RESUMO
The sonic hedgehog subtype of medulloblastoma (SHH MB) is associated with treatment failure and poor outcome. Current strategies utilizing whole brain radiation therapy result in deleterious off-target effects on the normal developing childhood brain. Most conventional chemotherapies remain limited by ineffective blood-brain barrier (BBB) penetrance. These challenges signify an unmet need for drug carriers that can cross the BBB and deliver drugs to targeted sites with high drug-loading efficiency and long-term stability. We herein leverage the enhanced stability and targeting ability of engineered high-density lipoprotein-mimetic nanoparticles (eHNPs) to cross the BBB and deliver a SHH inhibitor effectively to the cancer stem-like cell population in SHH MB. Our microfluidic technology enabled highly reproducible production of multicomponent eHNPs incorporated with apolipoprotein A1, anti-CD15, and a SHH inhibitor (LDE225). We demonstrate the dual-targeted delivery and enhanced therapeutic effect of eHNP-A1-CD15-LDE225 via scavenger receptor class B type 1 (SR-B1) and CD15 on brain SHH MB cells in vitro, ex vivo, and in vivo. Moreover, we show that eHNP-A1 not only serves as a stable drug carrier, but also has a therapeutic effect itself through SR-B1-mediated intracellular cholesterol depletion in SHH MB cells. Through the facilitated and targeted cellular uptake of drugs and direct therapeutic role of this engineered biomimetic nanocarrier in SHH MB, our multifunctional nanoparticle provides intriguing therapeutic promise as an effective and potent nanomedicine for the treatment of SHH MB.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Cerebelares/tratamento farmacológico , Portadores de Fármacos , Meduloblastoma/tratamento farmacológico , Nanopartículas/química , Animais , Materiais Biomiméticos , Barreira Hematoencefálica , Linhagem Celular Tumoral , HDL-Colesterol , Feminino , Proteínas Hedgehog/antagonistas & inibidores , Humanos , Camundongos Transgênicos , Terapia de Alvo Molecular , Gravidez , TamoxifenoRESUMO
Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-ß signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.
Assuntos
Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/genética , Genoma Humano/genética , Variação Estrutural do Genoma/genética , Meduloblastoma/classificação , Meduloblastoma/genética , Proteínas de Transporte/genética , Neoplasias Cerebelares/metabolismo , Criança , Variações do Número de Cópias de DNA/genética , Duplicação Gênica/genética , Genes myc/genética , Genômica , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/metabolismo , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas de Fusão Oncogênica/genética , Proteínas/genética , RNA Longo não Codificante , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Translocação Genética/genéticaRESUMO
R loops are RNA-DNA hybrid containing structures involved in diverse cellular processes, including DNA double-strand break (DSB) repair. R loop homeostasis involving the formation and resolution of R loops is critical for DSB repair, and its dysregulation leads to genome instability. Here we show that the HELZ helicase promotes R loop resolution to facilitate DSB repair by homologous recombination (HR). HELZ depletion causes hypersensitivity to DSB-inducing agents, and HELZ localizes and binds to DSBs. HELZ depletion further leads to genomic instability in a R loop dependent manner and the accumulation of R loops globally and at DSBs. HELZ binds to R loops in response to DSBs and promotes their resolution, thereby facilitating HR to promote genome integrity. Our findings thus define a role for HELZ in promoting the resolution of R loops critical for DSB repair by HR.
RESUMO
Y-box binding protein 1 (YBX1 or YB1) is a therapeutically relevant oncoprotein capable of RNA and DNA binding and mediating protein-protein interactions that drive proliferation, stemness, and resistance to platinum-based therapies. Given our previously published findings, the potential for YB1-driven cisplatin resistance in medulloblastoma (MB), and the limited studies exploring YB1-DNA repair protein interactions, we chose to investigate the role of YB1 in mediating radiation resistance in MB. MB, the most common pediatric malignant brain tumor, is treated with surgical resection, cranio-spinal radiation, and platinum-based chemotherapy, and could potentially benefit from YB1 inhibition. The role of YB1 in the response of MB to ionizing radiation (IR) has not yet been studied but remains relevant for determining potential anti-tumor synergy of YB1 inhibition with standard radiation therapy. We have previously shown that YB1 drives proliferation of cerebellar granular neural precursor cells (CGNPs) and murine Sonic Hedgehog (SHH) group MB cells. While others have demonstrated a link between YB1 and homologous recombination protein binding, functional and therapeutic implications remain unclear, particularly following IR-induced damage. Here we show that depleting YB1 in both SHH and Group 3 MB results not only in reduced proliferation but also synergizes with radiation due to differential response dynamics. YB1 silencing through shRNA followed by IR drives a predominantly NHEJ-dependent repair mechanism, leading to faster γH2AX resolution, premature cell cycle re-entry, checkpoint bypass, reduced proliferation, and increased senescence. These findings show that depleting YB1 in combination with radiation sensitizes SHH and Group 3 MB cells to radiation.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Células-Tronco Neurais , Proteína 1 de Ligação a Y-Box , Animais , Humanos , Camundongos , Neoplasias Encefálicas/metabolismo , Proliferação de Células , Neoplasias Cerebelares/patologia , Dano ao DNA , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Células-Tronco Neurais/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismoRESUMO
Sonic hedgehog (Shh) signaling is critical during development and its aberration is common across the spectrum of human malignancies. In the cerebellum, excessive activity of the Shh signaling pathway is associated with the devastating pediatric brain tumor medulloblastoma. We previously demonstrated that exaggerated de novo lipid synthesis is a hallmark of Shh-driven medulloblastoma and that hedgehog signaling inactivates the Rb/E2F tumor suppressor complex to promote lipogenesis. Indeed, such Shh-mediated metabolic reprogramming fuels tumor progression, in an E2F1- and FASN-dependent manner. Here, we show that the nutrient sensor PPARγ is a key component of the Shh metabolic network, particularly its regulation of glycolysis. Our data show that in primary cerebellar granule neural precursors (CGNPs), proposed medulloblastoma cells-of-origin, Shh stimulation elicits a marked induction of PPARγ alongside major glycolytic markers. This is also documented in the actively proliferating Shh-responsive CGNPs in the developing cerebellum, and PPARγ expression is strikingly elevated in Shh-driven medulloblastoma in vivo. Importantly, pharmacological blockade of PPARγ and/or Rb inactivation inhibits CGNP proliferation, drives medulloblastoma cell death and extends survival of medulloblastoma-bearing animals in vivo. This coupling of mitogenic Shh signaling to a major nutrient sensor and metabolic transcriptional regulator define a novel mechanism through which Shh signaling engages the nutrient sensing machinery in brain cancer, controls the cell cycle, and regulates the glycolytic index. This also reveals a dominant role of Shh in the etiology of glucose metabolism in medulloblastoma and underscores the function of the Shh â E2F1 â PPARγ axis in altering substrate utilization patterns in brain cancers in favor of tumor growth. These findings emphasize the value of PPARγ downstream of Shh as a global therapeutic target in hedgehog-dependent and/or Rb-inactivated tumors.
Assuntos
Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Células-Tronco Neurais/metabolismo , PPAR gama/metabolismo , Anilidas/farmacologia , Animais , Animais Recém-Nascidos , Compostos Azo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Células Cultivadas , Neoplasias Cerebelares/diagnóstico por imagem , Neoplasias Cerebelares/patologia , Cerebelo/citologia , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/genética , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/genética , Hexoquinase/metabolismo , Humanos , Antígeno Ki-67/metabolismo , Meduloblastoma/diagnóstico por imagem , Meduloblastoma/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuropeptídeos/genética , Tomografia por Emissão de Pósitrons , Hormônios Tireóideos/metabolismo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Transdução Genética/métodos , Proteínas de Ligação a Hormônio da TireoideRESUMO
Development of the cerebellum occurs postnatally and is marked by a rapid proliferation of cerebellar granule neuron precursors (CGNPs). CGNPs are the cells of origin for SHH-driven medulloblastoma, the most common malignant brain tumor in children. Here, we investigated the role of ERK, JNK, and p38 mitogen-activated protein kinases in CGNP proliferation. We found high levels of p38α in proliferating CGNPs. Concomitantly, members of the p38 pathway, such as ASK1, MKK3 and ATF-2, were also elevated. Inhibition of the Shh pathway or CGNP proliferation blunts p38α levels, irrespective of Shh treatment. Strikingly, p38α levels were high in vivo in the external granule layer of the postnatal cerebellum, Shh-dependent mouse medulloblastomas and human medulloblastomas of the SHH subtype. Finally, knocking down p38α by short hairpin RNA-carrying lentiviruses as well as the pharmacologically inhibiting of its kinase activity caused a marked decrease in CGNP proliferation, underscoring its requirement for Shh-dependent proliferation in CGNPs. The inhibition of p38α also caused a decrease in Gli1 and N-myc transcript levels, consistent with reduced proliferation. These findings suggest p38 inhibition as a potential way to increase the efficacy of treatments available for malignancies associated with deregulated SHH signaling, such as basal cell carcinoma and medulloblastoma.
Assuntos
Proliferação de Células/fisiologia , Cerebelo/enzimologia , Células-Tronco Neurais/enzimologia , Neurônios/enzimologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Encéfalo/enzimologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Meduloblastoma/enzimologia , Camundongos , Camundongos Transgênicos , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Medulloblastoma is heterogeneous, being characterized by molecular subgroups that demonstrate distinct gene expression profiles. Activation of the WNT or SHH signaling pathway characterizes two of these molecular subgroups, the former associated with low-risk disease and the latter potentially targeted by novel SHH pathway inhibitors. This manuscript reports the validation of a novel diagnostic immunohistochemical method to distinguish SHH, WNT, and non-SHH/WNT tumors and details their associations with clinical, pathological and cytogenetic variables. A cohort (n = 235) of medulloblastomas from patients aged 0.4-52 years was studied for expression of four immunohistochemical markers: GAB1, ß-catenin, filamin A, and YAP1. Immunoreactivity (IR) for GAB1 characterizes only SHH tumors and nuclear IR for ß-catenin only WNT tumors. IRs for filamin A and YAP1 identify SHH and WNT tumors. SHH, WNT, and non-SHH/WNT tumors contributed 31, 14, and 55% to the series. All desmoplastic/nodular (D/N) medulloblastomas were SHH tumors, while most WNT tumors (94%) had a classic phenotype. Monosomy 6 was strongly associated with WNT tumors, while PTCH1 loss occurred almost exclusively among SHH tumors. MYC or MYCN amplification and chromosome 17 imbalance occurred predominantly among non-SHH/WNT tumors. Among patients aged 3-16 years and entered onto the SIOP PNET3 trial, outcome was significantly better for children with WNT tumors, when compared to SHH or non-SHH/WNT tumors, which showed similar survival curves. However, high-risk factors (M+ disease, LC/A pathology, MYC amplification) significantly influenced survival in both SHH and non-SHH/WNT groups. We describe a robust method for detecting SHH, WNT, and non-SHH/WNT molecular subgroups in formalin-fixed medulloblastoma samples. In corroborating other studies that indicate the value of combining clinical, pathological, and molecular variables in therapeutic stratification schemes for medulloblastoma, we also provide the first outcome data based on a clinical trial cohort and novel data on how molecular subgroups are distributed across the range of disease.
Assuntos
Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Proteínas Hedgehog/metabolismo , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteínas Wnt/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Biomarcadores Tumorais/metabolismo , Criança , Pré-Escolar , Proteínas Contráteis/metabolismo , Feminino , Filaminas , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Lactente , Estimativa de Kaplan-Meier , Masculino , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Fosfoproteínas/metabolismo , Estudos Retrospectivos , Transdução de Sinais/fisiologia , Fatores de Transcrição , Proteínas de Sinalização YAP , Adulto Jovem , beta Catenina/metabolismoRESUMO
BACKGROUND: Medulloblastoma (MB) is the most common malignant pediatric brain tumor that originates in the cerebellum and brainstem. Frequent somatic mutations and deregulated expression of epigenetic regulators in MB highlight the substantial role of epigenetic alterations. 5-hydroxymethylcytosine (5hmC) is a highly abundant cytosine modification in the developing cerebellum and is regulated by ten-eleven translocation (TET) enzymes. RESULTS: We investigate the alterations of 5hmC and TET enzymes in MB and their significance to cerebellar cancer formation. We show total abundance of 5hmC is reduced in MB, but identify significant enrichment of MB-specific 5hmC marks at regulatory regions of genes implicated in stem-like properties and Nanog-binding motifs. While TET1 and TET2 levels are high in MBs, only knockout of Tet1 in the smoothened (SmoA1) mouse model attenuates uncontrolled proliferation, leading to a favorable prognosis. The pharmacological Tet1 inhibition reduces cell viability and platelet-derived growth factor signaling pathway-associated genes. CONCLUSIONS: These results together suggest a potential key role of 5hmC and indicate an oncogenic nature for TET1 in MB tumorigenesis, suggesting it as a potential therapeutic target for MBs.
Assuntos
Suscetibilidade a Doenças , Meduloblastoma/etiologia , Meduloblastoma/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , Animais , Biomarcadores Tumorais , Biologia Computacional/métodos , Ilhas de CpG , Metilação de DNA , Bases de Dados de Ácidos Nucleicos , Modelos Animais de Doenças , Progressão da Doença , Epigênese Genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/mortalidade , Meduloblastoma/patologia , Camundongos , Camundongos Transgênicos , Motivos de Nucleotídeos , PrognósticoRESUMO
Cerebellar development is a highly regulated process involving numerous factors acting with high specificity, both temporally and by location. Part of this process involves extensive proliferation of cerebellar granule neuron precursors (CGNPs) induced by Sonic Hedgehog (SHH) signaling, but downstream effectors of mitogenic signaling are still being elucidated. Using primary CGNP cultures, a well-established model for SHH-driven proliferation, we show that SHH-treated CGNPs feature high levels of hypoxia-inducible factor 1α (HIF1α), which is known to promote glycolysis, stemness, and angiogenesis. In CGNPs cultured under normoxic conditions, HIF1α is posttranslationally stabilized in a manner dependent upon reactive oxygen species (ROS) and NADPH oxidase (NOX), both of which are also upregulated in these cells. Inhibition of NOX activity resulted in HIF1α destabilization and reduced levels of cyclin D2, a marker of CGNP proliferation. As CGNPs are the putative cells of origin for the SHH subtype of medulloblastoma and aberrant SHH signaling is implicated in other neoplasms, these studies may also have future relevance in the context of cancer. Taken together, our findings suggest that a better understanding of nonhypoxic HIF1α stabilization through NOX-induced ROS generation can provide insights into normal cell proliferation in cerebellar development and SHH-driven cell proliferation in cancers with aberrant SHH signaling.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células-Tronco Neurais/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Neoplasias Cerebelares , Cerebelo/citologia , Cerebelo/metabolismo , Feminino , Proteínas Hedgehog/metabolismo , Hipóxia/metabolismo , Masculino , Camundongos , NADPH Oxidases/metabolismo , Células-Tronco Neurais/citologia , Neurônios/citologia , Neurônios/metabolismo , Transdução de Sinais , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Medulloblastoma, which is the most common malignant paediatric brain tumour, has a 70% survival rate, but standard treatments often lead to devastating life-long side effects and recurrence is fatal. One of the emerging strategies in the search for treatments is to determine the roles of tumour microenvironment cells in the growth and maintenance of tumours. The most attractive target is tumour-associated macrophages (TAMs), which are abundantly present in the Sonic Hedgehog (SHH) subgroup of medulloblastoma. Here, we report an unexpected beneficial role of TAMs in SHH medulloblastoma. In human patients, decreased macrophage number is correlated with significantly poorer outcome. We confirm macrophage anti-tumoural behaviour in both ex vivo and in vivo murine models of SHH medulloblastoma. Taken together, our findings suggest that macrophages play a positive role by impairing tumour growth in medulloblastoma, in contrast to the pro-tumoural role played by TAMs in glioblastoma, another common brain tumour.
Assuntos
Neoplasias Cerebelares/imunologia , Macrófagos/imunologia , Meduloblastoma/imunologia , Microambiente Tumoral/imunologia , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/genética , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Proteínas de Ligação ao Cálcio , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Proteínas Hedgehog/metabolismo , Humanos , Macrófagos/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Proteínas dos Microfilamentos , Microglia/imunologia , Células Mieloides/imunologia , Receptores CCR2/genética , Regulação para CimaRESUMO
Medulloblastoma is a malignant brain tumor that arises in the cerebellum in children, presumably from granule neuron precursors (GNP). Advances in patient treatment have been hindered by a paucity of animal models that accurately reflect the molecular pathogenesis of human tumors. Aberrant activation of the Sonic hedgehog (Shh) and insulin-like growth factor (IGF) pathways is associated with human medulloblastomas. Both pathways are essential regulators of GNP proliferation during cerebellar development. In cultured GNPs, IGF signaling stabilizes the oncogenic transcription factor N-myc by inhibiting glycogen synthase kinase 3beta-dependent phosphorylation and consequent degradation of N-myc. However, determinants of Shh and IGF tumorigenicity in vivo remain unknown. Here we report a high frequency of medulloblastoma formation in mice following postnatal overexpression of Shh in cooperation with N-myc. Overexpression of N-myc, alone or in combination with IGF signaling mediators or with the Shh target Gli1, did not cause tumors. Thus, Shh has transforming functions in addition to induction of N-myc and Gli1. This tumor model will be useful for testing novel medulloblastoma therapies and providing insight into mechanisms of hedgehog-mediated transformation.
Assuntos
Meduloblastoma/patologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Somatomedinas/fisiologia , Transativadores/fisiologia , Animais , Transformação Celular Neoplásica/patologia , Cerebelo/patologia , Modelos Animais de Doenças , Proteínas Hedgehog , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/patologia , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/fisiologia , Proteína GLI1 em Dedos de ZincoRESUMO
While deregulation of mitochondrial metabolism and cytosolic glycolysis has been well recognized in tumor cells, the role of coordinated mitochondrial oxidation and cytosolic fermentation of pyruvate, a key metabolite derived from glucose, in physiological processes is not well understood. Here, we report that knockout of PTPMT1, a mitochondrial phosphoinositide phosphatase, completely blocked postnatal cerebellar development. Proliferation of granule cell progenitors, the most actively replicating cells in the developing cerebellum, was only moderately decreased, and proliferation of Purkinje cell progenitors did not seem to be affected in knockout mice. In contrast, generation of functional Bergmann glia from multipotent precursor cells (radial glia), which is essential for cerebellar corticogenesis, was totally disrupted. Moreover, despite a low turnover rate, neural stem cells were impaired in self-renewal in knockout mice. Mechanistically, loss of PTPMT1 decreased mitochondrial aerobic metabolism by limiting utilization of pyruvate, which resulted in bioenergetic stress in neural precursor/stem cells but not in progenitor or mature cells, leading to cell cycle arrest through activation of the AMPK-p19/p21 pathway. This study suggests that mitochondrial oxidation of the carbohydrate fuel is required for postnatal cerebellar development, and identifies a bioenergetic stress-induced cell cycle checkpoint in neural precursor/stem cells.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Cerebelo/crescimento & desenvolvimento , Cerebelo/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/fisiologia , Animais , Animais Recém-Nascidos , Pontos de Checagem do Ciclo Celular/genética , Cerebelo/citologia , Feminino , Glicólise , Masculino , Camundongos Knockout , Células-Tronco Neurais/citologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Ácido Pirúvico/metabolismoRESUMO
microRNAs (miRNAs) have wide-ranging effects on large-scale gene regulation. As such, they play a vital role in dictating normal development, and their aberrant expression has been implicated in cancer. There has been a large body of research on the role of miRNAs in medulloblastoma, the most common malignant brain tumor of childhood. The identification of the 4 molecular subgroups with distinct biological, genetic, and transcriptional features has revolutionized the field of medulloblastoma research over the past 5 years. Despite this, the growing body of research on miRNAs in medulloblastoma has largely focused on the clinical entity of a single disease rather than the molecular subgroups. This review begins by highlighting the role of miRNAs in development and progresses to explore their myriad of implications in cancer. Medulloblastoma is characterized by increased proliferation, inhibition of apoptosis, and maintenance of stemness programs-features that are inadvertently regulated by altered expression patterns in miRNAs. This review aims to contextualize the large body of work on miRNAs within the framework of medulloblastoma subgroups. The goal of this review is to stimulate new areas of research, including potential therapeutics, within a rapidly growing field.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Cerebelares/diagnóstico , Regulação Neoplásica da Expressão Gênica , Meduloblastoma/diagnóstico , MicroRNAs/genética , Neoplasias Cerebelares/genética , Humanos , Meduloblastoma/genética , PrognósticoRESUMO
A number of studies have reported that donor cells consisting of serum starved cells, which are assumed to be at quiescence (G0), or non-starved confluent cells or mitotic cells obtained by shake-off, both of which are assumed to be at G1 phase, give better results in nuclear transfer (NT) than cells at other phases of the cell cycle. Whether G0 or G1 cells function better as donor cells is yet to be determined by detailed studies. The aims of this study were to analyze the cell cycle of goat transfected fibroblasts and determine the timing of transition from G0 to G1 by detecting G1-specific marker, cyclin D1 mRNA. Fluorescent-activated cell sorting (FACS) analyses of cells after 4 days of serum starvation showed that more that 90% of cells were in G0/G1. Additionally, detection of cyclin D1 mRNA by northern blot analysis showed that 4-day serum starved quiescent cells started entering G1 a few hours after addition of 10% serum to the medium. Taken together, the data indicated that serum starved transfected primary fibroblasts of adult goats experienced the G0 to G1 transition within 5 h of serum stimulation and were at the mid-G1 stage within 10 h of serum stimulation.
Assuntos
Ciclina D1/metabolismo , Fibroblastos/citologia , Fase G1/fisiologia , RNA Mensageiro/metabolismo , Fase de Repouso do Ciclo Celular/fisiologia , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultura Livres de Soro , Ciclina D1/genética , Fibroblastos/metabolismo , Citometria de Fluxo , CabrasRESUMO
During development and in cancer, tissue and cell growth control requires coordinated regulation of cell proliferation and apoptosis. The tumor suppressive Hippo pathway plays a key role in size regulation and cell-contact inhibition. During the past decade, this pathway has been delineated in Drosophila and now is starting to be better understood in mammals, where an increasing level of complexity and cell context specificity is becoming evident. As we discuss, dys-regulation of this pathway at any step can lead to uncontrolled growth and tumor formation. Indeed, a majority of the pathway components have been implicated in human cancers.
Assuntos
Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Inibição de Contato , HumanosRESUMO
Peripheral neuroblastic tumors (PNTs) share a common origin in the sympathetic nervous system, but manifest variable differentiation and growth potential. Malignant neuroblastoma (NB) and benign ganglioneuroma (GN) stand at opposite ends of the clinical spectrum. We hypothesize that a common PNT progenitor is driven to variable differentiation by specific developmental signaling pathways. To elucidate developmental pathways that direct PNTs along the differentiation spectrum, we compared the expression of genes related to neural crest development in GN and NB. In GNs, we found relatively low expression of sympathetic markers including adrenergic biosynthesis enzymes, indicating divergence from sympathetic fate. In contrast, GNs expressed relatively high levels of enteric neuropeptides and key constituents of the Hedgehog (HH) signaling pathway, including Dhh, Gli1 and Gli3. Predicted HH targets were also differentially expressed in GN, consistent with transcriptional response to HH signaling. These findings indicate that HH signaling is specifically active in GN. Together with the known role of HH activity in enteric neural development, these findings further suggested a role for HH activity in directing PNTs away from the sympathetic lineage toward a benign GN phenotype resembling enteric ganglia. We tested the potential for HH signaling to advance differentiation in PNTs by transducing NB cell lines with Gli1 and determining phenotypic and transcriptional response. Gli1 inhibited proliferation of NB cells, and induced a pattern of gene expression that resembled the differential pattern of gene expression of GN, compared to NB (p<0.00001). Moreover, the transcriptional response of SY5Y cells to Gli1 transduction closely resembled the transcriptional response to the differentiation agent retinoic acid (p<0.00001). Notably, Gli1 did not induce N-MYC expression in neuroblastoma cells, but strongly induced RET, a known mediator of RA effect. The decrease in NB cell proliferation induced by Gli1, and the similarity in the patterns of gene expression induced by Gli1 and by RA, corroborated by closely matched gene sets in GN tumors, all support a model in which HH signaling suppresses PNT growth by promoting differentiation along alternative neural crest pathways.
Assuntos
Sistema Nervoso Entérico/patologia , Ganglioneuroma/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/metabolismo , Crista Neural/citologia , Neuroblastoma/metabolismo , Animais , Catecolaminas/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Ganglioneuroma/patologia , Humanos , Camundongos , Neuropeptídeos/química , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-ret/biossíntese , Transdução de SinaisRESUMO
Gli signaling is critical for central nervous system development and is implicated in tumorigenesis. To monitor Gli signaling in gliomas in vivo, we created platelet-derived growth factor-induced gliomas in a Gli-luciferase reporter mouse. We find that Gli activation is found in gliomas and correlates with grade. In addition, we find that sonic hedgehog (SHH) is expressed in these tumors and also correlates with grade. We identify microvascular proliferation and pseudopalisades, elements that define high-grade gliomas as SHH-producing microenvironments. We describe two populations of SHH-producing stromal cells that reside in perivascular niche (PVN), namely low-cycling astrocytes and endothelial cells. Using the Ptc-LacZ knock-in mouse as a second Gli responsive reporter, we show beta-galactosidase activity in the PVN and in some tumors diffusely throughout the tumor. Lastly, we observe that SHH is similarly expressed in human gliomas and note that an intact tumor microenvironment or neurosphere conditions in vitro are required for Gli activity.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Regulação para Baixo , Genes Reporter , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/genética , Glioma/patologia , Proteínas Hedgehog/biossíntese , Proteínas Hedgehog/genética , Humanos , Camundongos , Camundongos Transgênicos , Oligodendroglioma/genética , Oligodendroglioma/metabolismo , Oligodendroglioma/patologia , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/genética , Proteína GLI1 em Dedos de Zinco , beta-Galactosidase/metabolismoAssuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Meduloblastoma/tratamento farmacológico , Neoplasias/tratamento farmacológico , Proteínas Hedgehog/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
D-cyclins (cyclins D1, D2, and D3) are components of the core cell cycle machinery. To directly test the ability of each D-cyclin to drive development of various lineages, we generated mice expressing only cyclin D1, or only cyclin D2, or only cyclin D3. We found that these "single-cyclin" embryos develop normally until late gestation. Our analyses revealed that in single-cyclin embryos, the tissue-specific expression pattern of D-cyclins was lost. Instead, mutant embryos ubiquitously expressed the remaining D-cyclin. These findings suggest that the functions of the three D-cyclins are largely exchangeable at this stage. Later in life, single-cyclin mice displayed focused abnormalities, resulting in premature mortality. "Cyclin D1-only" mice developed severe megaloblastic anemia, "cyclin D2-only" mice presented neurological abnormalities, and "cyclin D3-only" mice lacked normal cerebella. Analyses of the affected tissues revealed that these compartments failed to sufficiently up-regulate the remaining, intact D-cyclin. In particular, we found that in cerebellar granule neuron precursors, the N-myc transcription factor communicates with the cell cycle machinery via cyclins D1 and D2, but not D3, explaining the inability of D3-only mice to up-regulate cyclin D3 in this compartment. Hence, the requirement for a particular cyclin in a given tissue is likely caused by specific transcription factors, rather than by unique properties of cyclins.