RESUMO
Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60-65 d for cancer organoids generation and from 1-4 weeks for downstream applications. The protocol requires at least 3-6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.
Assuntos
Neoplasias Encefálicas , Neoplasias Cerebelares , Glioma , Células-Tronco Pluripotentes Induzidas , Meduloblastoma , Humanos , Criança , Animais , Camundongos , Meduloblastoma/genética , Meduloblastoma/patologia , Técnicas de Cocultura , Avaliação Pré-Clínica de Medicamentos , Glioma/patologia , Organoides , Prosencéfalo , Diferenciação Celular , Neoplasias Cerebelares/patologiaRESUMO
BACKGROUND: Medulloblastoma is the most frequent brain malignancy of childhood. The current multimodal treatment comes at the expense of serious and often long-lasting side effects. Drug repurposing is a strategy to fast-track anti-cancer therapy with low toxicity. Here, we showed the ability of ß-blockers to potentiate radiotherapy in medulloblastoma with bad prognosis. METHODS: Medulloblastoma cell lines, patient-derived xenograft cells, 3D spheroids and an innovative cerebellar organotypic model were used to identify synergistic interactions between ß-blockers and ionising radiations. Gene expression profiles of ß-adrenergic receptors were analysed in medulloblastoma samples from 240 patients. Signaling pathways were explored by RT-qPCR, RNA interference, western blotting and RNA sequencing. Medulloblastoma cell bioenergetics were evaluated by measuring the oxygen consumption rate, the extracellular acidification rate and superoxide production. FINDINGS: Low concentrations of ß-blockers significantly potentiated clinically relevant radiation protocols. Although patient biopsies showed detectable expression of ß-adrenergic receptors, the ability of the repurposed drugs to potentiate ionising radiations did not result from the inhibition of the canonical signaling pathway. We highlighted that the efficacy of the combinatorial treatment relied on a metabolic catastrophe that deprives medulloblastoma cells of their adaptive bioenergetics capacities. This led to an overproduction of superoxide radicals and ultimately to an increase in ionising radiations-mediated DNA damages. INTERPRETATION: These data provide the evidence of the efficacy of ß-blockers as potentiators of radiotherapy in medulloblastoma, which may help improve the treatment and quality of life of children with high-risk brain tumours. FUNDING: This study was funded by institutional grants and charities.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Criança , Metabolismo Energético , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/radioterapia , Qualidade de Vida , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos beta/uso terapêutico , SuperóxidosRESUMO
Medulloblastoma (MB) is a commonly occurring brain malignancy in adolescence. Currently, the combination of chemotherapy with subsequent irradiation is a regular therapeutic strategy. However, high dosage of chemotherapy is associated with drug resistance and side effects. The long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1), which is frequently overexpressed in diverse human tumors, is correlated with worse survival rate in cancer patients. Currently, the precise roles of NEAT1 in MB and chemoresistance remain unclear. Our study aimed to investigate the biological functions of NEAT1 in cisplatin-resistant medulloblastoma. We report that NEAT1 was significantly upregulated in medulloblastoma patient specimens. Silencing NEAT1 significantly suppressed MB cell proliferation and sensitized MB cells to cisplatin. In cisplatin-resistant MB cell line, DAOY Cis R, NEAT1 expression, and glutamine metabolism were remarkably upregulated in cisplatin-resistant cells. Under low glutamine supply, cisplatin-resistant cells displayed increased cisplatin sensitivity. Bioinformatical analysis and luciferase assay uncovered that NEAT1 functions as a ceRNA of miR-23a-3p to downregulate its expressions in MB cells. Moreover, miR-23a-3p was apparently downregulated in MB patient tissues and cisplatin resistant MB cells. We identified GLS (glutaminase), a glutamine metabolism enzyme, was directly targeted by miR-23a-3p in MB cells. Rescue experiments demonstrated restoration of miR-23a-3p in NEAT1-overexpressing DAOY cisplatin resistant cells successfully overcame the NEAT1-promoted cisplatin resistance by targeting GLS. In general, our results revealed new molecular mechanisms for the lncRNA-NEAT1-mediated cisplatin sensitivity of MB.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , MicroRNAs , RNA Longo não Codificante/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Glutaminase , Glutamina , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismoRESUMO
Medulloblastoma (MB), accounting for nearly 10% of all childhood brain tumors, are implicated with aberrant activation of the Hedgehog (Hh) signaling pathway. Saikosaponin B1 (SSB1) and Saikosaponin D (SSD), two bioactive constituents of Radix Bupleuri, are reported to have many biological activities including anticancer activities. In our work, we evaluated the inhibition of SSB1 and SSD on MB tumor growth in allograft mice and explored the underlying mechanisms. The associated biological activity was investigated in Shh Light II cells, an Hh-responsive fibroblast cell line, using the Dual-Glo® Luciferase Assay System. First, SSB1 (IC50, 241.8 nM) and SSD (IC50, 168.7 nM) inhibited GLI-luciferase activity in Shh Light II cells stimulated with ShhN CM, as well as Gli1 and Ptch1 mRNA expression. In addition, both compounds suppressed the Hh signaling activity provoked by smoothened agonist (SAG) or excessive Smoothened (SMO) expression. Meanwhile, SSB1 and SSD did not inhibit glioma-associated oncogene homolog (GLI) luciferase activity activated by abnormal expression of downstream molecules, suppressor of fuse (SUFU) knockdown or GLI2 overexpression. Consequently, SSB1 (30 mg/kg, ip) and SSD (10 mg/kg, ip) displayed excellent in vivo inhibitory activity in MB allografts, and the tumor growth inhibition ratios were approximately 50% and 70%, respectively. Our findings, thus, identify SSB1 and SSD significantly inhibit tumor growth in MB models by inhibiting the Hedgehog pathway through targeting SMO.
Assuntos
Neoplasias Cerebelares , Meduloblastoma , Aloenxertos/metabolismo , Aloenxertos/patologia , Animais , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Camundongos , Ácido Oleanólico/análogos & derivados , Saponinas , Transdução de Sinais , Proteína GLI1 em Dedos de Zinco/genética , Proteína GLI1 em Dedos de Zinco/metabolismoRESUMO
PURPOSE: Long non-coding RNA (LncRNA) HOX transcript antisense RNA (HOTAIR) and Yin Yang 1 (YY1) are reported to be involved in tumorigenesis. However, the effect and molecular mechanism of HOTAIR on YY1 expression remains poorly understood. The study aimed to investigate the functions and molecular mechanism of LncRNA HOTAIR in medulloblastoma progression. METHODS: qPCR was performed to detect HOTAIR and YY1 mRNA in tissues and cells, as well as that of miR-1 and miR-206 expression levels. Western blot assay was used to test YY1 and EMT-related biomarkers' protein levels. Cell proliferation was tested with CCK-8 assay and colony formation assay. Migration and invasion abilities were tested with Transwell migration and invasion assays. Tumor growth was tested with an in vivo animal study. Cell apoptosis was tested with an Annexin V-FITC/PI kit. Luciferase assay was used to test the luciferase intensity of YY1 and HOTAIR. RNA pull down assay was used to detect the combination between HOTAIR and miR-1/miR-206. RESULTS: In this study, we found that HOTAIR and YY1 were up-regulated in medulloblastoma tissues and cell lines, and HOTAIR increased YY1 expression. The molecular mechanism demonstrated that HOTAIR negatively regulated miR-1 and miR-206 expression, which can directly target YY1 in medulloblastoma cells. Moreover, HOTAIR increased YY1 expression through binding to miR-1 and miR-206. The functional experiments showed that HOTAIR knockdown suppressed medulloblastoma cell proliferation, tumor growth, migration and invasion, and promoted cell apoptosis via the modulation of the miR-1/miR-206-YY1 axis, as well as epithelial to mesenchymal transition (EMT). CONCLUSION: These data indicate that HOTAIR promotes medulloblastoma progression via acting as a competing endogenous RNA (ceRNA) to regulate YY1 expression through binding to miR-1 and miR-206.
Assuntos
Neoplasias Cerebelares/genética , Meduloblastoma/genética , RNA Longo não Codificante/genética , Fator de Transcrição YY1/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Cerebelares/patologia , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Meduloblastoma/patologia , Camundongos , Camundongos Nus , MicroRNAs/genética , Invasividade Neoplásica/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Medulloblastoma is associated with rare hereditary cancer predisposition syndromes; however, consensus medulloblastoma predisposition genes have not been defined and screening guidelines for genetic counselling and testing for paediatric patients are not available. We aimed to assess and define these genes to provide evidence for future screening guidelines. METHODS: In this international, multicentre study, we analysed patients with medulloblastoma from retrospective cohorts (International Cancer Genome Consortium [ICGC] PedBrain, Medulloblastoma Advanced Genomics International Consortium [MAGIC], and the CEFALO series) and from prospective cohorts from four clinical studies (SJMB03, SJMB12, SJYC07, and I-HIT-MED). Whole-genome sequences and exome sequences from blood and tumour samples were analysed for rare damaging germline mutations in cancer predisposition genes. DNA methylation profiling was done to determine consensus molecular subgroups: WNT (MBWNT), SHH (MBSHH), group 3 (MBGroup3), and group 4 (MBGroup4). Medulloblastoma predisposition genes were predicted on the basis of rare variant burden tests against controls without a cancer diagnosis from the Exome Aggregation Consortium (ExAC). Previously defined somatic mutational signatures were used to further classify medulloblastoma genomes into two groups, a clock-like group (signatures 1 and 5) and a homologous recombination repair deficiency-like group (signatures 3 and 8), and chromothripsis was investigated using previously established criteria. Progression-free survival and overall survival were modelled for patients with a genetic predisposition to medulloblastoma. FINDINGS: We included a total of 1022 patients with medulloblastoma from the retrospective cohorts (n=673) and the four prospective studies (n=349), from whom blood samples (n=1022) and tumour samples (n=800) were analysed for germline mutations in 110 cancer predisposition genes. In our rare variant burden analysis, we compared these against 53â105 sequenced controls from ExAC and identified APC, BRCA2, PALB2, PTCH1, SUFU, and TP53 as consensus medulloblastoma predisposition genes according to our rare variant burden analysis and estimated that germline mutations accounted for 6% of medulloblastoma diagnoses in the retrospective cohort. The prevalence of genetic predispositions differed between molecular subgroups in the retrospective cohort and was highest for patients in the MBSHH subgroup (20% in the retrospective cohort). These estimates were replicated in the prospective clinical cohort (germline mutations accounted for 5% of medulloblastoma diagnoses, with the highest prevalence [14%] in the MBSHH subgroup). Patients with germline APC mutations developed MBWNT and accounted for most (five [71%] of seven) cases of MBWNT that had no somatic CTNNB1 exon 3 mutations. Patients with germline mutations in SUFU and PTCH1 mostly developed infant MBSHH. Germline TP53 mutations presented only in childhood patients in the MBSHH subgroup and explained more than half (eight [57%] of 14) of all chromothripsis events in this subgroup. Germline mutations in PALB2 and BRCA2 were observed across the MBSHH, MBGroup3, and MBGroup4 molecular subgroups and were associated with mutational signatures typical of homologous recombination repair deficiency. In patients with a genetic predisposition to medulloblastoma, 5-year progression-free survival was 52% (95% CI 40-69) and 5-year overall survival was 65% (95% CI 52-81); these survival estimates differed significantly across patients with germline mutations in different medulloblastoma predisposition genes. INTERPRETATION: Genetic counselling and testing should be used as a standard-of-care procedure in patients with MBWNT and MBSHH because these patients have the highest prevalence of damaging germline mutations in known cancer predisposition genes. We propose criteria for routine genetic screening for patients with medulloblastoma based on clinical and molecular tumour characteristics. FUNDING: German Cancer Aid; German Federal Ministry of Education and Research; German Childhood Cancer Foundation (Deutsche Kinderkrebsstiftung); European Research Council; National Institutes of Health; Canadian Institutes for Health Research; German Cancer Research Center; St Jude Comprehensive Cancer Center; American Lebanese Syrian Associated Charities; Swiss National Science Foundation; European Molecular Biology Organization; Cancer Research UK; Hertie Foundation; Alexander and Margaret Stewart Trust; V Foundation for Cancer Research; Sontag Foundation; Musicians Against Childhood Cancer; BC Cancer Foundation; Swedish Council for Health, Working Life and Welfare; Swedish Research Council; Swedish Cancer Society; the Swedish Radiation Protection Authority; Danish Strategic Research Council; Swiss Federal Office of Public Health; Swiss Research Foundation on Mobile Communication; Masaryk University; Ministry of Health of the Czech Republic; Research Council of Norway; Genome Canada; Genome BC; Terry Fox Research Institute; Ontario Institute for Cancer Research; Pediatric Oncology Group of Ontario; The Family of Kathleen Lorette and the Clark H Smith Brain Tumour Centre; Montreal Children's Hospital Foundation; The Hospital for Sick Children: Sonia and Arthur Labatt Brain Tumour Research Centre, Chief of Research Fund, Cancer Genetics Program, Garron Family Cancer Centre, MDT's Garron Family Endowment; BC Childhood Cancer Parents Association; Cure Search Foundation; Pediatric Brain Tumor Foundation; Brainchild; and the Government of Ontario.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Cerebelares/genética , Metilação de DNA , Testes Genéticos/métodos , Mutação em Linhagem Germinativa , Meduloblastoma/genética , Modelos Genéticos , Adolescente , Adulto , Neoplasias Cerebelares/mortalidade , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/terapia , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Perfilação da Expressão Gênica , Predisposição Genética para Doença , Hereditariedade , Humanos , Lactente , Masculino , Meduloblastoma/mortalidade , Meduloblastoma/patologia , Meduloblastoma/terapia , Linhagem , Fenótipo , Valor Preditivo dos Testes , Intervalo Livre de Progressão , Estudos Prospectivos , Reprodutibilidade dos Testes , Estudos Retrospectivos , Fatores de Risco , Transcriptoma , Sequenciamento do Exoma , Adulto JovemRESUMO
BACKGROUND: Extraneural metastases are relatively rare manifestations of medulloblastoma. CASE PRESENTATION: We present the case of a young boy with group three MYCN-amplified medulloblastoma. He received multimodal chemotherapy consisting of gross total resection followed by postoperative craniospinal radiation and adjuvant chemotherapy. The patient developed extraneural metastases 4 months after the end of therapy. Literature review identifies the poor prognosis of MYCN-amplified medulloblastomas as well as extraneural metastases; we review the current limitations and future directions of medulloblastoma treatment options. CONCLUSION: To the best of our knowledge, this is the first molecularly characterized report of extraneural metastases of medulloblastoma in a child.
Assuntos
Meduloblastoma/genética , Proteína Proto-Oncogênica N-Myc/genética , Quimioterapia Adjuvante , Pré-Escolar , Radiação Cranioespinal , Ciclofosfamida/uso terapêutico , Relação Dose-Resposta à Radiação , Etoposídeo/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Humanos , Isotretinoína/uso terapêutico , Imageamento por Ressonância Magnética , Masculino , Meduloblastoma/tratamento farmacológico , Meduloblastoma/radioterapia , Período Pós-Operatório , Proteínas Proto-Oncogênicas c-myc/genética , Vincristina/uso terapêuticoRESUMO
BACKGROUND: Medulloblastoma (MB) is the most common pediatric brain tumor. Current treatment regimes consisting of primary surgery followed by radio- and chemotherapy, achieve 5-year overall survival rates of only about 60 %. Therapy-induced endocrine and neurocognitive deficits are common late adverse effects. Thus, improved antitumor strategies are urgently needed. In this study, we combined irradiation (IR) together with epigenetic modifiers and differentiation inducers in a multimodal approach to enhance the efficiency of tumor therapy in MB and also assessed possible late adverse effects on neurogenesis. METHODS: In three human MB cell lines (DAOY, MEB-Med8a, D283-Med) short-time survival (trypan blue exclusion assay), apoptosis, autophagy, cell cycle distribution, formation of gH2AX foci, and long-term reproductive survival (clonogenic assay) were analyzed after treatment with 5-aza-2'-deoxycytidine (5-azadC), valproic acid (VPA), suberanilohydroxamic acid (SAHA), abacavir (ABC), all-trans retinoic acid (ATRA) and resveratrol (RES) alone or combined with 5-aza-dC and/or IR. Effects of combinatorial treatments on neurogenesis were evaluated in cultured murine hippocampal slices from transgenic nestin-CFPnuc C57BL/J6 mice. Life imaging of nestin-positive neural stem cells was conducted at distinct time points for up to 28 days after treatment start. RESULTS: All tested drugs showed a radiosynergistic action on overall clonogenic survival at least in two-outof-three MB cell lines. This effect was pronounced in multimodal treatments combining IR, 5-aza-dC and a second drug. Hereby, ABC and RES induced the strongest reduction of clongenic survival in all three MB cell lines and led to the induction of apoptosis (RES, ABC) and/or autophagy (ABC). Additionally, 5-aza-dC, RES, and ABC increased the S phase cell fraction and induced the formation of gH2AX foci at least in oneout-of-three cell lines. Thereby, the multimodal treatment with 5-aza-dC, IR, and RES or ABC did not change the number of normal neural progenitor cells in murine slice cultures. CONCLUSION: In conclusion, the radiosensitizing capacities of epigenetic and differentiation-inducing drugs presented here suggest that their adjuvant administration might improve MB therapy. Thereby, the combination of 5-aza-dC/IR with ABC and RES seemed to be the most promising to enhance tumor control without affecting the normal neural precursor cells.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/radioterapia , Meduloblastoma/tratamento farmacológico , Meduloblastoma/radioterapia , Radiossensibilizantes/administração & dosagem , Animais , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Neoplasias Cerebelares/genética , Terapia Combinada , Decitabina , Didesoxinucleosídeos/administração & dosagem , Didesoxinucleosídeos/farmacologia , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/efeitos da radiação , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Ácidos Hidroxâmicos/farmacologia , Meduloblastoma/genética , Camundongos , Neurogênese/efeitos dos fármacos , Neurogênese/efeitos da radiação , Radiossensibilizantes/farmacologia , Resveratrol , Estilbenos/administração & dosagem , Estilbenos/farmacologia , Resultado do Tratamento , Tretinoína/administração & dosagem , Tretinoína/farmacologia , Ácido Valproico/administração & dosagem , Ácido Valproico/farmacologia , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although methotrexate (MTX) is the most well-known antifolate included in many standard therapeutic regimens, substantial toxicity limits its wider use, particularly in pediatric oncology. Our study focused on a detailed analysis of MTX effects in cell lines derived from two types of pediatric solid tumors: medulloblastoma and osteosarcoma. The main aim of this study was to analyze the effects of treatment with MTX at concentrations comparable to MTX plasma levels in patients treated with high-dose or low-dose MTX. The results showed that treatment with MTX significantly decreased proliferation activity, inhibited the cell cycle at S-phase and induced apoptosis in Daoy and Saos-2 reference cell lines, which were found to be MTX-sensitive. Furthermore, no difference in these effects was observed following treatment with various doses of MTX ranging from 1 to 40 µM. These findings suggest the possibility of achieving the same outcome with the application of low-dose MTX, an extremely important result, particularly for clinical practice. Another important aspect of treatment with high-dose MTX in clinical practice is the administration of leucovorin (LV) as an antidote to reduce MTX toxicity in normal cells. For this reason, the combined application of MTX and LV was also included in our experiments; however, this application of MTX together with LV did not elicit any detectable effect. The expression analysis of genes involved in the mechanisms of resistance to MTX was a final component of our study, and the results helped us to elucidate the mechanisms of the various responses to MTX among the cell lines included in our study.
Assuntos
Neoplasias Ósseas , Neoplasias Cerebelares , Antagonistas do Ácido Fólico/administração & dosagem , Meduloblastoma , Metotrexato/administração & dosagem , Osteossarcoma , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Cerebelares/enzimologia , Neoplasias Cerebelares/genética , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Citometria de Fluxo , Humanos , Meduloblastoma/enzimologia , Meduloblastoma/genética , Osteossarcoma/enzimologia , Osteossarcoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetra-Hidrofolato Desidrogenase/metabolismoRESUMO
BACKGROUND: New therapeutic targets are needed to eliminate cancer stem cells (CSCs). We hypothesize that direct comparison of paired CSCs and nonstem tumor cells (NSTCs) will facilitate identification of primary "driver" chromosomal aberrations that can serve as diagnostic markers and/or therapeutic targets. METHODS: We applied spectral karyotyping and G-banding to matched pairs of neurospheres (CSC-enriched cultures) and fetal bovine serum-based monolayer cultures (enriched with NSTCs) from 16 patient-derived orthotopic xenograft mouse models, including 9 medulloblastomas (MBs) and 7 high-grade gliomas (HGGs), followed by direct comparison of their numerical and structural abnormalities. RESULTS: Chromosomal aberrations were detected in neurospheres of all 16 models, and 82.0% numerical and 82.4% structural abnormalities were maintained in their matching monolayer cultures. Among the shared abnormalities, recurrent clonal changes were identified including gain of chromosomes 18 and 7 and loss of chromosome 10/10q (5/16 models), isochromosome 17q in 2 MBs, and a new breakpoint of 13q14 in 3 HGGs. Chromothripsis-like evidence was also observed in 3 HGG pairs. Additionally, we noted 20 numerical and 15 structural aberrations that were lost from the neurospheres and found 26 numerical and 23 structural aberrations that were only present in the NSTCs. Compared with MBs, the neurosphere karyotypes of HGG were more complex, with fewer chromosomal aberrations preserved in their matching NSTCs. CONCLUSION: Self-renewing CSCs in MBs and pediatric HGGs harbor recurrent numerical and structural aberrations that were maintained in the matching monolayer cultures. These primary chromosomal changes may represent new markers for anti-CSC therapies.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Cerebelares/genética , Aberrações Cromossômicas , Glioma/genética , Meduloblastoma/genética , Adolescente , Animais , Bovinos , Linhagem Celular Tumoral , Criança , Pré-Escolar , Análise Citogenética , Feminino , Humanos , Lactente , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Soroalbumina Bovina , Transplante HeterólogoRESUMO
Medulloblastoma is a cancer of the cerebellum, for which there is currently no approved targeted therapy. Recent transcriptomics approaches have demonstrated that medulloblastoma is composed of molecularly distinct subgroups, one of which is characterized by activation of the Hedgehog pathway, which in mouse models is sufficient to drive medulloblastoma development. There is thus considerable interest in targeting the Hedgehog pathway for therapeutic benefit in medulloblastoma, particularly given the recent approval of the Hedgehog pathway inhibitor vismodegib for metastatic and locally advanced basal cell carcinoma. Like other molecularly targeted therapies, however, there have been reports of acquired resistance to vismodegib, driven by secondary Hedgehog pathway mutations and potentially by activation of the phosphatidylinositol 3-kinase (PI3K) pathway. Given that acquired resistance to vismodegib may occur as a result of inappropriate PI3K pathway activation, we asked if loss of the PI3K pathway regulator, phosphatase and tensin homologue (Pten), which has been reported to occur in patients within the Hedgehog subgroup, would constitute a mechanism of innate resistance to vismodegib in Hedgehog-driven medulloblastoma. We find that Hedgehog pathway inhibition successfully restrains growth of Pten-deficient medulloblastoma in this mouse model, but does not drive tumor regression, as it does in Pten-wild-type medulloblastoma. Combined inhibition of the Hedgehog and PI3K pathways may lead to superior antitumor activity in PTEN-deficient medulloblastoma in the clinic.
Assuntos
Anilidas/uso terapêutico , Antineoplásicos/uso terapêutico , Neoplasias Cerebelares/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Meduloblastoma/tratamento farmacológico , PTEN Fosfo-Hidrolase/fisiologia , Piridinas/uso terapêutico , Anilidas/farmacologia , Animais , Antineoplásicos/farmacologia , Transformação Celular Neoplásica/genética , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Deleção de Genes , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/genética , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Camundongos Transgênicos , Gravidez , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Hereditary nevoid basal cell carcinoma syndrome (NBCCS) is caused by PTCH1 gene mutations that result in diverse neoplasms including medulloblastoma (MB). Epidemiological studies report reduced pediatric brain tumor risks associated with maternal intake of prenatal vitamins containing folic acid (FA) and FA supplements specifically. We hypothesized that low maternal FA intake during the perigestational period would increase MB incidence in a transgenic NBCCS mouse model, which carries an autosomal dominant mutation in the Ptch1 gene. Female wild-type C57BL/6 mice (n = 126) were randomized to 1 of 3 diets with differing FA amounts: 0.3 mg/kg (low), 2.0 mg/kg (control), and 8.0 mg/kg (high) 1 mo prior to mating with Ptch1 (+/-) C57BL/6 males. Females were maintained on the diet until pup weaning; the pups were then aged for tumor development. Compared to the control group, offspring MB incidence was significantly lower in the low FA group (Hazard Ratio = 0.47; 95% confidence interval 0.27-0.80) at 1 yr. No significant difference in incidence was observed between the control and high FA groups. Low maternal perigestational FA levels may decrease MB incidence in mice genetically predisposed to tumor development. Our results could have implications for prenatal FA intake recommendations in the presence of cancer syndromes.
Assuntos
Síndrome do Nevo Basocelular/tratamento farmacológico , Suplementos Nutricionais , Deficiência de Ácido Fólico/patologia , Ácido Fólico/administração & dosagem , Fenômenos Fisiológicos da Nutrição Materna , Meduloblastoma/tratamento farmacológico , Receptores de Superfície Celular/genética , Animais , Síndrome do Nevo Basocelular/complicações , Síndrome do Nevo Basocelular/genética , Modelos Animais de Doenças , Feminino , Deficiência de Ácido Fólico/complicações , Deficiência de Ácido Fólico/tratamento farmacológico , Predisposição Genética para Doença , Masculino , Meduloblastoma/complicações , Meduloblastoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Receptores Patched , Receptor Patched-1 , Gravidez , Receptores de Superfície Celular/metabolismoRESUMO
BACKGROUND: Medulloblastoma (MB) is the most common malignant brain tumor in childhood with a 5-year survival of approximately 60%. We have recently shown that treatment of human MB cells with 5-aza-2'-deoxycytidine (5-aza-dC) reduces the clonogenic survival significantly. Here, we tested combinatorial effects of 5-aza-dC with other epigenetic (valproic acid, SAHA) and differentiation-inducing drugs (resveratrol, abacavir, retinoic acid) on human MB cells in vitro to intensify the antitumor therapy further. METHODS: Three human MB cell lines were treated with 5-aza-dC alone or in combination for three or six days. Metabolic activity was measured by WST-1 assay. To determine long-term reproductive survival, clonogenic assays were performed. Induction of DNA double-strand break (DSB) repair was measured by γH2AX assay. RESULTS: The applied single drugs, except for ATRA, reduced the metabolic activity dose-dependently in all MB cell lines. Longer treatment times enhanced the reduction of metabolic activity by 5-aza-dC. Combinatorial treatments showed differential, cell line-dependent responses indicating an important impact of the genetic background. 5-Aza-dC together with resveratrol was found to exert the most significant inhibitory effects on metabolic activity in all cell lines. 5-aza-dC alone reduced the clonogenicity of MB cells significantly and induced DSB with no further changes after adjuvant administration of resveratrol. CONCLUSION: The observed significant decrease in metabolic activity by combinatorial treatment of MB cells with 5-aza-dC and resveratrol does not translate into long-term reproductive survival deficiency in vitro. Further studies in animal models are needed to clarify the resveratrol-mediated anticancer mechanisms in vivo.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Epigênese Genética/efeitos dos fármacos , Meduloblastoma/genética , Meduloblastoma/patologia , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Cerebelares/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Decitabina , Relação Dose-Resposta a Droga , Humanos , Meduloblastoma/metabolismo , Gradação de Tumores , Tretinoína/farmacologia , Ensaio Tumoral de Célula-Tronco , Ácido Valproico/farmacologiaRESUMO
Recent improvements in the understanding of brain tumor biology have opened the door to a number of rational therapeutic strategies targeting distinct oncogenic pathways. The successful translation of such "designer drugs" to clinical application depends heavily on effective and expeditious screening methods in relevant disease models. By recapitulating both the underlying genetics and the characteristic tumor-stroma microenvironment of brain cancer, genetically engineered mouse models (GEMMs) may offer distinct advantages over cell culture and xenograft systems in the preclinical testing of promising therapies. This review focuses on recently developed GEMMs for both glioma and medulloblastoma, and discusses their potential use in preclinical trials. Examples showcasing the use of GEMMs in the testing of molecularly targeted therapeutics are given, and relevant topics, such as stem cell biology, in vivo imaging technology and radiotherapy, are also addressed.
Assuntos
Neoplasias Encefálicas/patologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Engenharia Genética/métodos , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Humanos , Meduloblastoma/tratamento farmacológico , Meduloblastoma/genética , Meduloblastoma/patologia , Camundongos , Camundongos TransgênicosRESUMO
Certain risk groups among tumors of the central nervous system (CNS) in children take an almost inevitably fatal course. The elucidation of molecular mechanisms offers hope for improved therapy. Aberrant methylation is common in malignant brain tumors of childhood and may have implications for stratification and therapy. Methylation of p16 (INK4A), p14 (ARF), TIMP3, CDH1, p15 (INK4B )and DAPK1 in medulloblastoma (MB) and ependymoma has been discussed controversially in the literature. DUTT1 and SOCS1 have not previously been analyzed. We examined methylation in MB, sPNET and ependymoma using methylation-specific PCR (MSP), quantitative Combined Bisulfite Restriction Analysis (COBRA) and direct and clone sequencing of bisulfite PCR products. We detected methylation of p16 (INK4A) (17/43), p14 (ARF) (11/42) and TIMP3 (9/44) in MB and others by MSP. CDH1 was not only methylated in MB (31/41), but also in normal controls. Evaluation of MSP results by quantitative COBRA and sequencing yielded methylation between the detection limits of COBRA (1%) and MSP (0.1%). Only p16 (INK4A )and TIMP3 were methylated consistently in medulloblastomas (p16 (INK4A ) 14%, TIMP3 11%) and p16 (INK4A) also in anaplastic ependymomas (1/4 tumors). Methylation ranged from 1-5%. Evaluation of methylation using MSP has thus to be supplemented by quantitative methods. Our analyses raise the issue of the functional significance of low level methylation, which may disturb the delicate growth factor equilibrium within the cell. Therapeutic and diagnostic implications urge into depth analyses of methylation as a mechanism, which might fill some of the gaps of our understanding of brain tumor origin.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Cerebelares/genética , Metilação de DNA , Genes p16 , Meduloblastoma/genética , Tumores Neuroectodérmicos Primitivos/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Adolescente , Adulto , Idoso , Proteínas Reguladoras de Apoptose/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Criança , Pré-Escolar , Proteínas Quinases Associadas com Morte Celular , Feminino , Inativação Gênica , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Receptores Imunológicos/genética , Proteínas RoundaboutRESUMO
Medulloblastoma is an embryonal childhood malignancy with poor prognosis. By screening 4 medulloblastoma cDNA expression libraries (SEREX) with autologous sera, 15 different antigens were identified. These antigens were encoded by 3 novel genes, genes of unknown function (KIAA0445, KIAA1853, KIAA0665, FLJ13942, HSPC213), a proto-oncogene (rab18), candidate tumor suppressor genes (BAP1, PRDM13) and genes encoding a motor protein (kinesin-2), a histone (H2A1.2), the ankyrin residue-rich nasopharyngeal cancer susceptibility protein (NZ16) and the transcription factor TZP, which is homologous to the tumor-associated antigens HCA58 and GLEA2. In a consecutive analysis of serum antibody titers and tumor load, a more than 10-fold increase in serum antibodies against PRDM13 preceded the clinical diagnosis of recurrent tumor growth in a patient with aggressive large cell medulloblastoma. When sera of pediatric patients with cancer (n = 40) and healthy controls (n = 40) were tested for humoral responses against the SEREX-defined antigens, 5 antigens were exclusively recognized by sera from cancer patients. These antigens included a novel rab18 gene product translated from mRNA sequences formerly described as 3' untranslated region. Humoral responses against 2 of the remaining 10 antigens were found preferentially in cancer patients. Antibodies against these antigens were detected in 8/40 and 12/40 cancer patients, respectively, but in only 1 healthy control. The 2 antigens were characterized by a tumor-specific deletion and a tumor-specific mutation, respectively. These findings indicate that the humoral immune response against medulloblastoma is directed against diverse antigens that may be useful as diagnostic markers or targets for immunotherapy.
Assuntos
Anticorpos Antineoplásicos/análise , Antígenos de Neoplasias/imunologia , Autoanticorpos/análise , Neoplasias Cerebelares/genética , DNA Complementar/imunologia , Meduloblastoma/genética , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Adolescente , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Estudos de Casos e Controles , Neoplasias Cerebelares/imunologia , Pré-Escolar , Primers do DNA/química , DNA de Neoplasias/imunologia , Biblioteca Gênica , Histonas/genética , Histonas/metabolismo , Humanos , Lactente , Cinesinas/genética , Cinesinas/metabolismo , Meduloblastoma/imunologia , Dados de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , Homologia de Sequência de Aminoácidos , Estudos Soroepidemiológicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
DMBT1 has been implicated as a candidate tumor suppressor gene on chromosome 10q for brain, gastrointestinal and lung cancer. Homozygous deletion and lack of expression are 2 known mechanisms for inactivating DMBT1. We evaluated whether somatic mutation, which represents a major inactivation mechanism for most tumor suppressor genes, occurs in the DMBT1 gene. A total of 102 primary brain tumors, consisting of 25 glioblastoma multiforme, 24 medulloblastoma and 53 oligodendroglial tumors, were analyzed by conformation-sensitive gel electrophoresis in all 54 coding exons of DMBT1. Twelve different base substitutions were detected in 26 (25%) tumors. Eight base substitutions resulted in amino acid changes and 4 were silent. These base changes were also detected in tumor-matched blood samples, however, indicating that the base variations represent genetic polymorphisms. We also assessed homozygous deletions of the DMBT1 gene in the series and found that 16 of 95 (5 glioblastomas, 5 medulloblastomas, 6 oligodendroglial tumors; total 17%) tumors harbor such alteration. High-quality blood DNA samples were available in 5 tumors carrying homozygous deletion and, using long-range PCR, 3 of these blood samples showed germline hemizygous deletions in a region between introns 10 and 26 of DMBT1. Our results showed that mutation does not play a role in inactivation of DMBT1 in brain tumors. Intragenic homozygous deletion of DMBT1 is common in brain tumors and is likely a result of a germline deletion of 1 allele followed by loss of the second allele during tumor development.
Assuntos
Aglutininas , Neoplasias Encefálicas/genética , Glioblastoma/genética , Meduloblastoma/genética , Mutação , Oligodendroglioma/genética , Receptores de Superfície Celular/genética , Alelos , Proteínas de Ligação ao Cálcio , Cromossomos Humanos Par 10 , DNA/metabolismo , DNA Complementar/metabolismo , Proteínas de Ligação a DNA , Éxons , Deleção de Genes , Homozigoto , Humanos , Íntrons , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , Polimorfismo Genético , Temperatura , Proteínas Supressoras de TumorRESUMO
Medulloblastoma, a malignant tumor arising from the medullary velum, is the most common malignant brain tumor of childhood. Local extension into the cerebellar hemisphere, infiltration of the floor of the fourth ventricle, and seeding into the subarachnoid space are common. Early diagnosis and improved treatment consisting of surgery followed by radiation and chemotherapy for selected high-risk patients has contributed to a dramatic change in survival. This article reviews current treatment strategies and describes new therapies that have the potential to improve the outlook of children with medulloblastoma.