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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
Clin Nucl Med ; 44(6): e385-e387, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30985433

RESUMO

Von Hippel-Lindau disease is an inherited syndrome associated with several benign and malignant tumors such as central nervous system (CNS) hemangioblastoma. Herein, we report a known case of A Von Hippel-Lindau patient with a cerebral hemangioblastoma who was referred for further evaluation because of recent paraparesis. F-FDG PET/CT showed no focal uptake in the thoracic spine, which demonstrated increased Ga DOTATATE activity, owing to overexpression of somatostatin receptors, suggesting spinal cord hemangioblastoma. This case report indicates the significant role of Ga-labeled somatostatin receptor analogs in the diagnosis of hemangioblastoma.


Assuntos
Neoplasias Cerebelares/diagnóstico por imagem , Fluordesoxiglucose F18 , Hemangioblastoma/diagnóstico por imagem , Compostos Organometálicos , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Doença de von Hippel-Lindau/complicações , Adulto , Neoplasias Cerebelares/complicações , Neoplasias Cerebelares/metabolismo , Hemangioblastoma/complicações , Hemangioblastoma/metabolismo , Humanos , Masculino , Receptores de Somatostatina/metabolismo
10.
Cancer Res ; 79(8): 1967-1980, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30862721

RESUMO

A subset of group 3 medulloblastoma frequently harbors amplification or overexpression of MYC lacking additional focal aberrations, yet it remains unclear whether MYC overexpression alone can induce tumorigenesis and which cells give rise to these tumors. Here, we showed that astrocyte progenitors in the early postnatal cerebellum were susceptible to transformation by MYC. The resulting tumors specifically resembled human group 3 medulloblastoma based on histology and gene-expression profiling. Gene-expression analysis of MYC-driven medulloblastoma cells revealed altered glucose metabolic pathways with marked overexpression of lactate dehydrogenase A (LDHA). LDHA abundance correlated positively with MYC expression and was associated with poor prognosis in human group 3 medulloblastoma. Inhibition of LDHA significantly reduced growth of both mouse and human MYC-driven tumors but had little effect on normal cerebellar cells or SHH-associated medulloblastoma. By generating a new mouse model, we demonstrated for the first time that astrocyte progenitors can be transformed by MYC and serve as the cells of origin for group 3 medulloblastoma. Moreover, we identified LDHA as a novel, specific therapeutic target for this devastating disease. SIGNIFICANCE: Insights from a new model identified LDHA as a novel target for group 3 medulloblastoma, paving the way for the development of effective therapies against this disease.


Assuntos
Biomarcadores Tumorais/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Transdução de Sinais , Células-Tronco/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
11.
J Neurooncol ; 142(3): 435-444, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30830680

RESUMO

PURPOSE: Elongation of telomeres is necessary for tumor cell immortalization and senescence escape; neoplastic cells use to alternative pathways to elongate telomeres: telomerase reactivation or a telomerase-independent mechanism termed alternative lengthening of telomeres (ALT). Telomerase and ALT pathway has been explored in adult and pediatric gliomas and medulloblastomas (MDBs); however, these mechanisms were not previously investigated in MDBs metastatic at the onset. Therefore, we analyzed the activation of telomerase and ALT pathway in a homogenous cohort of 43 pediatric metastatic medulloblastomas, to investigate whether telomere elongation could play a role in the biology of metastatic MDB. METHODS: We evaluated telomeres length via telomere-specific fluorescence in situ hybridization (Telo-FISH); we assessed nuclear expression of ATRX by immunohistochemistry (IHC). H3F3A and TERT promoter mutations were analyzed by pyrosequencing, while UTSS methylation status was analyzed via methylation-specific-PCR (MS-PCR). RESULTS: H3F3A mutations were absent in all MDBs, 30% of samples showed ATRX nuclear loss, 18.2% of cases were characterized by TERT promoter mutations, while 60.9% harboured TERT promoter hyper-methylation in the UTSS region. Elongation of telomeres was found in 42.8% of cases. Metastatic MDBs control telomere elongation via telomerase activation (10.7%), induced by TERT promoter mutations in association with UTSS hyper-methylation, and ALT mechanism (32.1%), triggered by ATRX inactivation. Among non-metastatic MDBs, only 5.9% (1/17) showed ATRX nuclear loss with activation of ALT. CONCLUSIONS: Our metastatic cases frequently activate ALT pathway, suggesting that it is a common process for senescence escape in primary metastatic medulloblastomas. Furthermore, the activation of mechanisms for telomere elongation is not restricted to certain molecular subgroups in this high-risk group of MDBs.


Assuntos
Neoplasias Cerebelares/secundário , Meduloblastoma/patologia , Mutação , Regiões Promotoras Genéticas , Telomerase/metabolismo , Homeostase do Telômero , Telômero/genética , Adolescente , Adulto , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , Prognóstico , Telomerase/genética , Adulto Jovem
12.
J Neurooncol ; 142(3): 411-422, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30725256

RESUMO

PURPOSE: Pediatric brain cancer medulloblastoma (MB) standard-of-care results in numerous comorbidities. MB is comprised of distinct molecular subgroups. Group 3 molecular subgroup patients have the highest relapse rates and after standard-of-care have a 20% survival. Group 3 tumors have high expression of GABRA5, which codes for the α5 subunit of the γ-aminobutyric acid type A receptor (GABAAR). We are advancing a therapeutic approach for group 3 based on GABAAR modulation using benzodiazepine-derivatives. METHODS: We performed analysis of GABR and MYC expression in MB tumors and used molecular, cell biological, and whole-cell electrophysiology approaches to establish presence of a functional 'druggable' GABAAR in group 3 cells. RESULTS: Analysis of expression of 763 MB tumors reveals that group 3 tumors share high subgroup-specific and correlative expression of GABR genes, which code for GABAAR subunits α5, ß3 and γ2 and 3. There are ~ 1000 functional α5-GABAARs per group 3 patient-derived cell that mediate a basal chloride-anion efflux of 2 × 109 ions/s. Benzodiazepines, designed to prefer α5-GABAAR, impair group 3 cell viability by enhancing chloride-anion efflux with subtle changes in their structure having significant impact on potency. A potent, non-toxic benzodiazepine ('KRM-II-08') binds to the α5-GABAAR (0.8 µM EC50) enhancing a chloride-anion efflux that induces mitochondrial membrane depolarization and in response, TP53 upregulation and p53, constitutively phosphorylated at S392, cytoplasmic localization. This correlates with pro-apoptotic Bcl-2-associated death promoter protein localization. CONCLUSION: GABRA5 expression can serve as a diagnostic biomarker for group 3 tumors, while α5-GABAAR is a therapeutic target for benzodiazepine binding, enhancing an ion imbalance that induces apoptosis.


Assuntos
Benzodiazepinas/farmacologia , Neoplasias Cerebelares/patologia , Meduloblastoma/patologia , Receptores de GABA-A/química , Regulação Alostérica , Morte Celular/efeitos dos fármacos , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/metabolismo , Perfilação da Expressão Gênica , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/metabolismo , Receptores de GABA-A/metabolismo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
13.
Cancer Res ; 79(5): 905-917, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30674530

RESUMO

Medulloblastoma and central nervous system primitive neuroectodermal tumors (CNS-PNET) are aggressive, poorly differentiated brain tumors with limited effective therapies. Using Sleeping Beauty (SB) transposon mutagenesis, we identified novel genetic drivers of medulloblastoma and CNS-PNET. Cross-species gene expression analyses classified SB-driven tumors into distinct medulloblastoma and CNS-PNET subgroups, indicating they resemble human Sonic hedgehog and group 3 and 4 medulloblastoma and CNS neuroblastoma with FOXR2 activation. This represents the first genetically induced mouse model of CNS-PNET and a rare model of group 3 and 4 medulloblastoma. We identified several putative proto-oncogenes including Arhgap36, Megf10, and Foxr2. Genetic manipulation of these genes demonstrated a robust impact on tumorigenesis in vitro and in vivo. We also determined that FOXR2 interacts with N-MYC, increases C-MYC protein stability, and activates FAK/SRC signaling. Altogether, our study identified several promising therapeutic targets in medulloblastoma and CNS-PNET. SIGNIFICANCE: A transposon-induced mouse model identifies several novel genetic drivers and potential therapeutic targets in medulloblastoma and CNS-PNET.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Cerebelares/genética , Meduloblastoma/genética , Tumores Neuroectodérmicos Primitivos/genética , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Elementos de DNA Transponíveis/genética , Feminino , Fatores de Transcrição Forkhead/genética , Proteínas Ativadoras de GTPase/biossíntese , Proteínas Ativadoras de GTPase/genética , Humanos , Masculino , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Nus , Mutagênese Insercional/métodos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/patologia , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patologia , Prognóstico
14.
Dev Cell ; 48(2): 184-199.e5, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30695697

RESUMO

During cerebellar development, granule neuron progenitors (GNPs) proliferate by transducing Sonic Hedgehog (SHH) signaling via the primary cilium. Precise regulation of ciliogenesis, thus, ensures proper GNP pool expansion. Here, we report that Atoh1, a transcription factor required for GNPs formation, controls the presence of primary cilia, maintaining GNPs responsiveness to SHH. Loss of primary cilia abolishes the ability of Atoh1 to keep GNPs in a proliferative state. Mechanistically, Atoh1 promotes ciliogenesis by transcriptionally regulating Cep131, which facilitates centriolar satellite (CS) clustering to the basal body. Importantly, ectopic expression of Cep131 counteracts the effects of Atoh1 loss in GNPs by restoring proper localization of CS and ciliogenesis. This Atoh1-CS-primary cilium-SHH pro-proliferative pathway is also conserved in SHH-type medulloblastoma, a pediatric brain tumor arising from the GNPs. Together, our data reveal how Atoh1 modulates the primary cilium to regulate GNPs development.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular/fisiologia , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Neurônios/metabolismo , Animais , Neoplasias Encefálicas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Camundongos Transgênicos , Neurogênese
15.
DNA Repair (Amst) ; 74: 70-79, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30606609

RESUMO

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1+/- mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs-/-/Ptch1+/- mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation.


Assuntos
Neoplasias Cerebelares/genética , Reparo do DNA/efeitos da radiação , Meduloblastoma/genética , Neoplasias Induzidas por Radiação/genética , Receptor Patched-1/deficiência , Receptor Patched-1/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/efeitos da radiação , Linhagem Celular Tumoral , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/terapia , Dano ao DNA , Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Helicases/genética , Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Relação Dose-Resposta à Radiação , Recombinação Homóloga/efeitos da radiação , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Meduloblastoma/terapia , Camundongos , Terapia de Alvo Molecular , Mutação , Neoplasias Induzidas por Radiação/metabolismo , Neoplasias Induzidas por Radiação/patologia , Neoplasias Induzidas por Radiação/terapia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Risco , Raios X/efeitos adversos
16.
Cancer Lett ; 445: 24-33, 2019 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-30611741

RESUMO

Medulloblastoma is the most prevalent central nervous system tumor in children. Targeted treatment approaches for patients with high-risk medulloblastoma are needed as current treatment regimens are not curative in many cases and cause significant therapy-related morbidity. Medulloblastoma harboring MYC amplification have the most aggressive clinical course and worst outcome. Targeting the BET protein BRD4 has significant anti-tumor effects in preclinical models of MYC-amplified medulloblastoma, however, in most cases these are not curative. We here assessed the therapeutic efficacy of the orally bioavailable BRD4 inhibitor, MK-8628, in preclinical models of medulloblastoma. MK-8628 showed therapeutic efficacy against in vitro and in vivo models of MYC-amplified medulloblastoma by inducing apoptotic cell death and cell cycle arrest. Gene expression analysis of cells treated with MK-8628 showed that anti-tumor effects were accompanied by significant repression of MYC transcription as well as disruption of MYC-regulated transcriptional programs. Additionally, we found that targeting of MYC protein stability through pharmacological PLK1 inhibition showed synergistic anti-medulloblastoma effects when combined with MK-8628 treatment. Thus, MK-8628 is effective against preclinical high-risk medulloblastoma models and its effects can be enhanced through simultaneous targeting of PLK1.


Assuntos
Acetanilidas/administração & dosagem , Neoplasias Cerebelares/tratamento farmacológico , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Meduloblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-myc/química , Pteridinas/administração & dosagem , Acetanilidas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Sinergismo Farmacológico , Amplificação de Genes , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Pteridinas/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Oncogene ; 38(10): 1702-1716, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30348991

RESUMO

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor, representing 20% of newly diagnosed childhood central nervous system malignancies. Although advances in multimodal therapy yielded a 5-year survivorship of 80%, MB still accounts for the leading cause of childhood cancer mortality. In this work, we describe the epigenetic regulator BMI1 as a novel therapeutic target for the treatment of recurrent human Group 3 MB, a childhood brain tumor for which there is virtually no treatment option beyond palliation. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naive tumors will provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumor. Using a small molecule inhibitor against BMI1, PTC-028, we were able to demonstrate complete ablation of self-renewal of MB stem cells in vitro. When administered to mice xenografted with patient tumors, we observed significant reduction in tumor burden in both local and metastatic compartments and subsequent increased survival, without neurotoxicity. Strikingly, serial in vivo re-transplantation assays demonstrated a marked reduction in tumor initiation ability of recurrent MB cells upon re-transplantation of PTC-028-treated cells into secondary recipient mouse brains. As Group 3 MB is often metastatic and uniformly fatal at recurrence, with no current or planned trials of targeted therapy, an efficacious targeted agent would be rapidly transitioned to clinical trials.


Assuntos
Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Complexo Repressor Polycomb 1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/administração & dosagem , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Criança , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Complexo Repressor Polycomb 1/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Resultado do Tratamento , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Oncol Rep ; 41(1): 178-190, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30320358

RESUMO

Medulloblastomas are among the most frequently diagnosed pediatric solid tumors, and drug resistance remains as the principal cause of treatment failure. Hypoxia and the subsequent activation of hypoxia­inducible factor 1α (HIF­1α) are considered key factors in modulating drug antitumor effectiveness, but the underlying mechanisms in medulloblastomas have not yet been clearly understood. The aim of the present study was to determine whether hypoxia induces resistance to cyclophosphamide (CPA) and ifosfamide (IFA) in DAOY medulloblastoma cells, whether the mechanism is dependent on HIF­1α, and whether involves the modulation of the expression of cytochromes P450 (CYP)2B6, 3A4 and 3A5 and the control of cell proliferation. Monolayer cultures of DAOY medulloblastoma cells were exposed for 24 h to moderate (1% O2) or severe (0.1% O2) hypoxia, and protein expression was evaluated by immunoblotting. Cytotoxicity was studied with the MTT assay and by Annexin V/PI staining and flow cytometry. Cell proliferation was determined by the trypan­blue exclusion assay and cell cycle by propidium iodide staining and flow cytometry. Hypoxia decreased CPA and IFA cytotoxicity in medulloblastoma cells, which correlated with a reduction in the protein levels of CYP2B6, CYP3A4 and CYP3A5 and inhibition of cell proliferation. These responses were dependent on hypoxia­induced HIF­1α activation, as evidenced by chemical inhibition of its transcriptional activity with 2­methoxyestradiol (2­ME), which enhanced the cytotoxic activity of CPA and IFA and increased apoptosis. Our results indicate that by stimulating HIF­1α activity, hypoxia downregulates the expression of CYP2B6, CYP3A4 and CYP3A5, that in turn leads to decreased conversion of CPA and IFA into their active forms and thus to diminished cytotoxicity. These results support that the combination of HIF­1α inhibitors and canonical antineoplastic agents provides a potential therapeutic alternative against medulloblastoma.


Assuntos
Neoplasias Cerebelares/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Resistencia a Medicamentos Antineoplásicos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Meduloblastoma/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Ciclofosfamida/farmacologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ifosfamida/farmacologia
19.
Mol Cancer Res ; 17(1): 186-198, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30224541

RESUMO

Medulloblastomas, the most common malignant pediatric brain tumors, have been genetically defined into four subclasses, namely WNT-activated, Sonic Hedgehog (SHH)-activated, Group 3, and Group 4. Approximately 30% of medulloblastomas have aberrant SHH signaling and thus are referred to as SHH-activated medulloblastoma. The tumor suppressor gene TP53 has been recently recognized as a prognostic marker for patients with SHH-activated medulloblastoma; patients with mutant TP53 have a significantly worse outcome than those with wild-type TP53. It remains unknown whether p53 activity is impaired in SHH-activated, wild-type TP53 medulloblastoma, which is about 80% of the SHH-activated medulloblastomas. Utilizing the homozygous NeuroD2:SmoA1 mouse model with wild-type Trp53, which recapitulates human SHH-activated medulloblastoma, it was discovered that the endogenous Inhibitor 2 of Protein Phosphatase 2A (SET/I2PP2A) suppresses p53 function by promoting accumulation of phospho-MDM2 (S166), an active form of MDM2 that negatively regulates p53. Knockdown of I2PP2A in SmoA1 primary medulloblastoma cells reduced viability and proliferation in a p53-dependent manner, indicating the oncogenic role of I2PP2A. Importantly, this mechanism is conserved in the human medulloblastoma cell line ONS76 with wild-type TP53. Taken together, these findings indicate that p53 activity is inhibited by I2PP2A upstream of PP2A in SHH-activated and TP53-wildtype medulloblastomas. IMPLICATIONS: This study suggests that I2PP2A represents a novel therapeutic option and its targeting could improve the effectiveness of current therapeutic regimens for SHH-activated or other subclasses of medulloblastoma with wild-type TP53.


Assuntos
Neoplasias Cerebelares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Hedgehog/metabolismo , Chaperonas de Histonas/metabolismo , Meduloblastoma/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Adulto , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Técnicas de Silenciamento de Genes , Chaperonas de Histonas/antagonistas & inibidores , Chaperonas de Histonas/genética , Humanos , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Peptídeos/farmacologia , Proteína Supressora de Tumor p53/genética , Regulação para Cima
20.
Exp Neurol ; 312: 33-42, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30452905

RESUMO

The G-protein coupled receptor 37-like 1 (Gpr37l1) is specifically expressed in most astrocytic glial cells, including cerebellar Bergmann astrocytes and interacts with patched 1 (Ptch1), a co-receptor of the sonic hedgehog (Shh)-smoothened (Smo) signaling complex. Gpr37l1 null mutant mice exhibit precocious post-natal cerebellar development, with altered Shh-Smo mitogenic cascade and premature down-regulation of granule cell precursor (GCP) proliferation. Gpr37l1 expression is downregulated in medulloblastoma (MB) and upregulated in glioma and glioblastoma tumors. Shh-associated MBs originate postnatally, from dysregulated hyperproliferation of GCPs in developing cerebellum's external granular layer (EGL), as shown in heterozygous Ptch1+/- knock-out mouse strains that model human MB occurrence and progression. This study investigates cerebellar MB phenotypes in newly produced Gpr37l1, Ptch1 double mutant mice. Natural history analysis shows that Gpr37l1 genetic ablation, in Ptch1+/- model animals, results in marked deferment of post-natal tumor occurrence and decreased incidence of more aggressive tumor types. It is also associated with the delayed and diminished presence of more severe types of hyperplastic lesions in Ptch1+/- mice. Consistently, during early post-natal development Gpr37l1-/-;Ptch1+/- pups exhibit reduction in cerebellar GCP proliferation and EGL thickness and a precocious, sustained expression of wingless-type MMTV integration site member 3 (Wnt3), a specific inhibitor of Shh-induced neuronal mitogenesis, in comparison with Ptch1+/- heterozygous single mutants. These findings highlight the specific involvement of Gpr37l1 in modulating postnatal cerebellar Shh-Ptch1-Smo mitogenic signaling in both normal and pathological conditions. The novel Gpr37l1-/-;Ptch1+/- mouse models may thus be instrumental in the detailed characterization of the initial phases of Shh-associated MB insurgence and development.


Assuntos
Carcinogênese/metabolismo , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Receptor Patched-1/metabolismo , Receptores Acoplados a Proteínas-G/deficiência , Animais , Carcinogênese/genética , Proliferação de Células/fisiologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Feminino , Masculino , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor Patched-1/genética , Receptores Acoplados a Proteínas-G/genética
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