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1.
Nat Commun ; 11(1): 3627, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686664

RESUMO

OTX2 is a potent oncogene that promotes tumor growth in Group 3 medulloblastoma. However, the mechanisms by which OTX2 represses neural differentiation are not well characterized. Here, we perform extensive multiomic analyses to identify an OTX2 regulatory network that controls Group 3 medulloblastoma cell fate. OTX2 silencing modulates the repressive chromatin landscape, decreases levels of PRC2 complex genes and increases the expression of neurodevelopmental transcription factors including PAX3 and PAX6. Expression of PAX3 and PAX6 is significantly lower in Group 3 medulloblastoma patients and is correlated with reduced survival, yet only PAX3 inhibits self-renewal in vitro and increases survival in vivo. Single cell RNA sequencing of Group 3 medulloblastoma tumorspheres demonstrates expression of an undifferentiated progenitor program observed in primary tumors and characterized by translation/elongation factor genes. Identification of mTORC1 signaling as a downstream effector of OTX2-PAX3 reveals roles for protein synthesis pathways in regulating Group 3 medulloblastoma pathogenesis.


Assuntos
Carcinogênese/genética , Neoplasias Cerebelares , Meduloblastoma , Fatores de Transcrição Otx/metabolismo , Fator de Transcrição PAX3/genética , Animais , Carcinogênese/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Oncogenes , Fator de Transcrição PAX3/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Transdução de Sinais/genética
2.
PLoS One ; 15(7): e0235852, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32628717

RESUMO

The medulloblastoma (MB) microenvironment is diverse, and cell-cell interactions within this milieu is of prime importance. Astrocytes, a major component of the microenvironment, have been shown to impact primary tumor cell phenotypes and metastasis. Based on proximity of MB cells and astrocytes in the brain microenvironment, we investigated whether astrocytes may influence MB cell phenotypes directly. Astrocyte conditioned media (ACM) increased Daoy MB cell invasion, adhesion, and in vivo cellular protrusion formation. ACM conditioning of MB cells also increased CD133 surface expression, a key cancer stem cell marker of MB. Additional neural stem cell markers, Nestin and Oct-4A, were also increased by ACM conditioning, as well as neurosphere formation. By knocking down CD133 using short interfering RNA (siRNA), we showed that ACM upregulated CD133 expression in MB plays an important role in invasion, adhesion and neurosphere formation. Collectively, our data suggests that astrocytes influence MB cell phenotypes by regulating CD133 expression, a key protein with defined roles in MB tumorgenicity and survival.


Assuntos
Antígeno AC133/genética , Astrócitos/metabolismo , Meduloblastoma/metabolismo , Fenótipo , Antígeno AC133/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/fisiologia , Nestina/genética , Nestina/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Microambiente Tumoral , Peixe-Zebra
3.
Pharmacol Rev ; 72(3): 668-691, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571983

RESUMO

Eliminating cancer was once thought of as a war. This analogy is still apt today; however, we now realize that cancer is a much more formidable enemy than scientists originally perceived, and in some cases, it harbors a profound ability to thwart our best efforts to defeat it. However, before we were aware of the complexity of cancer, chemotherapy against childhood acute lymphoblastic leukemia (ALL) was successful because it applied the principles of pharmacology. Herein, we provide a historic perspective of the experience at St. Jude Children's Research Hospital. In 1962, when the hospital opened, fewer than 3% of patients experienced durable cure. Through judicious application of pharmacologic principles (e.g., combination therapy with agents using different mechanisms of action) plus appropriate drug scheduling, dosing, and pharmacodynamics, the survival of patients with ALL now exceeds 90%. We contrast this approach to treating ALL with the contemporary approach to treating medulloblastoma, in which genetics and molecular signatures are being used to guide the development of more-efficacious treatment strategies with minimal toxicity. Finally, we highlight the emerging technologies that can sustain and propel the collaborative efforts to squeeze the life out of these cancers. SIGNIFICANCE STATEMENT: Up until the early 1960s, chemotherapy for childhood acute lymphoblastic leukemia was mostly ineffective. This changed with the knowledge and implementation of rational approaches to combination therapy. Although the therapeutics of brain cancers such as medulloblastoma are not as refined (in part because of the blood-brain barrier obstacle), recent extraordinary advances in knowledge of medulloblastoma pathobiology has led to innovations in disease classification accompanied with strategies to improve therapeutic outcomes. Undoubtedly, additional novel approaches, such as immunological therapeutics, will open new avenues to further the goal of taming cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Encefálicas/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Neoplasias Encefálicas/metabolismo , Humanos , Meduloblastoma/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Ensaios Clínicos Controlados Aleatórios como Assunto
4.
Nat Neurosci ; 23(7): 842-853, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32424282

RESUMO

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-ß receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.


Assuntos
Neoplasias Cerebelares/imunologia , Meduloblastoma/imunologia , Evasão Tumoral/imunologia , Fator de Necrose Tumoral alfa/imunologia , Proteína Supressora de Tumor p53/imunologia , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Fator de Necrose Tumoral alfa/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
5.
Nature ; 580(7803): 396-401, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296180

RESUMO

Cancer genomics has revealed many genes and core molecular processes that contribute to human malignancies, but the genetic and molecular bases of many rare cancers remains unclear. Genetic predisposition accounts for 5 to 10% of cancer diagnoses in children1,2, and genetic events that cooperate with known somatic driver events are poorly understood. Pathogenic germline variants in established cancer predisposition genes have been recently identified in 5% of patients with the malignant brain tumour medulloblastoma3. Here, by analysing all protein-coding genes, we identify and replicate rare germline loss-of-function variants across ELP1 in 14% of paediatric patients with the medulloblastoma subgroup Sonic Hedgehog (MBSHH). ELP1 was the most common medulloblastoma predisposition gene and increased the prevalence of genetic predisposition to 40% among paediatric patients with MBSHH. Parent-offspring and pedigree analyses identified two families with a history of paediatric medulloblastoma. ELP1-associated medulloblastomas were restricted to the molecular SHHα subtype4 and characterized by universal biallelic inactivation of ELP1 owing to somatic loss of chromosome arm 9q. Most ELP1-associated medulloblastomas also exhibited somatic alterations in PTCH1, which suggests that germline ELP1 loss-of-function variants predispose individuals to tumour development in combination with constitutive activation of SHH signalling. ELP1 is the largest subunit of the evolutionarily conserved Elongator complex, which catalyses translational elongation through tRNA modifications at the wobble (U34) position5,6. Tumours from patients with ELP1-associated MBSHH were characterized by a destabilized Elongator complex, loss of Elongator-dependent tRNA modifications, codon-dependent translational reprogramming, and induction of the unfolded protein response, consistent with loss of protein homeostasis due to Elongator deficiency in model systems7-9. Thus, genetic predisposition to proteome instability may be a determinant in the pathogenesis of paediatric brain cancers. These results support investigation of the role of protein homeostasis in other cancer types and potential for therapeutic interference.


Assuntos
Neoplasias Cerebelares/metabolismo , Mutação em Linhagem Germinativa , Meduloblastoma/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Criança , Feminino , Humanos , Masculino , Meduloblastoma/genética , Linhagem , RNA de Transferência/metabolismo , Fatores de Elongação da Transcrição/genética
6.
J Clin Pathol ; 73(5): 243-249, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32034059

RESUMO

Medulloblastoma (MB) is the most common malignant primary intracranial neoplasm diagnosed in childhood. Although numerous efforts have been made during the past few years to exploit novel targeted therapies for this aggressive neoplasm, there still exist substantial hitches hindering successful management of MB. Lately, progress in cancer biology has shown evidence that a subpopulation of cells within the tumour, namely cancer stem cells (CSCs), are thought to be responsible for the resistance to most chemotherapeutic agents and radiation therapy, accounting for cancer recurrence. Hence, it is crucial to identify the molecular signatures and genetic aberrations that characterise those CSCs and develop therapies that specifically target them. In this review, we aim to give an overview of the main genetic and molecular cues that depict MB-CSCs and provide a synopsis of the novel therapeutic approaches that specifically target this population of cells to attain enhanced antitumorous effects and therefore overcome resistance to therapy.


Assuntos
Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Neoplasias Cerebelares , Resistencia a Medicamentos Antineoplásicos/fisiologia , Meduloblastoma , Células-Tronco Neoplásicas/fisiologia , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos
7.
Anticancer Res ; 40(1): 53-66, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31892552

RESUMO

BACKGROUND/AIM: Medulloblastoma (MB) accounts for ~20% of pediatric malignant central nervous system tumors. Treatment strategies, including surgery, radiation therapy and/or chemotherapy, are effective, but recurrence and metastasis frequently occur. Therefore, novel therapies are required. Herein, the effects of fibroblast growth factor receptor (FGFR) and phosphoinositide 3-kinase (PI3K) inhibitors on MB cells lines were evaluated. MATERIALS AND METHODS: MB cell lines (UW228-3, DAOY, Med8a, D425, D283) were tested for sensitivity to FGFR (AZD4547) and PI3K (BEZ235 and BYL719) inhibitors by viability, cytotoxicity, apoptosis, and proliferation assays. RESULTS: Single treatments with FGFR and PI3K inhibitors decreased viability and proliferation in a dose-dependent pattern in most cell lines. Combinination of the two type of drugs, increased sensitivity, especially of the most resistant cell line UW228-3. CONCLUSION: Combination treatments with FGFR and PI3K inhibitors were superior to single treatments with FGFR and PI3K inhibitors, especially with BEZ235, for MB cell lines.


Assuntos
Meduloblastoma/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Meduloblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Commun ; 11(1): 583, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996670

RESUMO

Medulloblastoma (MB) is the most common malignant brain tumor in children and among the subtypes, Group 3 MB has the worst outcome. Here, we perform an in vivo, patient-specific screen leading to the identification of Otx2 and c-MYC as strong Group 3 MB inducers. We validated our findings in human cerebellar organoids where Otx2/c-MYC give rise to MB-like organoids harboring a DNA methylation signature that clusters with human Group 3 tumors. Furthermore, we show that SMARCA4 is able to reduce Otx2/c-MYC tumorigenic activity in vivo and in human cerebellar organoids while SMARCA4 T910M, a mutant form found in human MB patients, inhibits the wild-type protein function. Finally, treatment with Tazemetostat, a EZH2-specific inhibitor, reduces Otx2/c-MYC tumorigenesis in ex vivo culture and human cerebellar organoids. In conclusion, human cerebellar organoids can be efficiently used to understand the role of genes found altered in cancer patients and represent a reliable tool for developing personalized therapies.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Organoides/metabolismo , Organoides/patologia , Benzamidas/antagonistas & inibidores , Carcinogênese , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fatores de Transcrição Otx/genética , Fatores de Transcrição Otx/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Piridonas/antagonistas & inibidores , Células-Tronco , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Cell ; 180(3): 502-520.e19, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-31983537

RESUMO

The tumor microenvironment (TME) is critical for tumor progression. However, the establishment and function of the TME remain obscure because of its complex cellular composition. Using a mouse genetic system called mosaic analysis with double markers (MADMs), we delineated TME evolution at single-cell resolution in sonic hedgehog (SHH)-activated medulloblastomas that originate from unipotent granule neuron progenitors in the brain. First, we found that astrocytes within the TME (TuAstrocytes) were trans-differentiated from tumor granule neuron precursors (GNPs), which normally never differentiate into astrocytes. Second, we identified that TME-derived IGF1 promotes tumor progression. Third, we uncovered that insulin-like growth factor 1 (IGF1) is produced by tumor-associated microglia in response to interleukin-4 (IL-4) stimulation. Finally, we found that IL-4 is secreted by TuAstrocytes. Collectively, our studies reveal an evolutionary process that produces a multi-lateral network within the TME of medulloblastoma: a fraction of tumor cells trans-differentiate into TuAstrocytes, which, in turn, produce IL-4 that stimulates microglia to produce IGF1 to promote tumor progression.


Assuntos
Astrócitos/metabolismo , Carcinogênese/metabolismo , Transdiferenciação Celular , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Comunicação Parácrina , Animais , Linhagem da Célula , Neoplasias Cerebelares/patologia , Modelos Animais de Doenças , Feminino , Proteínas Hedgehog/metabolismo , Xenoenxertos , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Masculino , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Microambiente Tumoral
10.
Nat Rev Cancer ; 20(1): 42-56, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31819232

RESUMO

Medulloblastoma, a malignant brain tumour primarily diagnosed during childhood, has recently been the focus of intensive molecular profiling efforts, profoundly advancing our understanding of biologically and clinically heterogeneous disease subgroups. Genomic, epigenomic, transcriptomic and proteomic landscapes have now been mapped for an unprecedented number of bulk samples from patients with medulloblastoma and, more recently, for single medulloblastoma cells. These efforts have provided pivotal new insights into the diverse molecular mechanisms presumed to drive tumour initiation, maintenance and recurrence across individual subgroups and subtypes. Translational opportunities stemming from this knowledge are continuing to evolve, providing a framework for improved diagnostic and therapeutic interventions. In this Review, we summarize recent advances derived from this continued molecular characterization of medulloblastoma and contextualize this progress towards the deployment of more effective, molecularly informed treatments for affected patients.


Assuntos
Suscetibilidade a Doenças , Meduloblastoma/diagnóstico , Meduloblastoma/etiologia , Animais , Biomarcadores Tumorais , Terapia Combinada , Gerenciamento Clínico , Epigenômica/métodos , Estudos de Associação Genética , Genômica/métodos , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/terapia , Técnicas de Diagnóstico Molecular , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Proteômica/métodos , Recidiva , Resultado do Tratamento , Microambiente Tumoral
11.
Mol Carcinog ; 59(3): 281-292, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31872914

RESUMO

Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-κB (NF-κB), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-κB target genes, NF-κB reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.


Assuntos
Neoplasias Cerebelares/metabolismo , Proteínas Correpressoras/metabolismo , Meduloblastoma/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Correpressoras/análise , Proteínas Correpressoras/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fatores de Transcrição/análise , Fatores de Transcrição/genética
12.
Cell Rep ; 29(12): 4036-4052.e10, 2019 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-31851932

RESUMO

The transition of neural progenitors to differentiated postmitotic neurons is mainly considered irreversible in physiological conditions. In the present work, we show that Shh pathway activation through SmoM2 expression promotes postmitotic neurons dedifferentiation, re-entering in the cell cycle and originating medulloblastoma in vivo. Notably, human adult patients present inactivating mutations of the chromatin reader BRPF1 that are associated with SMO mutations and absent in pediatric and adolescent patients. Here, we found that truncated BRPF1 protein, as found in human adult patients, is able to induce medulloblastoma in adult mice upon SmoM2 activation. Indeed, postmitotic neurons re-entered the cell cycle and proliferated as a result of chromatin remodeling of neurons by BRPF1. Our model of brain cancer explains the onset of a subset of human medulloblastoma in adult individuals where granule neuron progenitors are no longer present.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Hedgehog/metabolismo , Meduloblastoma/patologia , Mutação , Neurônios/patologia , Receptor Smoothened/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Animais , Apoptose , Proliferação de Células , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos , Feminino , Proteínas Hedgehog/genética , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Nus , Neurônios/metabolismo , Receptor Smoothened/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
13.
BMC Cancer ; 19(1): 1056, 2019 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-31694585

RESUMO

BACKGROUND: MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma. METHODS: Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666. RESULTS: We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity. CONCLUSION: Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.


Assuntos
Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Humanos , Isoquinolinas/farmacologia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Ligação Proteica , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Pirimidinas/farmacologia , Interferência de RNA , Análise de Sobrevida
14.
Gene Expr Patterns ; 34: 119068, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31437514

RESUMO

Cerebellar granule cell precursors (GCPs) and granule cells (GCs) constitute a good model system to investigate proliferation of neural precursors and differentiation of neurons. During development, GCPs proliferate in the outer external granule cell layer (outer EGL) and then exit the cell cycle in the inner EGL to become GCs, which inwardly migrate to the inner granule cell layer (IGL). Misregulation of GCP proliferation or GC differentiation leads to maldevelopment of the cerebellum and the formation of a cerebellar tumor, medulloblastoma. Despite many efforts in this field, the mechanisms underlying GC development remain elusive. In this study, we performed detailed immunostaining in the developing cerebellum, with particular focus on GCPs and GCs, looking at several transcription factors, signaling molecules, cell cycle regulators, some of which are known to regulate neural development. Interestingly, we found distinct distribution patterns of certain proteins within the outer and inner EGL, suggesting the existence of subpopulations of GCPs and GCs in those layers. This study provides a basis for future studies on the cerebellar GC development and medulloblastoma.


Assuntos
Cerebelo/metabolismo , Neurônios/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular , Diferenciação Celular/fisiologia , Divisão Celular , Proliferação de Células , Imuno-Histoquímica/métodos , Antígeno Ki-67/metabolismo , Meduloblastoma/metabolismo , Meduloblastoma/fisiopatologia , Camundongos , Camundongos Endogâmicos ICR , Neurogênese/fisiologia , Transdução de Sinais
15.
EMBO Mol Med ; 11(8): e9830, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31328883

RESUMO

Medulloblastoma (MB) is a pediatric tumor of the cerebellum divided into four groups. Group 3 is of bad prognosis and remains poorly characterized. While the current treatment involving surgery, radiotherapy, and chemotherapy often fails, no alternative therapy is yet available. Few recurrent genomic alterations that can be therapeutically targeted have been identified. Amplifications of receptors of the TGFß/Activin pathway occur at very low frequency in Group 3 MB. However, neither their functional relevance nor activation of the downstream signaling pathway has been studied. We showed that this pathway is activated in Group 3 MB with some samples showing a very strong activation. Beside genetic alterations, we demonstrated that an ActivinB autocrine stimulation is responsible for pathway activation in a subset of Group 3 MB characterized by high PMEPA1 levels. Importantly, Galunisertib, a kinase inhibitor of the cognate receptors currently tested in clinical trials for Glioblastoma patients, showed efficacy on orthotopically grafted MB-PDX. Our data demonstrate that the TGFß/Activin pathway is active in a subset of Group 3 MB and can be therapeutically targeted.


Assuntos
Comunicação Autócrina , Neoplasias Cerebelares/metabolismo , Subunidades beta de Inibinas/metabolismo , Meduloblastoma/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidades beta de Inibinas/genética , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Nus , Fosforilação , Pirazóis/farmacologia , Quinolinas/farmacologia , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta3/genética , Carga Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Nat Commun ; 10(1): 2410, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160587

RESUMO

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 Cima
17.
BMC Cancer ; 19(1): 571, 2019 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-31185958

RESUMO

BACKGROUND: Using a pathway-focused approach, we aimed to provide a subgroup-specific basis for finding novel therapeutic strategies and further refinement of the risk stratification in pediatric medulloblastoma. METHOD: Based on genome-wide Cox regression and Gene Set Enrichment Analysis, we investigated prognosis-related signaling pathways and core genes in pediatric medulloblastoma subgroups using 530 patient data from Medulloblastoma Advanced Genomic International Consortium (MAGIC) project. We further examined the relationship between expression of the prognostic core genes and frequent chromosome aberrations using broad range copy number change data. RESULTS: In SHH subgroup, relatively high expression of the core genes involved in p53, PLK1, FOXM1, and Aurora B signaling pathways are associated with poor prognosis, and their average expression synergistically increases with co-occurrence of losses of 17p, 14q, or 10q, or gain of 17q. In Group 3, in addition to high MYC expression, relatively elevated expression of PDGFRA, IGF1R, and FGF2 and their downstream genes in PI3K/AKT and MAPK/ERK pathways are related to poor survival outcome, and their average expression is increased with the presence of isochromosome 17q [i(17q)] and synergistically down-regulated with simultaneous losses of 16p, 8q, or 4q. In Group 4, up-regulation of the genes encoding various immune receptors and those involved in NOTCH, NF-κB, PI3K/AKT, or RHOA signaling pathways are associated with worse prognosis. Additionally, the expressions of Notch genes correlate with those of the prognostic immune receptors. Besides the Group 4 patients with previously known prognostic aberration, loss of chromosome 11, those with loss of 8q but without i(17q) show excellent survival outcomes and low average expression of the prognostic core genes whereas those harboring 10q loss, 1q gain, or 12q gain accompanied by i(17q) show bad outcomes. Finally, several metabolic pathways known to be reprogrammed in cancer cells are detected as prognostic pathways including glutamate metabolism in SHH subgroup, pentose phosphate pathway and TCA cycle in Group 3, and folate-mediated one carbon-metabolism in Group 4. CONCLUSIONS: The results underscore several subgroup-specific pathways for potential therapeutic interventions: SHH-GLI-FOXM1 pathway in SHH subgroup, receptor tyrosine kinases and their downstream pathways in Group 3, and immune and inflammatory pathways in Group 4.


Assuntos
Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Redes e Vias Metabólicas , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Cerebelares/diagnóstico , Criança , Saúde da Criança , Pré-Escolar , Feminino , Proteína Forkhead Box M1/genética , Proteína Forkhead Box M1/metabolismo , Expressão Gênica , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Humanos , Inflamação/metabolismo , Estimativa de Kaplan-Meier , Masculino , Meduloblastoma/diagnóstico , Prognóstico , Modelos de Riscos Proporcionais , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/genética , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismo
18.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035417

RESUMO

Glioblastoma multiforme and medulloblastoma are the most frequent high-grade brain tumors in adults and children, respectively. Standard therapies for these cancers are mainly based on surgical resection, radiotherapy, and chemotherapy. However, intrinsic or acquired resistance to treatment occurs almost invariably in the first case, and side effects are unacceptable in the second. Therefore, the development of new, effective drugs is a very important unmet medical need. A critical requirement for developing such agents is to identify druggable targets required for the proliferation or survival of tumor cells, but not of other cell types. Under this perspective, genes mutated in congenital microcephaly represent interesting candidates. Congenital microcephaly comprises a heterogeneous group of disorders in which brain volume is reduced, in the absence or presence of variable syndromic features. Genetic studies have clarified that most microcephaly genes encode ubiquitous proteins involved in mitosis and in maintenance of genomic stability, but the effects of their inactivation are particularly strong in neural progenitors. It is therefore conceivable that the inhibition of the function of these genes may specifically affect the proliferation and survival of brain tumor cells. Microcephaly genes encode for a few kinases, including CITK, PLK4, AKT3, DYRK1A, and TRIO. In this review, we summarize the evidence indicating that the inhibition of these molecules could exert beneficial effects on different aspects of brain cancer treatment.


Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Microcefalia/metabolismo , Microcefalia/patologia , Animais , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteínas Serina-Treonina Quinases/metabolismo
19.
J Neurooncol ; 143(3): 393-403, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31104222

RESUMO

INTRODUCTION: Molecular classification of medulloblastomas (MB) is prognostically and therapeutically relevant and helps in better risk-stratification. Translation of this subgrouping to routine practice still remains a challenge. The most pathologist accessible techniques for molecular subgrouping include immunohistochemistry (IHC), fluorescent in-situ hybridization (FISH) and NanoString. OBJECTIVES: (1) Molecular subgrouping of MBs by IHC and FISH, and NanoString assay (2) To compare their efficacy and cost for applicability in resource constrained centers. METHODS: Ninety-five cases of MB with adequate tissue were included. Molecular subgrouping was performed by IHC for ß-catenin, GAB1 and YAP1; FISH for MYC amplification, and sequencing for CTNNB1, and by NanoString Assay on the same set of MBs. A subset of cases was subjected to 850k DNA methylation array. RESULTS: IHC + FISH classified MBs into 15.8% WNT, 16.8% SHH, and 67.4% non-WNT/non-SHH subgroups; with MYC amplification identified in 20.3% cases of non-WNT/non-SHH. NanoString successfully classified 91.6% MBs into 25.3% WNT, 17.2% SHH, 23% Group 3 and 34.5% Group 4. However, NanoString assay failure was seen in eight cases, all of which were > 8-years-old formalin-fixed paraffin-embedded tissue blocks. Concordant subgroup assignment was noted in 88.5% cases, while subgroup switching was seen in 11.5% cases. Both methods showed prognostic correlation. Methylation profiling performed on discordant cases revealed 1 out of 4 extra WNT identified by NanoString to be WNT, others aligned with IHC subgroups; extra SHH by NanoString turned out to be SHH by methylation. CONCLUSIONS: Both IHC supplemented by FISH and NanoString are robust methods for molecular subgrouping, albeit with few disadvantages. IHC cannot differentiate between Groups 3 and 4, while NanoString cannot classify older-archived tumors, and is not available at most centres. Thus, both the methods complement each other and can be used in concert for high confidence allotment of molecular subgroups in clinical practice.


Assuntos
Neoplasias Cerebelares/classificação , Imuno-Histoquímica/métodos , Hibridização in Situ Fluorescente/métodos , Meduloblastoma/classificação , Nanotecnologia/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Adolescente , Adulto , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Criança , Pré-Escolar , Estudos de Coortes , Terapia Combinada , Metilação de DNA , Feminino , Seguimentos , Recursos em Saúde , Humanos , Lactente , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Adulto Jovem
20.
Gene ; 705: 67-76, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30991098

RESUMO

Medulloblastoma (MB) is characterized by highly invasive embryonal neuro-epithelial tumors that metastasize via cerebrospinal fluid. MB is difficult to treat and the chemotherapy is associated with significant toxicities and potential long-term disabilities. Previously, we showed that small molecule, clotam (tolfenamic acid: TA) inhibited MB cell proliferation and tumor growth in mice by targeting, survivin. Overexpression of survivin is associated with aggressiveness and poor prognosis in several cancers, including MB. The aim of this study was to test combination treatment involving Vincristine® (VCR), a standard chemotherapeutic drug for MB and TA against MB cells. DAOY and D283 MB cells were treated with 10 µg/mL TA or VCR (DAOY: 2 ng/mL; D283: 1 ng/mL) or combination (TA + VCR). These optimized doses were lower than individual IC50 values. The effect of single or combination treatment on cell viability (CellTiterGlo kit), Combination Index (Chou-Talalay method based on median-drug effect analysis), activation of apoptosis and cell cycle modulation (by flow cytometry using Annexin V and propidium iodide respectively) and the expression of associated markers including survivin (Western immunoblot) were determined. Combination Index showed moderate synergistic cytotoxic effect in both cells. When compared to individual agents, the combination of TA and VCR increased MB cell growth inhibition, induced apoptosis and caused cell cycle (G2/M phase) arrest. Survivin expression was also decreased by the combination treatment. TA is effective for inducing the anti-proliferative response of VCR in MB cells. MB has four distinct genetic/molecular subgroups. Experiments were conducted with MB cells representing two subgroups (DAOY: SHH group; D283: group 4/3). TA-induced inhibition of survivin expression potentially destabilizes mitotic microtubule assembly, sensitizing MB cells and enhancing the efficacy of VCR.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Survivina/metabolismo , Vincristina/farmacologia , ortoaminobenzoatos/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/tratamento farmacológico , Relação Dose-Resposta a Droga , Regulação para Baixo , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Meduloblastoma/tratamento farmacológico
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