring and invariably fatal primary brain tumor in adults. The vast majority of glioblastomas is characterized by chromosomal copy number alterations, including gain of whole chromosome 7 and loss of whole chromosome 10. Gain of whole chromosome 7 is an early event in gliomagenesis that occurs in proneural-like precursor cells, which give rise to all isocitrate dehydrogenase (IDH) wild-type glioblastoma transcriptional subtypes. Platelet-derived growth factor A (PDGFA) is one gene on chromosome 7 known to drive gliomagenesis, but, given its location near the end of 7p, there are likely several other genes located along chromosome 7 that select for its increased whole-chromosome copy number within glioblastoma cells. To identify other potential genes that could select for gain of whole chromosome 7, we developed an unbiased bioinformatics approach that identified homeobox A5 (HOXA5) as a gene whose expression correlated with gain of chromosome 7 and a more aggressive phenotype of the resulting glioma. High expression of HOXA5 in glioblastoma was associated with a proneural gene expression pattern and decreased overall survival in both human proneural and PDGF-driven mouse glioblastoma. Furthermore, HOXA5 overexpression promoted cellular proliferation and potentiated radioresistance. We also found enrichment of HOXA5 expression in recurrent human and mouse glioblastoma at first recurrence after radiotherapy. Overall, this study implicates HOXA5 as a chromosome 7-associated gene-level locus that promotes selection for gain of whole chromosome 7 and an aggressive phenotype in glioblastoma.
Assuntos
Neoplasias Encefálicas/genética , Cromossomos Humanos Par 7 , Glioblastoma/genética , Proteínas de Homeodomínio/metabolismo , Fosfoproteínas/metabolismo , Animais , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Proliferação de Células , Duplicação Cromossômica , Glioblastoma/mortalidade , Glioblastoma/patologia , Glioblastoma/radioterapia , Proteínas de Homeodomínio/genética , Humanos , Isocitrato Desidrogenase/genética , Camundongos , Recidiva Local de Neoplasia , Fosfoproteínas/genética , Tolerância a Radiação , Fatores de TranscriçãoRESUMO
In a recent issue of The Journal of Pathology, Chen and colleagues established novel patient-derived ex vivo models of NTRK fusion-positive soft tissue sarcoma to characterize resistance mechanisms against targeted therapy with tyrosine kinase inhibitors. Prolonged exposure to escalating concentrations of the tyrosine kinase inhibitor, entrectinib, ultimately led to the occurrence of resistant clones that harbored an inactivating mutation in the NF2 gene, not previously described in this context, accompanied by increased PI3K/AKT/mTOR and Ras/Raf/MEK/ERK signaling. Finally, an inhibitor screen identified, among others, MEK and mTOR inhibitors as potential combination agents. © 2024 The Pathological Society of Great Britain and Ireland.
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Gliomas harboring mutations in isocitrate dehydrogenase 1/2 (IDH1/2) have the CpG island methylator phenotype (CIMP) and significantly longer patient survival time than wild-type IDH1/2 (wtIDH1/2) tumors. Although there are many factors underlying the differences in survival between these two tumor types, immune-related differences in cell content are potentially important contributors. In order to investigate the role of IDH mutations in immune response, we created a syngeneic pair mouse model for mutant IDH1 (muIDH1) and wtIDH1 gliomas and demonstrated that muIDH1 mice showed many molecular and clinical similarities to muIDH1 human gliomas, including a 100-fold higher concentration of 2-hydroxygluratate (2-HG), longer survival time, and higher CpG methylation compared with wtIDH1. Also, we showed that IDH1 mutations caused down-regulation of leukocyte chemotaxis, resulting in repression of the tumor-associated immune system. Given that significant infiltration of immune cells such as macrophages, microglia, monocytes, and neutrophils is linked to poor prognosis in many cancer types, these reduced immune infiltrates in muIDH1 glioma tumors may contribute in part to the differences in aggressiveness of the two glioma types.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Glioma/genética , Glioma/imunologia , Sistema Imunitário/fisiopatologia , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Animais , Neoplasias Encefálicas/enzimologia , Quimiotaxia/genética , Metilação de DNA , Modelos Animais de Doenças , Glioma/enzimologia , Humanos , Antígenos Comuns de Leucócito/metabolismo , Leucócitos/patologia , Camundongos , Mutação , Infiltração de Neutrófilos/genética , Neutrófilos/patologiaRESUMO
YAP1 is a transcriptional co-activator whose activity is controlled by the Hippo signaling pathway. In addition to important functions in normal tissue homeostasis and regeneration, YAP1 has also prominent functions in cancer initiation, aggressiveness, metastasis, and therapy resistance. In this review we are discussing the molecular functions of YAP1 and its roles in cancer, with a focus on the different mechanisms of de-regulation of YAP1 activity in human cancers, including inactivation of upstream Hippo pathway tumor suppressors, regulation by intersecting pathways, miRNAs, and viral oncogenes. We are also discussing new findings on the function and biology of the recently identified family of YAP1 gene fusions, that constitute a new type of activating mutation of YAP1 and that are the likely oncogenic drivers in several subtypes of human cancers. Lastly, we also discuss different strategies of therapeutic inhibition of YAP1 functions.
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Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias/genética , Proteínas de Fusão Oncogênica/genética , Oncogenes/genética , Fosfoproteínas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
Paediatric high-grade gliomas (HGGs) account for the most brain tumour-related deaths in children and have a median survival of 12-15 months. One promising avenue of research is the development of novel therapies targeting the properties of non-neoplastic cell-types within the tumour such as tumour associated macrophages (TAMs). TAMs are immunosuppressive and promote tumour malignancy in adult HGG; however, in paediatric medulloblastoma, TAMs exhibit anti-tumour properties. Much is known about TAMs in adult HGG, yet little is known about them in the paediatric setting. This raises the question of whether paediatric HGGs possess a distinct constituency of TAMs because of their unique genetic landscapes. Using human paediatric HGG tissue samples and murine models of paediatric HGG, we demonstrate diffuse midline gliomas possess a greater inflammatory gene expression profile compared to hemispheric paediatric HGGs. We also show despite possessing sparse T-cell infiltration, human paediatric HGGs possess high infiltration of IBA1+ TAMs. CD31, PDGFRß, and PDGFB all strongly correlate with IBA1+ TAM infiltration. To investigate the TAM population, we used the RCAS/tv-a system to recapitulate paediatric HGG in newborn immunocompetent mice. Tumours are induced in Nestin-positive brain cells by PDGFA or PDGFB overexpression with Cdkn2a or Tp53 co-mutations. Tumours driven by PDGFB have a significantly lower median survival compared to PDGFA-driven tumours and have increased TAM infiltration. NanoString and quantitative PCR analysis indicates PDGFB-driven tumours have a highly inflammatory microenvironment characterized by high chemokine expression. In vitro bone marrow-derived monocyte and microglial cultures demonstrate bone marrow-derived monocytes are most responsible for the production of inflammatory signals in the tumour microenvironment in response to PDGFB stimulation. Lastly, using knockout mice deficient for individual chemokines, we demonstrate the feasibility of reducing TAM infiltration and prolonging survival in both PDGFA and PDGFB-driven tumours. We identify CCL3 as a potential key chemokine in these processes in both humans and mice. Together, these studies provide evidence for the potent inflammatory effects PDGFB has in paediatric HGGs.
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Neoplasias Encefálicas/imunologia , Encefalite/imunologia , Proteínas Proto-Oncogênicas c-sis/imunologia , Macrófagos Associados a Tumor/imunologia , Adolescente , Adulto , Animais , Neoplasias Encefálicas/genética , Células Cultivadas , Quimiocinas/genética , Criança , Pré-Escolar , Encefalite/genética , Feminino , Glioma , Humanos , Lactente , Recém-Nascido , Masculino , Camundongos Endogâmicos C57BL , Transcriptoma , Adulto JovemRESUMO
Gliomas are the most common primary intrinsic brain tumors occurring in adults. Of all malignant gliomas, glioblastoma (GBM) is considered the deadliest tumor type due to diffuse brain invasion, immune evasion, cellular, and molecular heterogeneity, and resistance to treatments resulting in high rates of recurrence. An extensive understanding of the genomic and microenvironmental landscape of gliomas gathered over the past decade has renewed interest in pursuing novel therapeutics, including immune checkpoint inhibitors, glioma-associated macrophage/microglia (GAMs) modulators, and others. In light of this, predictive animal models that closely recreate the conditions and findings found in human gliomas will serve an increasingly important role in identifying new, effective therapeutic strategies. Although numerous syngeneic, xenograft, and transgenic rodent models have been developed, few include the full complement of pathobiological features found in human tumors, and therefore few accurately predict bench-to-bedside success. This review provides an update on how genetically engineered rodent models based on the replication-competent avian-like sarcoma (RCAS) virus/tumor virus receptor-A (tv-a) system have been used to recapitulate key elements of human gliomas in an immunologically intact host microenvironment and highlights new approaches using this model system as a predictive tool for advancing translational glioma research.
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Neoplasias Encefálicas , Modelos Animais de Doenças , Glioma , Sarcoma , Animais , Vírus do Sarcoma Aviário/genética , Neoplasias Encefálicas/patologia , Glioma/patologia , Humanos , Vírus Oncogênicos , Receptores Virais , Microambiente TumoralRESUMO
Radiation therapy is part of the standard of care for gliomas and kills a subset of tumor cells, while also altering the tumor microenvironment. Tumor cells with stem-like properties preferentially survive radiation and give rise to glioma recurrence. Various techniques for enriching and quantifying cells with stem-like properties have been used, including the fluorescence activated cell sorting (FACS)-based side population (SP) assay, which is a functional assay that enriches for stem-like tumor cells. In these analyses, mouse models of glioma have been used to understand the biology of this disease and therapeutic responses, including the radiation response. We present combined SP analysis and single-cell RNA sequencing of genetically-engineered mouse models of glioma to show a time course of cellular response to radiation. We identify and characterize two distinct tumor cell populations that are inherently radioresistant and also distinct effects of radiation on immune cell populations within the tumor microenvironment.
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Neoplasias Encefálicas , Glioma , Células-Tronco , Animais , Neoplasias Encefálicas/radioterapia , Camundongos , Células-Tronco Neoplásicas , Análise de Célula Única , Microambiente TumoralRESUMO
Glioblastoma (GBM) is the most aggressive primary brain tumor. In addition to being genetically heterogeneous, GBMs are also immunologically heterogeneous. However, whether the differences in immune microenvironment are driven by genetic driver mutation is unexplored. By leveraging the versatile RCAS/tv-a somatic gene transfer system, we establish a mouse model for Classical GBM by introducing EGFRvIII expression in Nestin-positive neural stem/progenitor cells in adult mice. Along with our previously published Nf1-silenced and PDGFB-overexpressing models, we investigate the immune microenvironments of the three models of human GBM subtypes by unbiased multiplex profiling. We demonstrate that both the quantity and composition of the microenvironmental myeloid cells are dictated by the genetic driver mutations, closely mimicking what was observed in human GBM subtypes. These myeloid cells express high levels of the immune checkpoint protein PD-L1; however, PD-L1 targeted therapies alone or in combination with irradiation are unable to increase the survival time of tumor-bearing mice regardless of the driver mutations, reflecting the outcomes of recent human trials. Together, these results highlight the critical utility of immunocompetent mouse models for preclinical studies of GBM, making these models indispensable tools for understanding the resistance mechanisms of immune checkpoint blockade in GBM and immune cell-targeting drug discovery.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/imunologia , Glioblastoma/genética , Glioblastoma/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Mutação/fisiologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Tumorais CultivadasRESUMO
Glioblastoma is the most common and uncompromising primary brain tumour and is characterized by a dismal prognosis despite aggressive treatment regimens. At the cellular level, these tumours are composed of a mixture of neoplastic cells and non-neoplastic cells, including tumour-associated macrophages and endothelial cells. Cerebral oedema is a near-universal occurrence in patients afflicted with glioblastoma and it is almost exclusively managed with the corticosteroid dexamethasone despite significant drawbacks associated with its use. Here, we demonstrate that dexamethasone blocks interleukin-1 production in both bone marrow-derived and brain resident macrophage populations following stimulation with lipopolysaccharide and interferon gamma. Additionally, dexamethasone is shown to inhibit downstream effectors of interleukin-1 signalling in both macrophage populations. Co-culture of bone marrow-derived macrophages with organotypic tumour slices results in an upregulation of interleukin-1 cytokines, an effect that is absent in co-cultured microglia. Genetic ablation of interleukin-1 ligands or receptor in mice bearing RCAS/tv-a-induced platelet-derived growth factor B-overexpressing glioblastoma results in reduced oedema and partial restoration of the integrity of the blood-brain barrier, respectively; similar to results obtained with vascular endothelial growth factor neutralization. We establish that tumours from dexamethasone-treated mice exhibit reduced infiltration of cells of the myeloid and lymphoid compartments, an effect that should be considered during clinical trials for immunotherapy in glioblastoma patients. Additionally, we emphasize that caution should be used when immune profiling and single-cell RNA sequencing data are interpreted from fresh glioblastoma patient samples, as nearly all patients receive dexamethasone after diagnosis. Collectively, this evidence suggests that interleukin-1 signalling inhibition and dexamethasone treatment share therapeutic efficacies and establishes interleukin-1 signalling as an attractive and specific therapeutic target for the management of glioblastoma-associated cerebral oedema.
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Edema Encefálico/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Interleucina-1/metabolismo , Macrófagos/metabolismo , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Edema Encefálico/etiologia , Edema Encefálico/patologia , Neoplasias Encefálicas/complicações , Neoplasias Encefálicas/patologia , Dexametasona/farmacologia , Glioblastoma/complicações , Glioblastoma/patologia , Glucocorticoides/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Poststroke angiogenesis contributes to long-term recovery after stroke. Signal transducer and activator of transcription-3 (Stat3) is a key regulator for various inflammatory signals and angiogenesis. It was the aim of this study to determine its function in poststroke outcome. METHODS AND RESULTS: We generated a tamoxifen-inducible and endothelial-specific Stat3 knockout mouse model by crossbreeding Stat3(floxed/KO) and Tie2-Cre(ERT2) mice. Cerebral ischemia was induced by 30 minutes of middle cerebral artery occlusion. We demonstrated that endothelial Stat3 ablation did not alter lesion size 2 days after ischemia but did worsen functional outcome at 14 days and increase lesion size at 28 days. At this late time point vascular Stat3 expression and phosphorylation were still increased in wild-type mice. Gene array analysis of a CD31-enriched cell population of the neurovascular niche showed that endothelial Stat3 ablation led to a shift toward an antiangiogenic and axon growth-inhibiting micromilieu after stroke, with an increased expression of Adamts9. Remodeling and glycosylation of the extracellular matrix and microglia proliferation were increased, whereas angiogenesis was reduced. CONCLUSIONS: Endothelial Stat3 regulates angiogenesis, axon growth, and extracellular matrix remodeling and is essential for long-term recovery after stroke. It might serve as a potent target for stroke treatment after the acute phase by fostering angiogenesis and neuroregeneration.
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Endotélio Vascular/metabolismo , Infarto da Artéria Cerebral Média/fisiopatologia , Neovascularização Fisiológica/fisiologia , Plasticidade Neuronal/fisiologia , Fator de Transcrição STAT3/fisiologia , Proteínas ADAM/biossíntese , Proteínas ADAM/genética , Proteína ADAMTS9 , Animais , Axônios/fisiologia , Encéfalo/patologia , Microambiente Celular , Circulação Cerebrovascular , Convalescença , Proteínas da Matriz Extracelular/metabolismo , Perfilação da Expressão Gênica , Infarto da Artéria Cerebral Média/patologia , Camundongos , Camundongos Knockout , Microglia/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Processamento de Proteína Pós-Traducional , Recuperação de Função Fisiológica , Fator de Transcrição STAT3/deficiência , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologiaRESUMO
Glioblastoma (GBM) is the most aggressive brain tumor in adults. It is strongly infiltrated by microglia and peripheral monocytes that support tumor growth. In the present study we used RNA sequencing to compare the expression profile of CD11b(+) human glioblastoma-associated microglia/monocytes (hGAMs) to CD11b(+) microglia isolated from non-tumor samples. Hierarchical clustering and principal component analysis showed a clear separation of the two sample groups and we identified 334 significantly regulated genes in hGAMs. In comparison to human control microglia hGAMs upregulated genes associated with mitotic cell cycle, cell migration, cell adhesion, and extracellular matrix organization. We validated the expression of several genes associated with extracellular matrix organization in samples of human control microglia, hGAMs, and the hGAMs-depleted fraction via qPCR. The comparison to murine GAMs (mGAMs) showed that both cell populations share a significant fraction of upregulated transcripts compared with their respective controls. These genes were mostly related to mitotic cell cycle. However, in contrast to murine cells, human GAMs did not upregulate genes associated to immune activation. Comparison of human and murine GAMs expression data to several data sets of in vitro-activated human macrophages and murine microglia showed that, in contrast to mGAMs, hGAMs share a smaller overlap to these data sets in general and in particular to cells activated by proinflammatory stimulation with LPS + INFγ or TNFα. Our findings provide new insights into the biology of human glioblastoma-associated microglia/monocytes and give detailed information about the validity of murine experimental models. GLIA 2016 GLIA 2016;64:1416-1436.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Microglia/metabolismo , Monócitos/metabolismo , Animais , Antígeno CD11b/metabolismo , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA , TranscriptomaRESUMO
GPR34 is a Gi/o protein-coupled receptor (GPCR) of the nucleotide receptor P2Y12 -like group. This receptor is highly expressed in microglia, however, the functional relevance of GPR34 in these glial cells is unknown. Previous results suggested an impaired immune response in GPR34-deficient mice infected with Cryptococcus neoformans. Here we show that GPR34 deficiency results in morphological changes in retinal and cortical microglia. RNA sequencing analysis of microglia revealed a number of differentially expressed transcripts involved in cell motility and phagocytosis. We found no differences in microglial motility after entorhinal cortex lesion and in response to laser lesion. However, GPR34-deficient microglia showed reduced phagocytosis activity in both retina and acutely isolated cortical slices. Our study identifies GPR34 as an important signaling component controlling microglial function, morphology and phagocytosis.
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Regulação da Expressão Gênica/genética , Microglia/fisiologia , Fagocitose/genética , Receptores de Lisofosfolipídeos/deficiência , Animais , Encéfalo/citologia , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Receptor 1 de Quimiocina CX3C , Movimento Celular/genética , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Receptores de Lisofosfolipídeos/genética , Retina/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Recently, neurotransmitters/neurohormones have been identified as factors controlling the function of microglia, the immune competent cells of the central nervous system. In this study, we compared the responsiveness of microglia to neurotransmitters/neurohormones. We freshly isolated microglia from healthy adult C57Bl/6 mice and found that only a small fraction (1-20%) responded to the application of endothelin, histamine, substance P, serotonin, galanin, somatostatin, angiotensin II, vasopressin, neurotensin, dopamine, or nicotine. In cultured microglia from neonatal and adult mice, a similarly small population of cells responded to these neurotransmitters/neurohormones. To induce a proinflammatory phenotype, we applied lipopolysaccaride (LPS) or interferon-gamma (IFN-γ) to the cultures for 24 h. Several of the responding populations increased; however, there was no uniform pattern when comparing adult with neonatal microglia or LPS with IFN-γ treatment. IL-4 as an anti-inflammatory substance increased the histamine-, substance P-, and somatostatin-sensitive populations only in microglia from adult, but not in neonatal cells. We also found that the expression of different receptors was not strongly correlated, indicating that there are many different populations of microglia with a distinct set of receptors. Our results demonstrate that microglial cells are a heterogeneous population with respect to their sensitivity to neurotransmitters/neurohormones and that they are more responsive in defined activation states.
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Interferon gama/farmacologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurotransmissores/farmacologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Meningiomas, the most common primary brain tumors in adults, are often benign and curable by surgical resection. However, a subset is of higher grade, shows aggressive growth behavior as well as brain invasion, and often recurs even after several rounds of surgery. Increasing evidence suggests that tumor classification and grading primarily based on histopathology do not always accurately predict tumor aggressiveness and recurrence behavior. The underlying biology of aggressive treatment-resistant meningiomas and the impact of specific genetic aberrations present in these high-grade tumors is still only insufficiently understood. Therefore, an in-depth research into the biology of this tumor type is warranted. More recent studies based on large-scale molecular data such as whole exome/genome sequencing, DNA methylation sequencing, and RNA sequencing have provided new insights into the biology of meningiomas and have revealed new risk factors and prognostic subtypes. The most common genetic aberration in meningiomas is functional loss of NF2 and occurs in both low- and high-grade meningiomas, whereas NF2-wildtype meningiomas are enriched for recurrent mutations in TRAF7, KLF4, AKT1, PI3KCA, and SMO and are more frequently benign. Most meningioma mouse models are based on patient-derived xenografts and only recently have new genetically engineered mouse models of meningioma been developed that will aid in the systematic evaluation of specific mutations found in meningioma and their impact on tumor behavior. In this article, we review recent advances in the understanding of meningioma biology and classification and highlight the most common genetic mutations, as well as discuss new genetically engineered mouse models of meningioma.
Assuntos
Neoplasias Meníngeas , Meningioma , Adulto , Humanos , Animais , Camundongos , Meningioma/genética , Meningioma/patologia , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patologia , Neoplasias Meníngeas/cirurgia , Fator 4 Semelhante a Kruppel , Mutação , PrognósticoRESUMO
Hypertranscription is common in human cancers and predicts poor prognosis. However detection of hypertranscription is indirect, relying on accurately quantifying mRNA levels and estimating cell numbers. Previously, we introduced FFPE-CUTAC, a genome-wide method for mapping RNA Polymerase II (RNAPII) in formalin-fixed paraffin-embedded (FFPE) sections. Here we use FFPE-CUTAC to demonstrate genome-wide hypertranscription both in transgene-driven mouse gliomas and in assorted human tumors at active regulatory elements and replication-coupled histone genes with reduced mitochondrial DNA abundance. FFPE-CUTAC identified RNAPII-bound regulatory elements shared among diverse cancers and readily categorized human tumors despite using very small samples and low sequencing depths. Remarkably, RNAPII FFPE-CUTAC identified de novo and precisely mapped HER2 amplifications punctuated by likely selective sweeps including genes encoding direct positive regulators of RNAPII itself. Our results demonstrate that FFPE-CUTAC measurements of hypertranscription and classifications of tumors using small sections provides an affordable and sensitive genome-wide strategy for personalized medicine.
RESUMO
Meningiomas are the most common primary brain tumors in adults. Although generally benign, a subset of meningiomas is of higher grade, shows aggressive growth behavior and recurs even after multiple surgeries. Around half of all meningiomas harbor inactivating mutations in NF2. While benign low-grade NF2 mutant meningiomas exhibit few genetic events in addition to NF2 inactivation, aggressive high-grade NF2 mutant meningiomas frequently harbor a highly aberrant genome. We and others have previously shown that NF2 inactivation leads to YAP1 activation and that YAP1 acts as the pivotal oncogenic driver in benign NF2 mutant meningiomas. Using bulk and single-cell RNA-Seq data from a large cohort of human meningiomas, we show that aggressive NF2 mutant meningiomas harbor decreased levels YAP1 activity compared to their benign counterparts. Decreased expression levels of YAP target genes are significantly associated with an increased risk of recurrence. We then identify the increased expression of the YAP1 competitor VGLL4 as well as the YAP1 upstream regulators FAT3/4 as a potential mechanism for the downregulation of YAP activity in aggressive NF2 mutant meningiomas. High expression of these genes is significantly associated with an increased risk of recurrence. In vitro, overexpression of VGLL4 resulted in the downregulation of YAP activity in benign NF2 mutant meningioma cells, confirming the direct link between VGLL4 expression and decreased levels of YAP activity observed in aggressive NF2 mutant meningiomas. Our results shed new insight on the biology of benign and aggressive NF2 mutant meningiomas and may have important implications for the efficacy of therapies targeting oncogenic YAP1 activity in NF2 mutant meningiomas.