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This clinical trial evaluated whether whole exome sequencing (WES) and RNA sequencing (RNAseq) of paired normal and tumor tissues could be incorporated into a personalized treatment plan for newly diagnosed patients (<25 years of age) with diffuse intrinsic pontine glioma (DIPG). Additionally, whole genome sequencing (WGS) was compared to WES to determine if WGS would further inform treatment decisions, and whether circulating tumor DNA (ctDNA) could detect the H3K27M mutation to allow assessment of therapy response. Patients were selected across three Pacific Pediatric Neuro-Oncology Consortium member institutions between September 2014 and January 2016. WES and RNAseq were performed at diagnosis and recurrence when possible in a CLIA-certified laboratory. Patient-derived cell line development was attempted for each subject. Collection of blood for ctDNA was done prior to treatment and with each MRI. A specialized tumor board generated a treatment recommendation including up to four FDA-approved agents based upon the genomic alterations detected. A treatment plan was successfully issued within 21 business days from tissue collection for all 15 subjects, with 14 of the 15 subjects fulfilling the feasibility criteria. WGS results did not significantly deviate from WES-based therapy recommendations; however, WGS data provided further insight into tumor evolution and fidelity of patient-derived cell models. Detection of the H3F3A or HIST1H3B K27M (H3K27M) mutation using ctDNA was successful in 92% of H3K27M mutant cases. A personalized treatment recommendation for DIPG can be rendered within a multicenter setting using comprehensive next-generation sequencing technology in a clinically relevant timeframe.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Secuenciación del Exoma/métodos , Análisis de Secuencia de ARN/métodos , Secuenciación Completa del Genoma/métodos , Adolescente , Adulto , Neoplasias del Tronco Encefálico/genética , Niño , Preescolar , ADN Tumoral Circulante , Glioma Pontino Intrínseco Difuso/genética , Estudios de Factibilidad , Femenino , Histonas/genética , Humanos , Masculino , Terapia Molecular Dirigida/métodos , Proyectos Piloto , Medicina de Precisión , Adulto JovenRESUMEN
Balancing quiescence, self-renewal, and differentiation in adult stem cells is critical for tissue homeostasis. The underlying mechanisms, however, remain incompletely understood. Here we identify Fezf2 as a novel regulator of fate balance in adult zebrafish dorsal telencephalic neural stem cells (NSCs). Transgenic reporters show intermingled fezf2-GFP(hi) quiescent and fezf2-GFP(lo) proliferative NSCs. Constitutive or conditional impairment of fezf2 activity demonstrates its requirement for maintaining quiescence. Analyses of genetic chimeras reveal a dose-dependent role of fezf2 in NSC activation, suggesting that the difference in fezf2 levels directionally biases fate. Single NSC profiling coupled with genetic analysis further uncovers a fezf2-dependent gradient Notch activity that is high in quiescent and low in proliferative NSCs. Finally, fezf2-GFP(hi) quiescent and fezf2-GFP(lo) proliferative NSCs are observed in postnatal mouse hippocampus, suggesting possible evolutionary conservation. Our results support a model in which fezf2 heterogeneity patterns gradient Notch activity among neighbors that is critical to balance NSC fate.
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Células Madre Adultas/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Proteínas de Unión al ADN/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Células-Madre Neurales/metabolismo , Receptores Notch/metabolismo , Animales , Animales Modificados Genéticamente , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Transgénicos , Neurogénesis/fisiología , Pez CebraRESUMEN
MOTIVATION: Several cancer types consist of multiple genetically and phenotypically distinct subpopulations. The underlying mechanism for this intra-tumoral heterogeneity can be explained by the clonal evolution model, whereby growth advantageous mutations cause the expansion of cancer cell subclones. The recurrent phenotype of many cancers may be a consequence of these coexisting subpopulations responding unequally to therapies. Methods to computationally infer tumor evolution and subpopulation diversity are emerging and they hold the promise to improve the understanding of genetic and molecular determinants of recurrence. RESULTS: To address cellular subpopulation dynamics within human tumors, we developed a bioinformatic method, EXPANDS. It estimates the proportion of cells harboring specific mutations in a tumor. By modeling cellular frequencies as probability distributions, EXPANDS predicts mutations that accumulate in a cell before its clonal expansion. We assessed the performance of EXPANDS on one whole genome sequenced breast cancer and performed SP analyses on 118 glioblastoma multiforme samples obtained from TCGA. Our results inform about the extent of subclonal diversity in primary glioblastoma, subpopulation dynamics during recurrence and provide a set of candidate genes mutated in the most well-adapted subpopulations. In summary, EXPANDS predicts tumor purity and subclonal composition from sequencing data. AVAILABILITY AND IMPLEMENTATION: EXPANDS is available for download at http://code.google.com/p/expands (matlab version--used in this manuscript) and http://cran.r-project.org/web/packages/expands (R version).
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Frecuencia de los Genes , Glioblastoma/genética , Ploidias , Glioblastoma/patología , Humanos , Mutación , Neoplasias/genética , Probabilidad , RecurrenciaRESUMEN
Stem and progenitor cells are characterized by their ability to self-renew and produce differentiated progeny. A fine balance between these processes is achieved through controlled asymmetric divisions and is necessary to generate cellular diversity during development and to maintain adult tissue homeostasis. Disruption of this balance may result in premature depletion of the stem/progenitor cell pool, or abnormal growth. In many tissues, including the brain, dysregulated asymmetric divisions are associated with cancer. Whether there is a causal relationship between asymmetric cell division defects and cancer initiation is as yet not known. Here, we review the cellular and molecular mechanisms that regulate asymmetric cell divisions in the neural lineage and discuss the potential connections between this regulatory machinery and cancer.
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División Celular Asimétrica , Neoplasias/patología , Células Madre/patología , Animales , Homeostasis , Humanos , Neoplasias/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Células Madre/citología , Células Madre/metabolismoRESUMEN
Although malignant astrocytomas are a leading cause of cancer-related death in children, rational therapeutic strategies are lacking. We previously identified activating mutations of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) (BRAF(T1799A) encoding BRAF(V600E)) in association with homozygous cyclin-dependent kinase inhibitor 2A (CDKN2A, encoding p14ARF and p16Ink4a) deletions in pediatric infiltrative astrocytomas. Here we report that BRAF(V600E) expression in neural progenitors (NPs) is insufficient for tumorigenesis and increases NP cellular differentiation as well as apoptosis. In contrast, astrocytomas are readily generated from NPs with additional Ink4a-Arf deletion. The BRAF(V600E) inhibitor PLX4720 significantly increased survival of mice after intracranial transplant of genetically relevant murine or human astrocytoma cells. Moreover, combination therapy using PLX4720 plus the Cyclin-dependent kinase (CDK) 4/6-specific inhibitor PD0332991 further extended survival relative to either monotherapy. Our findings indicate a rational therapeutic strategy for treating a subset of pediatric astrocytomas with BRAF(V600E) mutation and CDKN2A deficiency.
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Astrocitoma/tratamiento farmacológico , Inhibidor p16 de la Quinasa Dependiente de Ciclina/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Apoptosis/efectos de los fármacos , Astrocitoma/genética , Astrocitoma/patología , Western Blotting , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Niño , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Inmunohistoquímica , Indoles/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Desnudos , Ratones SCID , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Piridinas/farmacología , Sulfonamidas/farmacologíaRESUMEN
Drosophila neuroblasts have served as a model to understand how the balance of stem cell self-renewal versus differentiation is achieved. Drosophila Numb protein regulates this process through its preferential segregation into the differentiating daughter cell. How Numb restricts the proliferation and self-renewal potentials of the recipient cell remains enigmatic. Here, we show that phosphorylation at conserved sites regulates the tumor suppressor activity of Numb. Enforced expression of a phospho-mimetic form of Numb (Numb-TS4D) or genetic manipulation that boosts phospho-Numb levels, attenuates endogenous Numb activity and causes ectopic neuroblast formation (ENF). This effect on neuroblast homeostasis occurs only in the type II neuroblast lineage. We identify Dronc caspase as a novel binding partner of Numb, and demonstrate that overexpression of Dronc suppresses the effects of Numb-TS4D in a non-apoptotic and possibly non-catalytic manner. Reduction of Dronc activity facilitates ENF induced by phospho-Numb. Our findings uncover a molecular mechanism that regulates Numb activity and suggest a novel role for Dronc caspase in regulating neural stem cell homeostasis.
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Caspasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Hormonas Juveniles/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis , Animales , Western Blotting , Caspasas/biosíntesis , Drosophila/fisiología , Proteínas de Drosophila/biosíntesis , Expresión Génica , Células HEK293 , Homeostasis , Humanos , Etiquetado Corte-Fin in Situ , Hormonas Juveniles/biosíntesis , Células-Madre Neurales/citología , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Low-grade glioma (LGG) is the most common brain tumor affecting pediatric patients (pLGG) and BRAF mutations constitute the most frequent genetic alterations. Within the spectrum of pLGGs, approximately 70%-80% of pediatric patients diagnosed with transforming pleomorphic xanthoastrocytoma (PXA) harbor the BRAF V600E mutation. However, the impact of glioma BRAF V600E cell regulation of tumor-infiltrating immune cells and their contribution to tumor progression remains unclear. Moreover, the efficacy of BRAF inhibitors in treating pLGGs is limited compared with their impact on BRAF-mutated melanoma. Here we report a novel immunocompetent RCAS-BRAF V600E murine glioma model. Pathological assessment indicates this model seems to be consistent with diffuse gliomas and morphological features of PXA. Our investigations revealed distinct immune cell signatures associated with increased trafficking and activation within the tumor microenvironment (TME). Intriguingly, immune system activation within the TME also generated a pronounced inflammatory response associated with dysfunctional CD8+ T cells, increased presence of immunosuppressive myeloid cells and regulatory T cells. Further, our data suggests tumor-induced inflammatory processes, such as cytokine storm. These findings suggest a complex interplay between tumor progression and the robust inflammatory response within the TME in preclinical BRAF V600E LGGs, which may significantly influence animal survival.
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Central nervous system (CNS) neoplasms are difficult to treat due to their sensitive location. Over the past two decades, the availability of patient tumor materials facilitated large scale genomic and epigenomic profiling studies, which have resulted in detailed insights into the molecular underpinnings of CNS tumorigenesis. Based on results from these studies, CNS tumors have high molecular and cellular intra-tumoral and inter-tumoral heterogeneity. CNS cancer models have yet to reflect the broad diversity of CNS tumors and patients and the lack of such faithful cancer models represents a major bottleneck to urgently needed innovations in CNS cancer treatment. Pediatric cancer model development is lagging behind adult tumor model development, which is why we focus this review on CNS tumors mutated for BRAFV600E which are more prevalent in the pediatric patient population. BRAFV600E-mutated CNS tumors exhibit high inter-tumoral heterogeneity, encompassing clinically and histopathological diverse tumor types. Moreover, BRAFV600E is the second most common alteration in pediatric low-grade CNS tumors, and low-grade tumors are notoriously difficult to recapitulate in vitro and in vivo. Although the mutation predominates in low-grade CNS tumors, when combined with other mutations, most commonly CDKN2A deletion, BRAFV600E-mutated CNS tumors are prone to develop high-grade features, and therefore BRAFV600E-mutated CNS are a paradigm for tumor progression. Here, we describe existing in vitro and in vivo models of BRAFV600E-mutated CNS tumors, including patient-derived cell lines, patient-derived xenografts, syngeneic models, and genetically engineered mouse models, along with their advantages and shortcomings. We discuss which research gaps each model might be best suited to answer, and identify those areas in model development that need to be strengthened further. We highlight areas of potential research focus that will lead to the heightened predictive capacity of preclinical studies, allow for appropriate validation, and ultimately improve the success of "bench to bedside" translational research.
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Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin ß1 and inhibits its downstream signaling, and Integrin ß1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin ß1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.
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Epitelio , Glicoproteínas , Integrina beta1 , Glándulas Mamarias Animales , Morfogénesis , Transducción de Señal , Animales , Movimiento Celular/genética , Polaridad Celular , Proliferación Celular , Regulación hacia Abajo , Células Epiteliales/metabolismo , Epitelio/crecimiento & desarrollo , Femenino , Regulación Neoplásica de la Expresión Génica , Glicoproteínas/metabolismo , Integrina beta1/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/metabolismo , Ratones Transgénicos , Modelos Biológicos , Unión ProteicaRESUMEN
Asymmetric cell divisions generate cellular diversity. In Drosophila, embryonic neuroblasts target cell fate determinants basally, rotate their spindles by 90 degrees to align with the apical-basal axis, and divide asymmetrically in a stem cell-like fashion. In this process, apically localized Bazooka recruits Inscuteable and other proteins to form an apical complex, which then specifies spindle orientation and basal localization of the cell fate determinants and their adapter proteins such as Miranda. Here we report that Miranda localization requires the unconventional myosin VI Jaguar (Jar). In jar null mutant embryos, Miranda is delocalized and the spindle is misoriented, but the Inscuteable crescent remains apical. Miranda directly binds to Jar, raising the possibility that Miranda and its associated proteins are translocated basally by this actin-based motor. Our studies demonstrate that a class VI myosin is necessary for basal protein targeting and spindle orientation in neuroblasts.
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Proteínas de Drosophila/fisiología , Drosophila melanogaster/embriología , Péptidos y Proteínas de Señalización Intracelular , Cadenas Pesadas de Miosina/fisiología , Neuronas/fisiología , Huso Acromático/fisiología , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , División Celular/fisiología , Movimiento Celular , Polaridad Celular , Proteínas del Citoesqueleto/metabolismo , Drosophila melanogaster/genética , Embrión no Mamífero/citología , Immunoblotting , Técnicas para Inmunoenzimas , Neuropéptidos , Interferencia de ARNRESUMEN
Glioblastoma multiforme (GBM) is one the most aggressive brain tumors due to the fast and invasive growth that is partly supported by the presence of extensive neovascularization. The matrix metalloproteinase MMP-2 has been associated with invasive and angiogenic properties in gliomas and is a marker of poor prognosis. Since MMP-2 is expressed in both tumor cells and endothelial cells in GBM, we generated genetically engineered MMP-2 knockout (MMP-2ko) GBM to examine the importance of the spatial expression of MMP-2 in tumor and/or normal host-derived cells. MMP-2-dependent effects appeared to be dose-dependent irrespective of its expression pattern. GBM completely devoid of MMP-2 exhibited markedly increased vascular density associated with vascular endothelial growth factor receptor 2 (VEGFR2) activation and enhanced vascular branching and sprouting. Surprisingly, despite the high vascular density, tumor cells were more prone to apoptosis, which led to prolonged survival of tumor-bearing mice, suggesting that the increased vascularity is not functional. Congruently, tumor vessels were poorly perfused, exhibited lower levels of VEGFR2, and did not undergo proper maturation because pericytes of MMP-2ko tumors were not activated and were less abundant. As a result of impaired and dysfunctional angiogenesis, MMP-2ko GBM became more invasive, predominantly by migrating along blood vessels into the brain parenchyma.
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Neoplasias Encefálicas/enzimología , Glioblastoma/enzimología , Metaloproteinasa 2 de la Matriz/metabolismo , Invasividad Neoplásica , Neovascularización Patológica/enzimología , Animales , Western Blotting , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/patología , Supervivencia Celular/fisiología , Citometría de Flujo , Glioblastoma/irrigación sanguínea , Glioblastoma/patología , Inmunohistoquímica , Hibridación in Situ , Metaloproteinasa 2 de la Matriz/genética , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells. METHODS: We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting. RESULTS: Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas (World Health Organization grade III) and glioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100 beta-v-erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins. CONCLUSION: microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioblastoma/genética , Glioblastoma/patología , MicroARNs/metabolismo , Neuronas/patología , Oligodendroglioma/genética , Oligodendroglioma/patología , Animales , Ciclo Celular/genética , Diferenciación Celular/genética , Regulación hacia Abajo , Expresión Génica , Humanos , Ratones , Células Madre Neoplásicas , Transfección , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
Oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to generate one OPC and one differentiating oligodendrocyte (OL) progeny. Loss of pro-mitotic proteoglycan and OPC marker NG2 in the OL progeny is the earliest immunophenotypic change of unknown mechanism that indicates differentiation commitment. Here, we report that expression of the mouse homolog of Drosophila tumor suppressor Lethal giant larvae 1 (Lgl1) is induced during OL differentiation. Lgl1 conditional knockout OPC progeny retain NG2 and show reduced OL differentiation, while undergoing more symmetric self-renewing divisions at the expense of asymmetric divisions. Moreover, Lgl1 and hemizygous Ink4a/Arf knockouts in OPC synergistically induce gliomagenesis. Time lapse and total internal reflection microscopy reveals a critical role for Lgl1 in NG2 endocytic routing and links aberrant NG2 recycling to failed differentiation. These data establish Lgl1 as a suppressor of gliomagenesis and positive regulator of asymmetric division and differentiation in the healthy and demyelinated murine brain.
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Glicoproteínas/metabolismo , Oligodendroglía/citología , Oligodendroglía/metabolismo , Proteoglicanos/metabolismo , Animales , División Celular Asimétrica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Técnica del Anticuerpo Fluorescente , Glicoproteínas/genética , Immunoblotting , Ratones , Monensina/farmacología , Oligodendroglía/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
Mice lacking the epidermal growth factor receptor (EGFR) develop an early postnatal degeneration of the frontal cortex and olfactory bulbs and show increased cortical astrocyte apoptosis. The poor health and early lethality of EGFR-/- mice prevented the analysis of mechanisms responsible for the neurodegeneration and function of the EGFR in the adult brain. Here, we show that postnatal EGFR-deficient neural stem cells are impaired in their self-renewal potential and lack clonal expansion capacity in vitro. Mice lacking the EGFR in the brain (EGFRΔbrain ) show low penetrance of cortical degeneration compared to EGFR-/- mice despite genetic recombination of the conditional allele. Adult EGFRΔ mice establish a proper blood-brain barrier and perform reactive astrogliosis in response to mechanical and infectious brain injury, but are more sensitive to Kainic acid-induced epileptic seizures. EGFR-deficient cortical astrocytes, but not midbrain astrocytes, have reduced expression of glutamate transporters Glt1 and Glast, and show reduced glutamate uptake in vitro, illustrating an excitotoxic mechanism to explain the hypersensitivity to Kainic acid and region-specific neurodegeneration observed in EGFR-deficient brains.
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Astrocitos/patología , Encéfalo/patología , Receptores ErbB/fisiología , Ácido Glutámico/metabolismo , Hipersensibilidad/complicaciones , Células-Madre Neurales/patología , Convulsiones/etiología , Animales , Astrocitos/metabolismo , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/metabolismo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Convulsiones/patologíaRESUMEN
Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity. For example, long-term memory in adult Drosophila requires Pumilio (Pum), an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis. Whether dendrite morphogenesis requires similar translational regulation is unknown. Here we report that nos and pum control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis.
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Dendritas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión al ARN/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila/genética , Proteínas Fluorescentes Verdes , Inmunohistoquímica , Larva/crecimiento & desarrollo , Larva/metabolismo , Proteínas Luminiscentes , Microscopía Fluorescente , MorfogénesisRESUMEN
Stem and progenitor cells are characterized by their abilities to self-renew and produce differentiated progeny. The balance between self-renewal and differentiation is achieved through control of cell division mode, which can be either asymmetric or symmetric. Failure to properly control cell division mode may result in premature depletion of the stem/progenitor cell pool or abnormal growth and impaired differentiation. In many tissues, including the brain, stem cells and progenitor cells undergo asymmetric cell division through the establishment of cell polarity. Cell polarity proteins are therefore potentially critical regulators of asymmetric cell division. Decrease or loss of asymmetric cell division can be associated with reduced differentiation common during aging or impaired remyelination as seen in demyelinating diseases. Progenitor-like glioma precursor cells show decreased asymmetric cell division rates and increased symmetric divisions, which suggests that asymmetric cell division suppresses brain tumor formation. Cancer stem cells, on the other hand, still undergo low rates of asymmetric cell division, which may provide them with a survival advantage during therapy. These findings led to the hypotheses that asymmetric cell divisions are not always tumor suppressive but can also be utilized to maintain a cancer stem cell population. Proper control of cell division mode is therefore not only deemed necessary to generate cellular diversity during development and to maintain adult tissue homeostasis but may also prevent disease and determine disease progression. Since brain cancer is most common in the adult and aging population, we review here the current knowledge on molecular mechanisms that regulate asymmetric cell divisions in the neural and oligodendroglial lineage during development and in the adult brain.
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División Celular Asimétrica/fisiología , Células Madre Neoplásicas/citología , Células-Madre Neurales/citología , Animales , HumanosRESUMEN
Cell death is essential for eliminating excess cells during development as well as removing damaged cells. While multiple conserved apoptosis pathways involving different cascades of caspases, which are cysteine proteases, have been identified, their regulation in the context of a developing organism is not very well understood. Expression of the Drosophila caspase-9 homolog, DRONC, can be induced by ecdysone, a steroid hormone, which induces metamorphosis. To elucidate the functional role of DRONC during metamorphosis and for cell death during development we have generated and analyzed two loss-of-function alleles of DRONC. We report that DRONC is required for developmentally induced neuroblast cell death and apoptosis in response to X irradiation. DRONC mutants show reduced pupariation even in the presence of high levels of ecdysone and impaired cell death of larval midgut. The levels of ecdysone-inducible transcripts such as E75A and Reaper (Rpr) are normal in the absence of DRONC, suggesting that DRONC acts downstream of these genes. In addition, Reaper and Grim, but not Hid induced apoptosis is sensitive to a reduction of DRONC levels. Our study places DRONC at a central point of convergence for multiple cell death pathways and for the ecdysone pathway regulating metamorphosis.
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Apoptosis/efectos de la radiación , Caspasas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Metamorfosis Biológica , Alelos , Animales , Animales Modificados Genéticamente , Caspasas/deficiencia , Caspasas/genética , Proteínas de Drosophila/deficiencia , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/efectos de la radiación , Ecdisona/farmacología , Regulación del Desarrollo de la Expresión Génica , Larva/efectos de los fármacos , Larva/genética , Larva/crecimiento & desarrollo , Larva/metabolismo , Metamorfosis Biológica/efectos de la radiación , Neuropéptidos/metabolismo , Pupa/genética , Pupa/crecimiento & desarrollo , Pupa/metabolismo , Factores de TiempoRESUMEN
Intratumor heterogeneity (ITH) drives neoplastic progression and therapeutic resistance. We used the bioinformatics tools 'expanding ploidy and allele frequency on nested subpopulations' (EXPANDS) and PyClone to detect clones that are present at a ≥10% frequency in 1,165 exome sequences from tumors in The Cancer Genome Atlas. 86% of tumors across 12 cancer types had at least two clones. ITH in the morphology of nuclei was associated with genetic ITH (Spearman's correlation coefficient, ρ = 0.24-0.41; P < 0.001). Mutation of a driver gene that typically appears in smaller clones was a survival risk factor (hazard ratio (HR) = 2.15, 95% confidence interval (CI): 1.71-2.69). The risk of mortality also increased when >2 clones coexisted in the same tumor sample (HR = 1.49, 95% CI: 1.20-1.87). In two independent data sets, copy-number alterations affecting either <25% or >75% of a tumor's genome predicted reduced risk (HR = 0.15, 95% CI: 0.08-0.29). Mortality risk also declined when >4 clones coexisted in the sample, suggesting a trade-off between the costs and benefits of genomic instability. ITH and genomic instability thus have the potential to be useful measures that can universally be applied to all cancers.
Asunto(s)
Biología Computacional , Heterogeneidad Genética , Neoplasias/genética , Forma del Núcleo Celular/genética , Células Clonales , Variaciones en el Número de Copia de ADN/genética , Frecuencia de los Genes , Inestabilidad Genómica/genética , Humanos , Mutación/genética , Neoplasias/mortalidad , Modelos de Riesgos ProporcionalesRESUMEN
Inhibitors of BRAFV600E kinase are currently under investigations in preclinical and clinical studies involving BRAFV600E glioma. Studies demonstrated clinical response to such individualized therapy in the majority of patients whereas in some patients tumors continue to grow despite treatment. To study resistance mechanisms, which include feedback activation of mitogen-activated protein kinase (MAPK) signaling in melanoma, we developed a luciferase-modified cell line (2341luc) from a BrafV600E mutant and Cdkn2a- deficient murine high-grade glioma, and analyzed its molecular responses to BRAFV600E- and MAPK kinase (MEK)-targeted inhibition. Immunocompetent, syngeneic FVB/N mice with intracranial grafts of 2341luc were tested for effects of BRAFV600E and MEK inhibitor treatments, with bioluminescence imaging up to 14-days after start of treatment and survival analysis as primary indicators of inhibitor activity. Intracranial injected tumor cells consistently generated high-grade glioma-like tumors in syngeneic mice. Intraperitoneal daily delivery of BRAFV600E inhibitor dabrafenib only transiently suppressed MAPK signaling, and rather increased Akt signaling and failed to extend survival for mice with intracranial 2341luc tumor. MEK inhibitor trametinib delivered by oral gavage daily suppressed MAPK pathway more effectively and had a more durable anti-growth effect than dabrafenib as well as a significant survival benefit. Compared with either agent alone, combined BRAFV600E and MEK inhibitor treatment was more effective in reducing tumor growth and extending animal subject survival, as corresponding to sustained MAPK pathway inhibition. Results derived from the 2341luc engraftment model application have clinical implications for the management of BRAFV600E glioma.