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Background: Atypical teratoid rhabdoid tumors (ATRT) are highly aggressive pediatric brain tumors. The available treatments rely on toxic chemotherapy and radiotherapy, which themselves can cause poor outcomes in young patients. Poly (ADP-ribose) polymerases (PARP), multifunctional enzymes which play an important role in DNA damage repair and genome stability have emerged as a new target in cancer therapy. An FDA-approved drug screen revealed that Rucaparib, a PARP inhibitor, is important for ATRT cell growth. This study aims to investigate the effect of Rucaparib treatment in ATRT. Methods: This study utilized cell viability, colony formation, flow cytometry, western blot, immunofluorescence, and immunohistochemistry assays to investigate Rucaparib's effectiveness in BT16 and MAF737 ATRT cell lines. In vivo, intracranial orthotopic xenograft model of ATRT was used. BT16 cell line was transduced with a luciferase-expressing vector and injected into the cerebellum of athymic nude mice. Animals were treated with Rucaparib by oral gavaging and irradiated with 2 Gy of radiation for 3 consecutive days. Tumor growth was monitored using In Vivo Imaging System. Results: Rucaparib treatment decreased ATRT cell growth, inhibited clonogenic potential of ATRT cells, induced cell cycle arrest and apoptosis, and led to DNA damage accumulation as shown by increased expression of γH2AX. In vivo, Rucaparib treatment decreased tumor growth, sensitized ATRT cells to radiation and significantly increased mice survival. Conclusion: We demonstrated that Rucaparib has potential to be a new therapeutic strategy for ATRT as seen by its ability to decrease ATRT tumor growth both in vitro and in vivo.
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Atypical teratoid rhabdoid tumor (ATRT) is a highly aggressive pediatric brain tumor. Despite radiation, aggressive chemotherapy and autologous stem cell rescue, children usually have a poor survival time. In the present study, the role of TP53/MDM2 interaction in ATRT was investigated. A functional genomic screen identified the TP53/MDM2 axis as a therapeutic target in the central nervous system (CNS) ATRT. Gene expression analysis revealed that all ATRT subgroups expressed high levels of MDM2, which is a negative regulator of TP53. Using cell viability, colony formation and methylcellulose assays it was found that genetic MDM2 inhibition with short hairpin RNA or chemical MDM2 inhibition with small molecule inhibitors, Nutlin3 and idasanutlin (RG7388) decreased the growth of ATRT cell lines. Furthermore, idasanutlin significantly decreased the growth of intracranial orthotopic ATRT brain tumors, as evaluated using T2 MRI, and prolonged survival time relative to control animals. MRI of intracranial tumors showed that diffusion coefficient, an effective marker for successful treatment, significantly increased with idasanutlin treatment showing tumor necrosis/apoptosis. Immunohistochemistry revealed an increased number of caspase3positive cells in the idasanutlin treatment group, confirming the induction of apoptosis in vivo. Using flow cytometry and western blot analysis we show that inhibition of MDM2 enhanced radiation sensitivity in vitro by potentiating DNA damage via the induction of the TP53/Bax/Puma proapoptotic axis. Furthermore, DNA damage was associated with increased mitochondrial reactive oxygen species accumulation. The present study demonstrated that MDM2 expression level was increased in ATRT patient samples and MDM2 inhibition suppressed ATRT cell growth in vitro, and leads to apoptosis in vivo. MDM2 inhibition potentiates DNA damage and sensitizes ATRT cells to radiation. These findings highlight the TP53/MDM2 axis as a rational therapeutic target in CNS ATRT.
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Proteínas Proto-Oncogénicas c-mdm2/efectos de los fármacos , Tolerancia a Radiación/efectos de los fármacos , Tumor Rabdoide/radioterapia , Proteína p53 Supresora de Tumor/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Proliferación Celular/efectos de los fármacos , Colorado , Humanos , Tolerancia a Radiación/genética , Teratoma/radioterapiaRESUMEN
BACKGROUND: Group 3 medulloblastoma (MB) is often accompanied by MYC amplification. PLK1 is an oncogenic kinase that controls cell cycle and proliferation and has been preclinically validated as a cancer therapeutic target. Onvansertib (PCM-075) is a novel, orally available PLK1 inhibitor, which shows tumor growth inhibition in various types of cancer. We aim to explore the effect of onvansertib on MYC-driven medulloblastoma as a monotherapy or in combination with radiation. METHODS: Crisper-Cas9 screen was used to discover essential genes for MB tumor growth. Microarray and immunohistochemistry on pediatric patient samples were performed to examine the expression of PLK1. The effect of onvansertib in vitro was measure by cell viability, colony-forming assays, extreme limiting dilution assay, and RNA-Seq. ALDH activity, cell-cycle distribution, and apoptosis were analyzed by flow cytometry. DNA damage was assessed by immunofluorescence staining. Medulloblastoma xenografts were generated to explore the monotherapy or radio-sensitizing effect. RESULTS: PLK1 is overexpressed in Group 3 MB. The IC50 concentrations of onvansertib in Group 3 MB cell lines were in a low nanomolar range. Onvansertib reduced colony formation, cell proliferation, stem cell renewal and induced G2/M arrest in vitro. Moreover, onvansertib in combination with radiation increased DNA damage and apoptosis compared with radiation treatment alone. The combination radiotherapy resulted in marked tumor regression in xenografts. CONCLUSIONS: These findings demonstrate the efficacy of a novel PLK1 inhibitor onvansertib in vitro and in xenografts of Group 3 MB, which suggests onvansertib is an effective strategy as monotherapy or in combination with radiotherapy in MB.
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Neoplasias Cerebelosas , Meduloblastoma , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Neoplasias Cerebelosas/patología , Niño , Puntos de Control de la Fase G2 del Ciclo Celular , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Meduloblastoma/radioterapia , Piperazinas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Pirazoles , QuinazolinasRESUMEN
Diffuse intrinsic pontine glioma (DIPG) is an incurable brain tumor of childhood characterized by histone mutations at lysine 27, which results in epigenomic dysregulation. There has been a failure to develop effective treatment for this tumor. Using a combined RNAi and chemical screen targeting epigenomic regulators, we identify the polycomb repressive complex 1 (PRC1) component BMI1 as a critical factor for DIPG tumor maintenance in vivo. BMI1 chromatin occupancy is enriched at genes associated with differentiation and tumor suppressors in DIPG cells. Inhibition of BMI1 decreases cell self-renewal and attenuates tumor growth due to induction of senescence. Prolonged BMI1 inhibition induces a senescence-associated secretory phenotype, which promotes tumor recurrence. Clearance of senescent cells using BH3 protein mimetics co-operates with BMI1 inhibition to enhance tumor cell killing in vivo.
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Envejecimiento/genética , Glioma Pontino Intrínseco Difuso/genética , Complejo Represivo Polycomb 1/metabolismo , Astrocitoma/genética , Neoplasias del Tronco Encefálico/tratamiento farmacológico , Neoplasias del Tronco Encefálico/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Niño , Preescolar , Cromatina/genética , Glioma Pontino Intrínseco Difuso/tratamiento farmacológico , Glioma Pontino Intrínseco Difuso/metabolismo , Epigenómica , Femenino , Glioma/tratamiento farmacológico , Glioma/genética , Glioma/patología , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Mutación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Complejo Represivo Polycomb 1/antagonistas & inhibidores , Complejo Represivo Polycomb 1/genéticaRESUMEN
Loss of SMARCB1 is the hallmark genetic event that characterizes rhabdoid tumors in children. Rhabdoid tumors of the brain (ATRT) occur in young children and are particularly challenging with poor long-term survival. SMARCB1 is a member of the SWI/SNF chromatin remodeling complex that is responsible for determining cellular pluripotency and lineage commitment. The mechanisms by which SMARCB1 deletion results in tumorigenesis remain unclear. Recent studies demonstrate that ATRT consists of 3 genomic subgroups with a subset of poor outcome tumors expressing high BMP and MYC pathway activation. Here we show that MYC occupies distinct promoter loci in ATRT compared to embryonic stem (ES) cells. Furthermore, using human ATRT cell lines, patient-derived cell culture, ex vivo patient-derived tumor, and orthotopic xenograft models, we show that MYC inhibition is a molecular vulnerability in SMARCB1-deleted tumors and that such inhibition effectively suppresses BMP and pluripotency-associated genomic programs, attenuates tumor cell self-renewal, promotes senescence, and inhibits ATRT tumor growth in vivo. Transgenic expression of Omomyc (a bona-fide MYC dominant negative) or chemical inhibition of MYC transcriptomic programs with the BET inhibitor JQ1 phenocopy genetic depletion of MYC, effectively restricting ATRT tumor growth and opening a promising therapeutic avenue for rhabdoid tumors in children.
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Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Tumor Rabdoide/genética , Proteína SMARCB1/genética , Teratoma/genética , Animales , Azepinas/farmacología , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Línea Celular Tumoral , Autorrenovación de las Células/efectos de los fármacos , Autorrenovación de las Células/genética , Senescencia Celular/efectos de los fármacos , Senescencia Celular/genética , Cromatina/genética , Cromatina/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-myc/genética , Tumor Rabdoide/patología , Teratoma/patología , Triazoles/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Atypical teratoid rhabdoid tumor (ATRT) is an aggressive and malignant pediatric brain tumor. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and essential for mitosis. Overexpression of PLK1 promotes chromosome instability and aneuploidy by overriding the G2-M DNA damage and spindle checkpoints. Recent studies suggest that targeting PLK1 by small molecule inhibitors is a promising approach to tumor therapy. We investigated the effect of PLK1 inhibition in ATRT. Gene expression analysis showed that PLK1 was overexpressed in ATRT patient samples and tumor cell lines. Genetic inhibition of PLK1 with shRNA potently suppressed ATRT cell growth in vitro. Treatment with the PLK1 inhibitor BI 6727 (Volasertib) significantly decreased cell growth, inhibited clonogenic potential, and induced apoptosis. BI6727 treatment led to G2-M phase arrest, consistent with PLK1's role as a critical regulator of mitosis. Moreover, inhibition of PLK1 by BI6727 suppressed the tumor-sphere formation of ATRT cells. Treatment also significantly decreased levels of the DNA damage proteins Ku80 and RAD51 and increased γ-H2AX expression, indicating that BI 6727 can induce DNA damage. Importantly, BI6727 significantly enhanced radiation sensitivity of ATRT cells. In vivo, BI6727 slowed growth of ATRT tumors and prolonged survival in a xenograft model. PLK1 inhibition is a compelling new therapeutic approach for treating ATRT, and the use of BI6727 should be evaluated in clinical studies.
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Medulloblastoma is the most common type of malignant brain tumor that affects children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients perform poorly with significant morbidity. Gene expression profiling has revealed that monopolar spindle 1 (MPS1) (TTK1) is highly expressed in medulloblastoma patient samples compared to that noted in normal cerebellum. MPS1 is a key regulator of the spindle assembly checkpoint (SAC), a mitotic mechanism specifically required for proper chromosomal alignment and segregation. The SAC can be activated in aneuploid cancer cells and MPS1 is overexpressed in many types of cancers. A previous study has demonstrated the effectiveness of inhibiting MPS1 with small-molecule inhibitors, but the role of MPS1 in medulloblastoma is unknown. In the present study, we demonstrated that MPS1 inhibition by shRNA or with a small-molecule drug, NMS-P715, resulted in decreased cell growth, inhibition of clonogenic potential and induction of apoptosis in cells belonging to both the Shh and group 3 medulloblastoma genomic signature. These findings highlight MPS1 as a rational therapeutic target for medulloblastoma.
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Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/biosíntesis , Meduloblastoma/genética , Terapia Molecular Dirigida , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/patología , Mitosis/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Pirazoles/administración & dosificación , Quinazolinas/administración & dosificaciónRESUMEN
BACKGROUND: Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive, fatal, childhood tumors that arise in the brainstem. DIPGs have no effective treatment, and their location and diffuse nature render them inoperable. Radiation therapy remains the only standard of care for this devastating disease. New therapeutic targets are needed to develop novel therapy for DIPG. METHODS: We examined the expression of PLK1 mRNA in DIPG tumor samples through microarray analysis and found it to be up regulated versus normal pons. Using the DIPG tumor cells, we inhibited PLK1 using a clinically relevant specific inhibitor BI 6727 and evaluated the effects on, proliferation, apoptosis, induction of DNA damage and radio sensitization of the DIPG tumor cells. RESULTS: Treatment of DIPG cell lines with BI 6727, a new generation, highly selective inhibitor of PLK1, resulted in decreased cell proliferation and a marked increase in cellular apoptosis. Cell cycle analysis showed a significant arrest in G2-M phase and a substantial increase in cell death. Treatment also resulted in an increased γH2AX expression, indicating induction of DNA damage. PLK1 inhibition resulted in radiosensitization of DIPG cells. CONCLUSION: These findings suggest that targeting PLK1 with small-molecule inhibitors, in combination with radiation therapy, will hold a novel strategy in the treatment of DIPG that warrants further investigation.
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Neoplasias Encefálicas/genética , Proteínas de Ciclo Celular/genética , Glioma/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Pteridinas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Daño del ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Análisis de Secuencia de ADN/métodos , Regulación hacia Arriba/efectos de los fármacos , Quinasa Tipo Polo 1RESUMEN
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.
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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
Medulloblastoma is a pediatric brain tumor with a variable prognosis due to clinical and genomic heterogeneity. Among the 4 major genomic sub-groups, patients with MYC amplified tumors have a particularly poor prognosis despite therapy with surgery, radiation and chemotherapy. Targeting the MYC oncogene has traditionally been problematic. Here we report that MYC driven medulloblastoma can be targeted by inhibition of the bromodomain protein BRD4. We show that bromodomain inhibition with JQ1 restricts c-MYC driven transcriptional programs in medulloblastoma, suppresses medulloblastoma cell growth and induces a cell cycle arrest. Importantly JQ1 suppresses stem cell associated signaling in medulloblastoma cells and inhibits medulloblastoma tumor cell self-renewal. Additionally JQ1 also promotes senescence in medulloblastoma cells by activating cell cycle kinase inhibitors and inhibiting activity of E2F1. Furthermore BRD4 inhibition displayed an anti-proliferative, pro-senescence effect in a medulloblastoma model in vivo. In clinical samples we found that transcriptional programs suppressed by JQ1 are associated with adverse risk in medulloblastoma patients. Our work indicates that BRD4 inhibition attenuates stem cell signaling in MYC driven medulloblastoma and demonstrates the feasibility BET domain inhibition as a therapeutic approach in vivo.
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Proliferación Celular , Neoplasias Cerebelosas/prevención & control , Meduloblastoma/prevención & control , Células Madre Neoplásicas/patología , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Animales , Apoptosis , Azepinas/farmacología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/mortalidad , Neoplasias Cerebelosas/patología , Niño , Perfilación de la Expresión Génica , Humanos , Meduloblastoma/genética , Meduloblastoma/mortalidad , Meduloblastoma/patología , Ratones , Ratones Desnudos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Triazoles/farmacología , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although recent advances in chemotherapy and radiation have improved outcomes, high-risk patients do poorly with significant morbidity. METHODS: To identify new molecular targets, we performed an integrated genomic analysis using structural and functional methods. Gene expression profiling in 16 medulloblastoma patient samples and subsequent gene set enrichment analysis indicated that cell cycle-related kinases were associated with disease development. In addition a kinome-wide small interfering RNA (siRNA) screen was performed to identify kinases that, when inhibited, could prevent cell proliferation. The two genome-scale analyses were combined to identify key vulnerabilities in medulloblastoma. The inhibition of one of the identified targets was further investigated using RNAi and a small molecule inhibitor. RESULTS: Combining the two analyses revealed that mitosis-related kinases were critical determinants of medulloblastoma cell proliferation. RNA interference (RNAi)-mediated knockdown of WEE1 kinase and other mitotic kinases was sufficient to reduce medulloblastoma cell proliferation. These data prompted us to examine the effects of inhibiting WEE1 by RNAi and by a small molecule inhibitor of WEE1, MK-1775, in medulloblastoma cell lines. MK-1775 inhibited the growth of medulloblastoma cell lines, induced apoptosis and increased DNA damage at nanomolar concentrations. Further, MK-1775 was synergistic with cisplatin in reducing medulloblastoma cell proliferation and resulted in an associated increase in cell death. In vivo MK-1775 suppressed medulloblastoma tumor growth as a single agent. CONCLUSIONS: Taken together, these findings highlight mitotic kinases and, in particular, WEE1 as a rational therapeutic target for medulloblastoma.
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Proteínas de Ciclo Celular/biosíntesis , Meduloblastoma/genética , Terapia Molecular Dirigida , Proteínas Nucleares/biosíntesis , Proteínas Tirosina Quinasas/biosíntesis , Apoptosis/efectos de los fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Preescolar , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genoma Humano , Genómica , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patología , Proteínas Nucleares/genética , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Tirosina Quinasas/genética , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , PirimidinonasRESUMEN
Medulloblastoma accounts for 20 % of all primary pediatric intracranial tumors. Current treatment cures 50-80 % of patients but is associated with significant long-term morbidity and thus new therapeutic targets are needed. One such target is cyclin-dependent kinase 6 (CDK6), a serine/threonine kinase that plays a vital role in cell cycle progression and differentiation. CDK6 is overexpressed in medulloblastoma patients and is associated with an adverse prognosis. To investigate the role of CDK6 in medulloblastoma, we assayed the effect of CDK6 inhibition on proliferation by depleting expression with RNA interference (RNAi) or by inhibiting kinase function with a small molecule inhibitor, PD0332991. Cell proliferation was assessed by colony focus assay or by the xCELLigence system. We then investigated the impact of CDK6 inhibition on differentiation of murine neural stem cells by immunofluorescence of relevant markers. Finally we evaluated the effects of PD0332991 treatment on medulloblastoma cell cycle and radiosensitivity using colony focus assays. Gene expression analysis revealed that CDK6 mRNA expression is higher than normal cerebellum in fifteen out of sixteen medulloblastoma patient samples. Inhibition of CDK6 by RNAi significantly decreased medulloblastoma cell proliferation and colony forming potential. Interestingly, CDK6 inhibition by RNAi increased differentiation in murine neural stem cells. PD0332991 treatment significantly decreased medulloblastoma cell proliferation and led to a G0/G1 cell cycle arrest. Furthermore, PD0332991 pretreatment sensitized medulloblastoma cells to ionizing radiation. Our findings suggest that targeting CDK6 with small molecule inhibitors may prove beneficial in the treatment of medulloblastoma, especially when combined with radiation.
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Neoplasias Cerebelosas/patología , Quinasa 6 Dependiente de la Ciclina/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Meduloblastoma/patología , Tolerancia a Radiación/fisiología , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/efectos de la radiación , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Preescolar , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Masculino , Meduloblastoma/tratamiento farmacológico , Ratones , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/fisiología , Piperazinas/farmacología , Piridinas/farmacología , Interferencia de ARN/fisiología , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Radiación Ionizante , Tubulina (Proteína)/metabolismoRESUMEN
INTRODUCTION: Overexpression of the Polycomb repressive complex 2 (PRC2) subunit Enhancer of Zeste 2 (EZH2) occurs in several malignancies, including prostate cancer, breast cancer, medulloblastoma, and glioblastoma multiforme. Recent evidence suggests that EZH2 may also have a role in rhabdoid tumors. Atypical teratoid/rhabdoid tumor (ATRT) is a rare, high-grade embryonal brain tumor that occurs most commonly in young children and carries a very poor prognosis. ATRTs are characterized by absence of the chromatin remodeling protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes, we investigated the role of EZH2 in ATRT. METHODS: Microarray analysis was used to evaluate expression of EZH2 in ATRT tumor samples. We used shRNA and a chemical inhibitor of EZH2 to examine the impact of EZH2 inhibition on cell growth, proliferation, and tumor cell self-renewal. RESULTS: Here, we show that targeted disruption of EZH2 by RNAi or pharmacologic inhibition strongly impairs ATRT cell growth, suppresses tumor cell self-renewal, induces apoptosis, and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity, we found the cyclin D1-E2F axis to be repressed after EZH2 depletion in ATRT cells. CONCLUSIONS: Our observations provide evidence that EZH2 disruption alters cell cycle progression and may be an important new therapeutic target, particularly in combination with radiation, in ATRT.
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Biomarcadores de Tumor/genética , Proliferación Celular , Senescencia Celular , Complejo Represivo Polycomb 2/metabolismo , Tolerancia a Radiación , Tumor Rabdoide/patología , Teratoma/patología , Adolescente , Apoptosis , Biomarcadores de Tumor/metabolismo , Western Blotting , Ciclo Celular , Cerebelo/metabolismo , Cerebelo/patología , Niño , Proteína Potenciadora del Homólogo Zeste 2 , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Complejo Represivo Polycomb 2/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tumor Rabdoide/metabolismo , Tumor Rabdoide/radioterapia , Esferoides Celulares , Teratoma/metabolismo , Teratoma/radioterapia , Células Tumorales CultivadasRESUMEN
Aberrant expression of microRNAs has been implicated in many cancers. We recently demonstrated differential expression of several microRNAs in medulloblastoma. In this study, the regulation and function of microRNA 218 (miR-218), which is significantly underexpressed in medulloblastoma, was evaluated. Re-expression of miR-218 resulted in a significant decrease in medulloblastoma cell growth, cell colony formation, cell migration, invasion, and tumor sphere size. We used C17.2 neural stem cells as a model to show that increased miR-218 expression results in increased cell differentiation and also decreased malignant transformation when transfected with the oncogene REST. These results suggest that miR-218 acts as a tumor suppressor in medulloblastoma. MicroRNAs function by down-regulating translation of target mRNAs. Targets are determined by imperfect base pairing of the microRNA to the 3'-UTR of the mRNA. To comprehensively identify actual miR-218 targets, medulloblastoma cells overexpressing miR-218 and control cells were subjected to high throughput sequencing of RNA isolated by cross-linking immunoprecipitation, a technique that identifies the mRNAs bound to the RNA-induced silencing complex component protein Argonaute 2. High throughput sequencing of mRNAs identified 618 genes as targets of miR-218 and included both previously validated targets and many targets not predicted computationally. Additional work further confirmed CDK6, RICTOR, and CTSB (cathepsin B) as targets of miR-218 and examined the functional role of one of these targets, CDK6, in medulloblastoma.
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Neoplasias Cerebelosas/genética , Cerebelo/metabolismo , Regulación Neoplásica de la Expresión Génica , Meduloblastoma/genética , MicroARNs/genética , ARN Mensajero/antagonistas & inhibidores , Regiones no Traducidas 3' , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catepsina B/genética , Catepsina B/metabolismo , Línea Celular Tumoral , Movimiento Celular , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patología , Cerebelo/patología , Preescolar , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patología , Ratones , MicroARNs/metabolismo , Invasividad Neoplásica , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Fenotipo , ARN Mensajero/biosíntesis , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteínas Represoras , Transducción de SeñalRESUMEN
INTRODUCTION: There is a growing scientific and public interest in the development of new antiaging drugs for the purposes of extending mean and/or maximum life span, maintaining normal physiological function, and alleviating the onset and severity of age-associated diseases. This review looks at the current screening approaches used to evaluate the efficacy of such compounds, with a particular focus on those that extend life span. AREAS COVERED: This article reviews the current preclinical approaches for assessing longevity therapy including the assessment of antiaging drugs (aging reversal) and geroprotectors (drugs that prevent premature aging and/or slowdown or postpone aging). This article also discusses the methods and the importance in evaluating the anticarcinogenic potential and safety of antitumor drugs. EXPERT OPINION: Based on more than 30 years of experience in the field, the authors believe that the standard testing protocols for antiaging drugs should include the simultaneous evaluation of the drug's safety, as well as its antitumor and anticarcinogenic activity potential. The authors also believe that the principles of international programs for the expert critical evaluation of pharmacological interventions should be created to improve the range of antiaging interventions available for human studies.
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Envejecimiento/efectos de los fármacos , Diseño de Fármacos , Evaluación Preclínica de Medicamentos/métodos , Envejecimiento/fisiología , Animales , Anticarcinógenos/efectos adversos , Anticarcinógenos/farmacología , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Modelos Animales de Enfermedad , Humanos , Longevidad , Ratones , RatasRESUMEN
BACKGROUND: Medulloblastoma is the most common malignant brain tumor in children and remains a therapeutic challenge due to its significant therapy-related morbidity. Polo-like kinase 1 (PLK1) is highly expressed in many cancers and regulates critical steps in mitotic progression. Recent studies suggest that targeting PLK1 with small molecule inhibitors is a promising approach to tumor therapy. METHODS: We examined the expression of PLK1 mRNA in medulloblastoma tumor samples using microarray analysis. The impact of PLK1 on cell proliferation was evaluated by depleting expression with RNA interference (RNAi) or by inhibiting function with the small molecule inhibitor BI 2536. Colony formation studies were performed to examine the impact of BI 2536 on medulloblastoma cell radiosensitivity. In addition, the impact of depleting PLK1 mRNA on tumor-initiating cells was evaluated using tumor sphere assays. RESULTS: Analysis of gene expression in two independent cohorts revealed that PLK1 mRNA is overexpressed in some, but not all, medulloblastoma patient samples when compared to normal cerebellum. Inhibition of PLK1 by RNAi significantly decreased medulloblastoma cell proliferation and clonogenic potential and increased cell apoptosis. Similarly, a low nanomolar concentration of BI 2536, a small molecule inhibitor of PLK1, potently inhibited cell growth, strongly suppressed the colony-forming ability, and increased cellular apoptosis of medulloblastoma cells. Furthermore, BI 2536 pretreatment sensitized medulloblastoma cells to ionizing radiation. Inhibition of PLK1 impaired tumor sphere formation of medulloblastoma cells and decreased the expression of SRY (sex determining region Y)-box 2 (SOX2) mRNA in tumor spheres indicating a possible role in targeting tumor initiating cells. CONCLUSIONS: Our data suggest that targeting PLK1 with small molecule inhibitors, in combination with radiation therapy, is a novel strategy in the treatment of medulloblastoma that warrants further investigation.
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Proteínas de Ciclo Celular/antagonistas & inhibidores , Neoplasias Cerebelosas/radioterapia , Meduloblastoma/radioterapia , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Tolerancia a Radiación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Western Blotting , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias Cerebelosas/enzimología , Neoplasias Cerebelosas/patología , Niño , Preescolar , Estudios de Cohortes , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Masculino , Meduloblastoma/enzimología , Meduloblastoma/patología , Análisis por Micromatrices , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Pteridinas/farmacología , ARN/metabolismo , ARN Mitocondrial , Quinasa Tipo Polo 1RESUMEN
Enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2 that catalyzes the trimethylation of histone H3 on Lys 27, and represses gene transcription. EZH2 enhances cancer-cell proliferation and regulates stem cell maintenance and differentiation. Here, we demonstrate that EZH2 is highly expressed in medulloblastoma, a highly malignant brain tumor of childhood, and this altered expression is correlated with genomic gain of chromosome 7 in a subset of medulloblastoma. Inhibition of EZH2 by RNAi suppresses medulloblastoma tumor cell growth. We show that 3-deazaneplanocin A, a chemical inhibitor of EZH2, can suppress medulloblastoma cell growth partially by inducing apoptosis. Suppression of EZH2 expression diminishes the ability of tumor cells to form spheres in culture and strongly represses the ability of known oncogenes to transform neural stem cells. These findings establish a role of EZH2 in medulloblastoma and identify EZH2 as a potential therapeutic target especially in high-risk tumors.
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Transformación Celular Neoplásica/patología , Neoplasias Cerebelosas/patología , Proteínas de Unión al ADN/metabolismo , Meduloblastoma/patología , Células Madre Neoplásicas/patología , ARN Interferente Pequeño/genética , Factores de Transcripción/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Adolescente , Apoptosis/efectos de los fármacos , Western Blotting , Estudios de Casos y Controles , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Cerebelo/metabolismo , Niño , Ensayo de Unidades Formadoras de Colonias , Variaciones en el Número de Copia de ADN , Metilación de ADN , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteína Potenciadora del Homólogo Zeste 2 , Citometría de Flujo , Histonas/metabolismo , Humanos , Luciferasas/metabolismo , Meduloblastoma/genética , Meduloblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Complejo Represivo Polycomb 2 , Esferoides Celulares , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Células Tumorales CultivadasRESUMEN
Atypical teratoid/rhabdoid tumors (ATRT) are rare, highly malignant, embryonal CNS tumors with a poor prognosis. Therapy relies on highly toxic chemotherapy and radiotherapy. To improve outcomes and decrease morbidity, more targeted therapy is required. Gene expression analysis revealed elevated expression of multiple kinases in ATRT tissues. Aurora Kinase A was one of the candidate kinases. The objective of this study was to evaluate the impact of Aurora Kinase A inhibition in ATRT cell lines. Our analysis revealed that inhibition of Aurora Kinase A induces cell death in ATRT cells and the small molecule inhibitor MLN 8237 sensitizes these cells to radiation. Furthermore, inhibition of Aurora Kinase A resulted in decreased activity of pro-proliferative signaling pathways. These data indicate that inhibition of Aurora Kinase A is a promising small molecule target for ATRT therapy.
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Neoplasias del Sistema Nervioso Central/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Tolerancia a Radiación/genética , Tumor Rabdoide/enzimología , Teratoma/enzimología , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Aurora Quinasa A , Aurora Quinasas , Azepinas/farmacología , Western Blotting , Neoplasias del Sistema Nervioso Central/genética , Inhibidores Enzimáticos/farmacología , Perfilación de la Expresión Génica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Serina-Treonina Quinasas/genética , Pirimidinas/farmacología , Reacción en Cadena en Tiempo Real de la Polimerasa , Tumor Rabdoide/genética , Teratoma/genética , Células Tumorales CultivadasRESUMEN
Atypical teratoid/rhabdoid tumor (ATRT) is a highly malignant central nervous system neoplasm that primarily occurs in children less than 3 years of age. Because of poor outcomes with intense and toxic multimodality treatment, new therapies are urgently needed. Histone deacetylase inhibitors (HDIs) have been evaluated as novel agents for multiple malignancies and have been shown to function as radiosensitizers. They act as epigenetic modifiers and lead to re-expression of inappropriately repressed genes, proteins, and cellular functions. Because of the underlying chromatin remodeling gene mutation in ATRT, HDIs are ideal candidates for therapeutic evaluation. To evaluate the role of HDIs against ATRT in vitro, we assessed the effect of drug treatment on proliferation, apoptosis, and gene expression. In addition, we examined HDI pretreatment as a radiosensitization strategy for ATRT. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium with phenazine methosulfate electron coupling reagent (MTS) and clonogenic assays demonstrated that HDI treatment significantly reduces the proliferative capacity of BT-12 and BT-16 ATRT cells. In addition, the HDI SNDX-275 was able to induce apoptosis in both cell lines and induced p21(Waf1/Cip1) protein expression as measured by Western blot. Evaluation of differential gene expression by microarray and pathway analysis after HDI treatment demonstrated alterations of several key ATRT cellular functions. Finally, we showed that HDI pretreatment effectively potentiates the effect of ionizing radiation on ATRT cells as measured by clonogenic assay. Our findings suggest that the addition of HDIs to ATRT therapy may prove to be beneficial, especially when administered in combination with current treatment modalities, such as radiation.
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Inhibidores de Histona Desacetilasas/farmacología , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones/farmacología , Tumor Rabdoide/terapia , Acetilación/efectos de los fármacos , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Benzamidas/farmacología , Línea Celular Tumoral , Neoplasias del Sistema Nervioso Central/tratamiento farmacológico , Terapia Combinada , Inhibidores Enzimáticos/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Piridinas/farmacología , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Tumor Rabdoide/tratamiento farmacológico , Tumor Rabdoide/radioterapiaRESUMEN
BACKGROUND: MicroRNAs (miRNAs) are a class of short non-coding RNAs that regulate cell homeostasis by inhibiting translation or degrading mRNA of target genes, and thereby can act as tumor suppressor genes or oncogenes. The role of microRNAs in medulloblastoma has only recently been addressed. We hypothesized that microRNAs differentially expressed during normal CNS development might be abnormally regulated in medulloblastoma and are functionally important for medulloblastoma cell growth. METHODOLOGY AND PRINCIPAL FINDINGS: We examined the expression of microRNAs in medulloblastoma and then investigated the functional role of one specific one, miR-128a, in regulating medulloblastoma cell growth. We found that many microRNAs associated with normal neuronal differentiation are significantly down regulated in medulloblastoma. One of these, miR-128a, inhibits growth of medulloblastoma cells by targeting the Bmi-1 oncogene. In addition, miR-128a alters the intracellular redox state of the tumor cells and promotes cellular senescence. CONCLUSIONS AND SIGNIFICANCE: Here we report the novel regulation of reactive oxygen species (ROS) by microRNA 128a via the specific inhibition of the Bmi-1 oncogene. We demonstrate that miR-128a has growth suppressive activity in medulloblastoma and that this activity is partially mediated by targeting Bmi-1. This data has implications for the modulation of redox states in cancer stem cells, which are thought to be resistant to therapy due to their low ROS states.