RESUMEN
Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum. Although abnormal GABAergic receptor activation has been described in MB, studies have not yet elucidated the contribution of receptor-independent GABA metabolism to MB pathogenesis. We find primary MB tumors globally display decreased expression of GABA transaminase (ABAT), the protein responsible for GABA metabolism, compared with normal cerebellum. However, less aggressive WNT and SHH subtypes express higher ABAT levels compared with metastatic G3 and G4 tumors. We show that elevated ABAT expression results in increased GABA catabolism, decreased tumor cell proliferation, and induction of metabolic and histone characteristics mirroring GABAergic neurons. Our studies suggest ABAT expression fluctuates depending on metabolite changes in the tumor microenvironment, with nutrient-poor conditions upregulating ABAT expression. We find metastatic MB cells require ABAT to maintain viability in the metabolite-scarce cerebrospinal fluid by using GABA as an energy source substitute, thereby facilitating leptomeningeal metastasis formation.
Asunto(s)
4-Aminobutirato Transaminasa/metabolismo , Neoplasias Cerebelosas/líquido cefalorraquídeo , Neoplasias Cerebelosas/enzimología , Meduloblastoma/líquido cefalorraquídeo , Meduloblastoma/enzimología , Meninges/patología , Microambiente Tumoral , Acetilación , Animales , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Femenino , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Neoplasias Meníngeas/secundario , Ratones Desnudos , Mitocondrias/metabolismo , Neuronas/metabolismo , Fosforilación Oxidativa , Fenotipo , Ratas , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Medulloblastoma is the most common malignant pediatric brain tumor. Tumors having high levels of c-MYC have the worst clinical prognosis, with only a minority of patients surviving. To address this unmet clinical need, we generated a human neural stem cell model of medulloblastoma that recapitulated the most aggressive subtype phenotypically and by mRNA expression profiling. An in silico analysis of these cells identified mTOR inhibitors as potential therapeutic agents. We hypothesized that the orally bioavailable TORC1/2 kinase inhibitor TAK228 would have activity against MYC-driven medulloblastoma. TAK228 inhibited mTORC1/2, decreased cell growth and caused apoptosis in high-MYC medulloblastoma cell lines. Comprehensive metabolic profiling of medulloblastoma orthotopic xenografts showed upregulation of glutathione compared to matched normal brain. TAK228 suppressed glutathione production. Because glutathione is required to detoxify platinum-containing chemotherapy, we hypothesized that TAK228 would cooperate with carboplatin in medulloblastoma. TAK228 synergized with carboplatin to inhibit cell growth and induce apoptosis and extended survival in orthotopic xenografts of high-MYC medulloblastoma. Brain-penetrant TORC1/2 inhibitors and carboplatin may be an effective combination therapy for high-risk medulloblastoma.
Asunto(s)
Antineoplásicos/uso terapéutico , Carboplatino/uso terapéutico , Proliferación Celular/fisiología , Neoplasias Cerebelosas/patología , Glutatión/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Diana Mecanicista del Complejo 2 de la Rapamicina/antagonistas & inhibidores , Meduloblastoma/patología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-myc/fisiología , Animales , Antineoplásicos/farmacología , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/metabolismo , Femenino , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/enzimología , Meduloblastoma/metabolismo , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
MYC amplification is common in Group 3 medulloblastoma and is associated with poor survival. Group 3 and Group 4 medulloblastomas are also known to have elevated levels of histone H3-lysine 27-tri-methylation (H3K27me3), at least in part due to high expression of the H3K27 methyltransferase enhancer of zest homologue 2 (EZH2), which can be regulated by MYC. We therefore examined whether MYC expression is associated with elevated EZH2 and H3K27me3 in medulloblastoma, and if high-MYC medulloblastomas are particularly sensitive to pharmacological EZH2 blockade. Western blot analysis of low (DAOY, UW228, CB SV40) and high (DAOY-MYC, UW228-MYC, CB-MYC, D425) MYC cell lines showed that higher levels of EZH2 and H3K27me3 were associated with elevated MYC. In fixed medulloblastoma samples examined using immunohistochemistry, most MYC positive tumors also had high H3K27me3, but many MYC negative ones did as well, and the correlation was not statistically significant. All high MYC lines tested were sensitive to the EZH2 inhibitor EPZ6438. Many low MYC lines also grew more slowly in the presence of EPZ6438, although DAOY-MYC cells responded more strongly than parent DAOY cultures with lower MYC levels. We find that higher MYC levels are associated with increased EZH2, and pharmacological blockade of EZH2 is a potential therapeutic strategy for aggressive medulloblastoma with elevated MYC.
Asunto(s)
Neoplasias Cerebelosas/enzimología , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Meduloblastoma/enzimología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Neoplasias Cerebelosas/tratamiento farmacológico , Técnicas de Silenciamiento del Gen , Humanos , Meduloblastoma/tratamiento farmacológicoRESUMEN
BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children. Despite improvement in overall survival rate, it still lacks an effective targeted treatment strategy. The Janus family of cytoplasmic tyrosine kinases (JAKs) and Src kinases, upstream protein kinases of signal transducer and activator of transcription 3 (STAT3), play important roles in medulloblastoma pathogenesis and therefore represent potential therapeutic targets. METHODS: In this report, we examined the inhibitory efficacy of the JAK1/2 inhibitor, ruxolitinib, the JAK3 inhibitor, tofacitinib and two Src inhibitors, KX2-391 and dasatinib. RESULTS: These small molecule drugs significantly reduce cell viability and inhibit cell migration and colony formation in human medulloblastoma cells in vitro. Src inhibitors have more potent efficacy than JAK inhibitors in inhibiting medulloblastoma cell migration ability. The Src inhibitors can inhibit both phosphorylation of STAT3 and Src while JAK inhibitors reduce JAK/STAT3 phosphorylation. We also investigated the combined effect of the Src inhibitor, dasatinib with cisplatin. The results show that dasatinib exerts synergistic effects with cisplatin in human medulloblastoma cells through the inhibition of STAT3 and Src. CONCLUSION: Our results suggest that the small molecule inhibitors of STAT3 upstream kinases, ruxolitinib, tofacitinib, KX2-391, and dasatinib could be novel and attractive candidate drugs for the treatment of human medulloblastoma.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Cerebelosas/tratamiento farmacológico , Sinergismo Farmacológico , Meduloblastoma/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidores , Familia-src Quinasas/antagonistas & inhibidores , Apoptosis , Movimiento Celular , Proliferación Celular , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/patología , Cisplatino/administración & dosificación , Dasatinib/administración & dosificación , Humanos , Meduloblastoma/enzimología , Meduloblastoma/patología , Nitrilos , Piperidinas/administración & dosificación , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Pirroles/administración & dosificación , Células Tumorales CultivadasRESUMEN
Overactivation of the Hedgehog (HH) signaling pathway is implicated in many cancers. In this study, we demonstrate that the small molecule RITA, a p53 activator, effectively downregulates HH signaling in human medulloblastoma and rhabdomyosarcoma cells irrespective of p53. This is mediated by a ROS-independent activation of the MAP kinase JNK. We also show that in vitro RITA sensitized cells to the GLI antagonist GANT61, as co-administration of the two drugs had more pronounced effects on cell proliferation and apoptosis. In vivo administration of RITA or GANT61 suppressed rhabdomyosarcoma xenograft growth in nude mice; however, co-administration did not further enhance tumor suppression, even though cell proliferation was decreased. RITA was more potent than GANT61 in downregulating HH target gene expression; surprisingly, this suppressive effect was almost completely eliminated when the two drugs were administered together. Notably, RNA-seq demonstrated a broader response of pathways involved in cancer cell growth in the combination treatment, providing a plausible interpretation for tumor reduction in the absence of HH signaling downregulation.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Cerebelosas/tratamiento farmacológico , Furanos/farmacología , Proteínas Hedgehog/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Meduloblastoma/tratamiento farmacológico , Rabdomiosarcoma/tratamiento farmacológico , Proteína p53 Supresora de Tumor/metabolismo , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Femenino , Proteínas Hedgehog/genética , Humanos , Meduloblastoma/enzimología , Meduloblastoma/genética , Meduloblastoma/patología , Ratones Desnudos , Piridinas/farmacología , Pirimidinas/farmacología , Rabdomiosarcoma/enzimología , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína con Dedos de Zinc GLI1/análisis , Proteína con Dedos de Zinc GLI1/genéticaAsunto(s)
Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/genética , Sistema de Señalización de MAP Quinasas/genética , Neoplasias Neuroepiteliales/enzimología , Neoplasias Neuroepiteliales/genética , Adulto , Encéfalo/diagnóstico por imagen , Encéfalo/patología , Neoplasias Cerebelosas/diagnóstico por imagen , Neoplasias Cerebelosas/patología , Niño , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias Neuroepiteliales/diagnóstico por imagen , Neoplasias Neuroepiteliales/patología , Adulto JovenRESUMEN
Aerobic glycolysis supports proliferation through unresolved mechanisms. We have previously shown that aerobic glycolysis is required for the regulated proliferation of cerebellar granule neuron progenitors (CGNP) and for the growth of CGNP-derived medulloblastoma. Blocking the initiation of glycolysis via deletion of hexokinase-2 (Hk2) disrupts CGNP proliferation and restricts medulloblastoma growth. Here, we assessed whether disrupting pyruvate kinase-M (Pkm), an enzyme that acts in the terminal steps of glycolysis, would alter CGNP metabolism, proliferation, and tumorigenesis. We observed a dichotomous pattern of PKM expression, in which postmitotic neurons throughout the brain expressed the constitutively active PKM1 isoform, while neural progenitors and medulloblastomas exclusively expressed the less active PKM2. Isoform-specific Pkm2 deletion in CGNPs blocked all Pkm expression. Pkm2-deleted CGNPs showed reduced lactate production and increased SHH-driven proliferation. 13C-flux analysis showed that Pkm2 deletion reduced the flow of glucose carbons into lactate and glutamate without markedly increasing glucose-to-ribose flux. Pkm2 deletion accelerated tumor formation in medulloblastoma-prone ND2:SmoA1 mice, indicating the disrupting PKM releases CGNPs from a tumor-suppressive effect. These findings show that distal and proximal disruptions of glycolysis have opposite effects on proliferation, and that efforts to block the oncogenic effect of aerobic glycolysis must target reactions upstream of PKM. Cancer Res; 77(12); 3217-30. ©2017 AACR.
Asunto(s)
Neoplasias Cerebelosas/enzimología , Cerebelo/enzimología , Meduloblastoma/enzimología , Células-Madre Neurales/enzimología , Neurogénesis/fisiología , Piruvato Quinasa/metabolismo , Animales , Western Blotting , Proliferación Celular , Neoplasias Cerebelosas/patología , Cromatografía Liquida , Humanos , Inmunohistoquímica , Espectrometría de Masas , Meduloblastoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Células-Madre Neurales/patología , Reacción en Cadena de la PolimerasaRESUMEN
PURPOSE: Medulloblastoma (MB) is the most common malignant tumor of the central nervous system (CNS) in children. Despite its relative good survival rates, treatment can cause long time sequels and may impair patients' lifespan and quality, making the search for new treatment options still necessary. Polo like kinases (PLKs) constitute a five-member serine/threonine kinases family (PLK 1-5) that regulates different stages during cell cycle. Abnormal PLKs expression has been observed in several cancer types, including MB. As gene regulators, miRNAs have also been described with variable expression in cancer. METHODS: We evaluated gene expression profiles of all PLK family members and related miRNAs (miR-100, miR-126, miR-219, and miR-593*) in MB cell lines and tumor samples. RESULTS: RT-qPCR analysis revealed increased levels of PLK1-4 in all cell lines and in most MB samples, while PLK5 was found underexpressed. In parallel, miR-100 was also found upregulated while miR-129, miR-216, and miR-593* were decreased in MB cell lines. Variable miRNAs expression patterns were observed in MB samples. However, a correlation between miR-100 and PLK4 expression was observed, and associations between miR-100, miR-126, and miR-219 expression and overall and event free survival were also evinced in our cohort. Moreover, despite the lack of association with clinico-pathological features, when comparing primary tumors to those relapsed, we found a consistent decrease on PLK2, miR-219, and miR-598* and an increase on miR-100 and miR-126. CONCLUSION: Specific dysregulation on PLKs and associated miRNAs may be important in MB and can be used to predict prognosis. Although miRNAs sequences are fundamental to predict its target, the cell type may also be consider once that mRNA repertoire can define different roles for specific miRNA in a given cell.
Asunto(s)
Proteínas de Ciclo Celular/genética , Neoplasias Cerebelosas/genética , Regulación Neoplásica de la Expresión Génica/genética , Meduloblastoma/genética , MicroARNs/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Adolescente , Adulto , Factores de Edad , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/mortalidad , Neoplasias Cerebelosas/patología , Niño , Femenino , Humanos , Masculino , Meduloblastoma/enzimología , Meduloblastoma/mortalidad , Meduloblastoma/patología , MicroARNs/genética , Pronóstico , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Estadísticas no Paramétricas , Análisis de Supervivencia , Quinasa Tipo Polo 1RESUMEN
In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development. Our previous studies showed that the ISR is activated in medulloblastoma in both human patients and animal models, and that the decreased ISR via PERK heterozygous deficiency attenuates medulloblastoma formation in Patched1 heterozygous deficient (Ptch1+/-) mice by enhancing apoptosis of pre-malignant granule cell precursors (GCPs) during cell transformation. We showed here that GADD34 heterozygous mutation moderately enhanced the ISR and noticeably increased the incidence of medulloblastoma in adult Ptch1+/- mice. Surprisingly, GADD34 homozygous mutation strongly enhanced the ISR, but significantly decreased the incidence of medulloblastoma in adult Ptch1+/- mice. Intriguingly, GADD34 homozygous mutation significantly enhanced pre-malignant GCP apoptosis in cerebellar hyperplastic lesions and reduced the lesion numbers in young Ptch1+/- mice. Nevertheless, neither GADD34 heterozygous mutation nor GADD34 homozygous mutation had a significant effect on medulloblastoma cells in adult Ptch1+/- mice. Collectively, these data imply the dual role of the ISR, promoting and inhibiting, in medulloblastoma tumorigenesis by regulating apoptosis of pre-malignant GCPs during the course of malignant transformation.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Neoplasias Cerebelosas/enzimología , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/metabolismo , Meduloblastoma/enzimología , Proteína Fosfatasa 1/metabolismo , eIF-2 Quinasa/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Activación Enzimática , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Homocigoto , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Neovascularización Patológica , Receptor Patched-1/deficiencia , Receptor Patched-1/genética , Fenotipo , Fosforilación , Proteína Fosfatasa 1/deficiencia , Proteína Fosfatasa 1/genética , Transducción de Señal , Factores de TiempoRESUMEN
Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.
Asunto(s)
Neoplasias Cerebelosas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Meduloblastoma/genética , Adolescente , Adulto , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/patología , Niño , Preescolar , ADN/genética , ADN (Citosina-5-)-Metiltransferasa 1 , Metilación de ADN/fisiología , ADN Metiltransferasa 3A , Femenino , Humanos , Inmunohistoquímica/métodos , Lactante , Masculino , Meduloblastoma/diagnóstico , Meduloblastoma/enzimología , Meduloblastoma/patología , Adulto Joven , ADN Metiltransferasa 3BRESUMEN
Checkpoint kinase 1 (CHK1) is an integral component of the cell cycle as well as the DNA Damage Response (DDR) pathway. Previous work has demonstrated the effectiveness of inhibiting CHK1 with small-molecule inhibitors, but the role of CHK1 mediated DDR in medulloblastoma is unknown. CHK1, both at the mRNA and protein level, is highly expressed in medulloblastoma and elevated CHK1 expression in Group3 medulloblastoma is an adverse prognostic marker. CHK1 inhibition with the small-molecule drug AZD7762, results in decreased cell growth, increased DNA damage and cell apoptosis. Furthermore, AZD7762 acts in synergy with cisplatin in reducing cell proliferation in medulloblastoma. Similar phenotypic changes were observed with another CHK1 inhibitor, PF477736, as well as genetic knockdown using siRNA against CHK1. Treatments with small-molecule inhibitors of CHK1 profoundly modulated the expression of both upstream and downstream target proteins within the CHK1 signaling pathways. This suggests the presence of a feedback loop in activating CHK1. Overall, our results demonstrate that small-molecule inhibition of CHK1 in combination with, cisplatin, is more advantageous than either treatment alone, especially for Group 3 medulloblastoma, and therefore this combined therapeutic approach serves as an avenue for further investigation.
Asunto(s)
Biomarcadores de Tumor/análisis , Neoplasias Cerebelosas/patología , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/biosíntesis , Meduloblastoma/patología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Benzodiazepinonas/farmacología , Biomarcadores de Tumor/metabolismo , Supervivencia Celular/efectos de los fármacos , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/mortalidad , Cisplatino/farmacología , Supervivencia sin Enfermedad , Genes myc , Humanos , Estimación de Kaplan-Meier , Meduloblastoma/enzimología , Meduloblastoma/mortalidad , Pronóstico , Pirazoles/farmacología , Tiofenos/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum. Here, we determined the role of PERK signaling on medulloblastoma tumorigenesis by assessing its effects on premalignant GCPs and tumor cells. We found that PERK signaling was activated in both premalignant GCPs in young Ptch1(+/-) mice and medulloblastoma cells in adult mice. We demonstrated that PERK haploinsufficiency reduced the incidence of medulloblastomas in Ptch1(+/-) mice. Interestingly, PERK haploinsufficiency enhanced apoptosis of premalignant GCPs in young Ptch1(+/-) mice but had no significant effect on medulloblastoma cells in adult mice. Moreover, we showed that the PERK pathway was activated in medulloblastomas in humans. These results suggest that PERK signaling promotes medulloblastoma tumorigenesis by attenuating apoptosis of premalignant GCPs during the course of malignant transformation.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Neoplasias Cerebelosas/patología , Meduloblastoma/patología , Células-Madre Neurales/patología , eIF-2 Quinasa/metabolismo , Adulto , Animales , Apoptosis , Western Blotting , Carcinogénesis/metabolismo , Carcinogénesis/patología , Transformación Celular Neoplásica/patología , Neoplasias Cerebelosas/enzimología , Niño , Preescolar , Modelos Animales de Enfermedad , Activación Enzimática/fisiología , Femenino , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Lactante , Masculino , Meduloblastoma/enzimología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Mutantes , Neuronas/patología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Chromothripsis is a recently discovered form of genomic instability, characterized by tens to hundreds of clustered DNA rearrangements resulting from a single dramatic event. Telomere dysfunction has been suggested to play a role in the initiation of this phenomenon, which occurs in a large number of tumor entities. Here, we show that telomere attrition can indeed lead to catastrophic genomic events, and that telomere patterns differ between cells analyzed before and after such genomic catastrophes. Telomere length and telomere stabilization mechanisms diverge between samples with and without chromothripsis in a given tumor subtype. Longitudinal analyses of the evolution of chromothriptic patterns identify either stable patterns between matched primary and relapsed tumors, or loss of the chromothriptic clone in the relapsed specimen. The absence of additional chromothriptic events occurring between the initial tumor and the relapsed tumor sample points to telomere stabilization after the initial chromothriptic event which prevents further shattering of the genome.
Asunto(s)
Neoplasias Cerebelosas/genética , Inestabilidad Genómica , Meduloblastoma/genética , Homeostasis del Telómero , Estudios de Casos y Controles , Neoplasias Cerebelosas/enzimología , Trastornos de los Cromosomas/enzimología , Trastornos de los Cromosomas/genética , Ependimoma/enzimología , Ependimoma/genética , Expresión Génica , Humanos , Meduloblastoma/enzimología , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
The protein phosphatase 2A (PP2A) inhibitor, LB100, has been shown in pre-clinical studies to be an effective chemo- and radio-sensitizer for treatment of various cancers. We investigated effects associated with LB100 treatment alone and in combination with cisplatin for medulloblastoma (MB) in vitro and in vivo in an intracranial xenograft model. We demonstrated that LB100 had a potent effect on MB cells. By itself, LB100 inhibited proliferation and induced significant apoptosis in a range of pediatric MB cell lines. It also attenuated MB cell migration, a pre-requirement for invasion. When used in combination, LB100 enhanced cisplatin-mediated cytotoxic effects. Cell viability in the presence of 1 uM cisplatin alone was 61% (DAOY), 100% (D341), and 58% (D283), but decreased with the addition of 2 µM of LB100 to 26% (DAOY), 67% (D341), and 27% (D283), (p < 0.005). LB100 suppressed phosphorylation of the STAT3 protein and several STAT3 downstream targets. Also, LB100 directly increased cisplatin uptake and overcame cisplatin-resistance in vitro. Finally, LB100 exhibited potent in vivo anti-neoplastic activity in combination with cisplatin in an intracranial xenograft model.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Neoplasias Cerebelosas/tratamiento farmacológico , Cisplatino/farmacología , Meduloblastoma/tratamiento farmacológico , Piperazinas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Línea Celular Tumoral , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/patología , Cisplatino/administración & dosificación , Resistencia a Antineoplásicos , Sinergismo Farmacológico , Humanos , Meduloblastoma/enzimología , Meduloblastoma/patología , Ratones , Ratones SCID , Piperazinas/administración & dosificación , Proteína Fosfatasa 2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Medulloblastomas are the most common pediatric malignant primary brain tumor. To our knowledge, there are no known critical and druggable tyrosine kinases in medulloblastomas, precluding the use of established tyrosine kinase inhibitors that have shown efficacy in other tumor types. We studied the expression of anaplastic lymphoma kinase (ALK), a well-characterized tyrosine kinase and drug target, in a cohort of medulloblastomas by immunohistochemistry, and identified three ALK-positive cases. Mutational analyses did not reveal a definite underlying genetic mechanism for the ALK expression, although one of the cases showed increased ALK copy number. Our findings have clinical implications and warrant further pharmacological and functional studies, as well as evaluation in larger patient cohorts, to fully characterize the value of ALK as a prognostic and predictive therapeutic marker in medulloblastomas.
Asunto(s)
Biomarcadores de Tumor/biosíntesis , Neoplasias Cerebelosas/enzimología , Meduloblastoma/enzimología , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Adolescente , Quinasa de Linfoma Anaplásico , Biomarcadores de Tumor/genética , Neoplasias Cerebelosas/diagnóstico , Neoplasias Cerebelosas/genética , Niño , Preescolar , Estudios de Cohortes , Femenino , Humanos , Masculino , Meduloblastoma/diagnóstico , Meduloblastoma/genética , Pronóstico , Proteínas Tirosina Quinasas Receptoras/genéticaRESUMEN
We present the case of a 72-year old female with a right cerebellar pilocytic astrocytoma WHO grade I with an Isocitrate dehydrogenase 1 (IDH1) R132H mutation. The patient is recurrence-free 6 years after the initial diagnosis. Only one single case with strikingly similar clinicopathological features has been reported before. Otherwise, IDH1/2 mutations are not seen in pilocytic astrocytomas. The clinical implications of these findings are discussed.
Asunto(s)
Astrocitoma/genética , Biomarcadores de Tumor/genética , Neoplasias Cerebelosas/genética , Isocitrato Deshidrogenasa/genética , Mutación , Anciano , Astrocitoma/enzimología , Astrocitoma/patología , Astrocitoma/cirugía , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/cirugía , Análisis Mutacional de ADN , Femenino , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Resultado del TratamientoRESUMEN
The expression of members of the Eph family of receptor tyrosine kinases and their ephrin ligands is frequently dysregulated in medulloblastomas. We assessed the expression and functional role of EphB1 in medulloblastoma cell lines and engineered mouse models. mRNA and protein expression profiling showed expression of EphB1 receptor in the human medulloblastoma cell lines DAOY and UW228. EphB1 downregulation reduced cell growth and viability, decreased the expression of important cell cycle regulators, and increased the percentage of cells in G1 phase of the cell cycle. It also modulated the expression of proliferation, and cell survival markers. In addition, EphB1 knockdown in DAOY cells resulted in significant decrease in migration, which correlated with decreased ß1-integrin expression and levels of phosphorylated Src. Furthermore, EphB1 knockdown enhanced cellular radiosensitization of medulloblastoma cells in culture and in a genetically engineered mouse medulloblastoma model. Using genetically engineered mouse models, we established that genetic loss of EphB1 resulted in a significant delay in tumor recurrence following irradiation compared to EphB1-expressing control tumors. Taken together, our findings establish that EphB1 plays a key role in medulloblastoma cell growth, viability, migration, and radiation sensitivity, making EphB1 a promising therapeutic target.
Asunto(s)
Neoplasias Cerebelosas/patología , Meduloblastoma/patología , Proteínas de Neoplasias/fisiología , Receptor EphB1/fisiología , Animales , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/genética , Supervivencia sin Enfermedad , Fase G1 , Humanos , Integrina beta1/biosíntesis , Integrina beta1/genética , Meduloblastoma/enzimología , Meduloblastoma/genética , Meduloblastoma/radioterapia , Ratones , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Tolerancia a Radiación , Receptor EphB1/deficiencia , Receptor EphB1/genéticaRESUMEN
BACKGROUND: Medulloblastoma is a malignant, invasive embryonal tumor of the cerebellum and accounts for 20% of intracranial tumors in children. QSOX1, whose functions include formation of disulphide bridges, which are needed for correct protein folding and stability, formation of the extracellular matrix, regulation of the redox status and cell cycle control, appears to be involved in apoptosis in pathological states such as cancer. Thus, the aim of this study was to investigate the immunohistochemical expression of QSOX1 in medulloblastomas and nonneoplastic cerebellum. METHODS: Histology blocks of pediatric medulloblastomas were separated and two representative areas of the tumors and non-neoplastic cerebellum samples were used to construct tissue microarrays (TMAs) that were stained with an anti-QSOX1 antibody, and the slides were read using image analysis software. RESULTS: QSOX1 immunoexpression was observed in the non-neoplastic cerebellum samples and the medulloblastoma samples. There was no statistically significant relationship between QSOX1 immunopositivity in the medulloblastoma samples and the clinical and pathological variables. CONCLUSIONS: Although QSOX1 did not prove useful for stratifying patients into risk groups, tumor cells and the fibrillar extracellular matrix were positive for this marker, indicating that this enzyme may be involved in the pathogenesis of medulloblastoma. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1822040654139436.
Asunto(s)
Neoplasias Cerebelosas/enzimología , Meduloblastoma/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Adolescente , Apoptosis/fisiología , Línea Celular Tumoral , Neoplasias Cerebelosas/patología , Niño , Preescolar , Matriz Extracelular/enzimología , Matriz Extracelular/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica/métodos , Lactante , Meduloblastoma/patologíaRESUMEN
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.
Asunto(s)
Neoplasias Óseas , Neoplasias Cerebelosas , Antagonistas del Ácido Fólico/administración & dosificación , Meduloblastoma , Metotrexato/administración & dosificación , Osteosarcoma , Neoplasias Óseas/enzimología , Neoplasias Óseas/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/genética , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Citometría de Flujo , Humanos , Meduloblastoma/enzimología , Meduloblastoma/genética , Osteosarcoma/enzimología , Osteosarcoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tetrahidrofolato Deshidrogenasa/metabolismoRESUMEN
Deregulation of the Phosphoinositide 3-kinase (PI3K)/AKT signalling network is a hallmark of oncogenesis. Also medulloblastoma, the most common malignant brain tumor in children, is characterized by high levels of AKT phosphorylation and activated PI3K signalling in medulloblastoma is associated with enhanced cellular motility, survival and chemoresistency underscoring its role of as a potential therapeutic target. Here we demonstrate that GDC-0941, a highly specific PI3K inhibitor with good clinical tolerability and promising anti-neoplastic activity in adult cancer, also displays anti-proliferative and pro-apoptotic effects in pediatric human medulloblastoma cell lines. Loss in cell viability is accompanied by reduced phosphorylation of AKT, a downstream target of PI3K. Furthermore, we show that GDC-0941 attenuates the migratory capacity of medulloblastoma cells and targets subpopulations expressing the stem cell marker CD133. GDC-0941 also synergizes with the standard medulloblastoma chemotherapeutic etoposide. In an orthotopic xenograft model of the most aggressive human medulloblastoma variant we document that oral adminstration of GDC-0941 impairs tumor growth and significantly prolongs survival. These findings provide a rational to further investigate GDC-0941 alone and in combination with standard chemotherapeutics for medulloblastoma treatment.