RESUMO
Epigenetic mechanisms are essential to regulate gene expression during normal development. However, they are often disrupted in pathological conditions including tumours, where they contribute to their formation and maintenance through altered gene expression. In recent years, next generation genomic techniques has allowed a remarkable advancement of our knowledge of the genetic and molecular landscape of paediatric brain tumours and have highlighted epigenetic deregulation as a common hallmark in their pathogenesis. This review describes the main epigenetic dysregulations found in paediatric brain tumours, including at DNA methylation and histone modifications level, in the activity of chromatin-modifying enzymes and in the expression of non-coding RNAs. How these altered processes influence tumour biology and how they can be leveraged to dissect the molecular heterogeneity of these tumours and contribute to their classification is also addressed. Finally, the availability and value of preclinical models as well as the current clinical trials exploring targeting key epigenetic mediators in paediatric brain tumours are discussed.
Assuntos
Neoplasias Encefálicas , Epigênese Genética , Neoplasias Encefálicas/genética , Criança , Cromatina , Metilação de DNA , Epigenômica , HumanosRESUMO
The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases. Stem Cells 2017;35:725-738.
Assuntos
Processamento Alternativo/genética , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Regeneração , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Hipertrofia , Fatores de Transcrição MEF2/química , Fatores de Transcrição MEF2/genética , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mioblastos/metabolismo , Células NIH 3T3 , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Ligação Proteica , Domínios Proteicos , Células Satélites de Músculo Esquelético/metabolismo , Serina/metabolismoRESUMO
Glioblastomas are the most common malignant brain tumors in adults; they are highly aggressive and heterogeneous and show a high degree of plasticity. Here, we show that methyltransferase-like 7B (METTL7B) is an essential regulator of lineage specification in glioblastoma, with an impact on both tumor size and invasiveness. Single-cell transcriptomic analysis of these tumors and of cerebral organoids derived from expanded potential stem cells overexpressing METTL7B reveal a regulatory role for the gene in the neural stem cell-to-astrocyte differentiation trajectory. Mechanistically, METTL7B downregulates the expression of key neuronal differentiation players, including SALL2, via post-translational modifications of histone marks.
Assuntos
Diferenciação Celular , Linhagem da Célula , Glioblastoma , Metiltransferases , Glioblastoma/patologia , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Metiltransferases/metabolismo , Metiltransferases/genética , Linhagem da Célula/genética , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Regulação Neoplásica da Expressão Gênica , Camundongos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Linhagem Celular Tumoral , Astrócitos/metabolismo , Astrócitos/patologia , Organoides/metabolismo , Organoides/patologiaRESUMO
BACKGROUND: Epigenetic changes play a key role in the pathogenesis of medulloblastoma (MB), the most common malignant pediatric brain tumor. METHODS: We explore the therapeutic potential of BMI1 and MAPK/ERK inhibition in BMI1High;CHD7Low MB cells and in a preclinical xenograft model. RESULTS: We identify a synergistic vulnerability of BMI1High;CHD7Low MB cells to a combination treatment with BMI1 and MAPK/ERK inhibitors. Mechanistically, CHD7-dependent binding of BMI1 to MAPK-regulated genes underpins the CHD7-BMI1-MAPK regulatory axis responsible of the antitumour effect of the inhibitors in vitro and in a preclinical mouse model. Increased ERK1 and ERK2 phosphorylation activity is found in BMI1High;CHD7Low G4 MB patients, raising the possibility that they could be amenable to a similar therapy. CONCLUSIONS: The molecular dissection of the CHD7-BMI1-MAPK regulatory axis in BMI1High;CHD7Low MB identifies this signature as a proxy to predict MAPK functional activation, which can be effectively drugged in preclinical models, and paves the way for further exploration of combined BMI1 and MAPK targeting in G4 MB patients.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Meduloblastoma , Inibidores de Proteínas Quinases , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/genética , Humanos , Meduloblastoma/genética , Camundongos , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/genéticaRESUMO
Deregulation of chromatin modifiers plays an essential role in the pathogenesis of medulloblastoma, the most common paediatric malignant brain tumour. Here, we identify a BMI1-dependent sensitivity to deregulation of inositol metabolism in a proportion of medulloblastoma. We demonstrate mTOR pathway activation and metabolic adaptation specifically in medulloblastoma of the molecular subgroup G4 characterised by a BMI1High;CHD7Low signature and show this can be counteracted by IP6 treatment. Finally, we demonstrate that IP6 synergises with cisplatin to enhance its cytotoxicity in vitro and extends survival in a pre-clinical BMI1High;CHD7Low xenograft model.
Assuntos
Adaptação Fisiológica , Neoplasias Cerebelares/genética , Epigênese Genética , Inositol/farmacologia , Meduloblastoma/genética , Adaptação Fisiológica/efeitos dos fármacos , Animais , Contagem de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Proteínas de Ligação a DNA/metabolismo , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neurais/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas com Domínio T , Serina-Treonina Quinases TOR/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog-/-zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs, we found that myog-/- myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, Pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog-/-myofibres instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an 'alerted' state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life.
Assuntos
Músculo Esquelético/crescimento & desenvolvimento , Miofibrilas/fisiologia , Miogenina/fisiologia , Células-Tronco/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Técnicas de Inativação de Genes , Homeostase , Músculo Esquelético/metabolismo , Miofibrilas/metabolismo , Miogenina/metabolismo , Células-Tronco/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Glioblastoma (GBM) is the most common and most aggressive intrinsic brain tumour in adults. Integrated transcriptomic and epigenomic analyses of glioblastoma initiating cells (GIC) in a mouse model uncovered a novel epigenetic regulation of EfnA5. In this model, Bmi1 enhances H3K27me3 at the EfnA5 locus and reinforces repression of selected target genes in a cellular context-dependent fashion. EfnA5 mediates Bmi1-dependent proliferation and invasion in vitro and tumour formation in an allograft model. Importantly, we show that this novel Polycomb feed-forward loop is also active in human GIC and we provide pre-clinical evidence of druggability of the EFNA5 signalling pathway in GBM xenografts overexpressing Bmi1.
Assuntos
Efrina-A5/metabolismo , Glioblastoma/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Animais , Anti-Hipertensivos/farmacologia , Proliferação de Células , Doxazossina/farmacologia , Sistemas de Liberação de Medicamentos , Efrina-A5/antagonistas & inibidores , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Histonas/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Camundongos Transgênicos , Invasividade Neoplásica , Células-Tronco Neurais/metabolismo , Neurogênese , Complexo Repressor Polycomb 1/genética , Células Tumorais CultivadasRESUMO
Established cell lines have been extensively used in cancer research. They are easy to obtain and expand and are composed of a relatively uniform population of cells. When experimental conditions are kept standard, these cells allow a high reproducibility of experimental findings from independent research groups. However, because these cell lines have been propagated in culture for decades, additional genetic lesions may be acquired leading to modification of their characteristics as compared to the original tumor. Primary cultures represent a valid alternative. Here, we describe standardized protocols to establish medulloblastoma (MB) patient-derived primary cultures from fresh tumor samples. MB primary cells grow as an adherent culture on a laminin coating and can be propagated in vitro for a limited number of passages, therefore reducing the chances to accumulate molecular alterations compared to long-term cultures. Consequently, they better resemble the original tumor both in terms of biological behavior and molecular characteristics. Low-passage MB primary cells can be used as an in vitro model for biochemical studies and functional assays, representing a useful tool to dissect the contribution of molecular pathways to MB pathogenesis. They can also represent a useful screening tool for potential therapeutic agents in preclinical studies.
Assuntos
Neoplasias Encefálicas/patologia , Técnicas de Cultura de Células/métodos , Meduloblastoma/patologia , Neoplasias Encefálicas/cirurgia , Criopreservação , Humanos , Lentivirus/metabolismo , Meduloblastoma/cirurgia , Esferoides Celulares/patologia , Transdução Genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Medulloblastoma (MB) is the most common malignant solid paediatric brain tumour. The standard treatment for MB is surgical resection of the tumour, radiation and chemotherapy. This therapy is associated with high morbidity and adverse side effects. Hence, more targeted and less toxic therapies are vitally needed to improve the quality of life of survivors. NPI-0052 is a novel proteasome inhibitor that irreversibly binds the 20S proteasome subunit. This compound has anti-tumour activity in metastatic solid tumours, glioblastoma and multiple myeloma with a good safety profile. Importantly, NPI-0052 has a lipophilic structure and can penetrate the blood-brain barrier, making it a suitable treatment for brain tumours. In the present study, we performed an in silico gene expression analysis to evaluate the proteasome subunit expression in MB. To evaluate the anticancer activity of NPI-0052, we used a range of MB patient-derived MB cells and cell lines. The synergistic cell death of NPI-0052 with γ-radiation was evaluated in tumour organoids derived from patient-derived MB cells. We show that high expression of proteasome subunits is a poor prognostic factor for MB patients. Also, our preclinical work demonstrated that NPI-0052 can inhibit proteasome activity and activate apoptosis in MB cells. Moreover, we observe that NPI-0052 has a synergistic apoptotic effect with γ-radiation, a component of the current MB therapy. Here, we present compelling preclinical evidence that NPI-0052 can be used as an adjuvant treatment for p53-family-expressing MB tumours.
Assuntos
Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/radioterapia , Raios gama/uso terapêutico , Lactonas/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/radioterapia , Pirróis/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Neoplasias Cerebelares/patologia , Quimiorradioterapia , Humanos , Meduloblastoma/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologiaRESUMO
Each skeletal muscle acquires its unique size before birth, when terminally differentiating myocytes fuse to form a defined number of multinucleated myofibres. Although mice in which the transcription factor Myogenin is mutated lack most myogenesis and die perinatally, a specific cell biological role for Myogenin has remained elusive. Here we report that loss of function of zebrafish myog prevents formation of almost all multinucleated muscle fibres. A second, Myogenin-independent, fusion pathway in the deep myotome requires Hedgehog signalling. Lack of Myogenin does not prevent terminal differentiation; the smaller myotome has a normal number of myocytes forming more mononuclear, thin, albeit functional, fast muscle fibres. Mechanistically, Myogenin binds to the myomaker promoter and is required for expression of myomaker and other genes essential for myocyte fusion. Adult myog mutants display reduced muscle mass, decreased fibre size and nucleation. Adult-derived myog mutant myocytes show persistent defective fusion ex vivo. Myogenin is therefore essential for muscle homeostasis, regulating myocyte fusion to determine both muscle fibre number and size.
Assuntos
RNA Mensageiro/metabolismo , Peixe-Zebra/metabolismo , Animais , Células Cultivadas , Imunoprecipitação da Cromatina , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Masculino , Células Musculares/citologia , Células Musculares/metabolismo , Miogenina/metabolismo , NADH Tetrazólio Redutase/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We describe molecular convergence between BMI1 and CHD7 in the initiation of medulloblastoma. Identified in a functional genomic screen in mouse models, a BMI1High;CHD7Low expression signature within medulloblastoma characterizes patients with poor overall survival. We show that BMI1-mediated repression of the ERK1/2 pathway leads to increased proliferation and tumor burden in primary human MB cells and in a xenograft model, respectively. We provide evidence that repression of the ERK inhibitor DUSP4 by BMI1 is dependent on a more accessible chromatin configuration in G4 MB cells with low CHD7 expression. These findings extend current knowledge of the role of BMI1 and CHD7 in medulloblastoma pathogenesis, and they raise the possibility that pharmacological targeting of BMI1 or ERK may be particularly indicated in a subgroup of MB with low expression levels of CHD7.
Assuntos
Proteínas de Ligação a DNA/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Meduloblastoma/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Western Blotting , Proliferação de Células/genética , Proliferação de Células/fisiologia , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/genética , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Masculino , Meduloblastoma/genética , Camundongos , Complexo Repressor Polycomb 1/genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation.