RESUMO
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.
Assuntos
Envelhecimento/genética , Glioma Pontino Intrínseco Difuso/genética , Complexo Repressor Polycomb 1/metabolismo , Astrocitoma/genética , Neoplasias do Tronco Encefálico/tratamento farmacológico , Neoplasias do Tronco Encefálico/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Criança , Pré-Escolar , Cromatina/genética , Glioma Pontino Intrínseco Difuso/tratamento farmacológico , Glioma Pontino Intrínseco Difuso/metabolismo , Epigenômica , Feminino , Glioma/tratamento farmacológico , Glioma/genética , Glioma/patologia , Histonas/metabolismo , Humanos , Lisina/metabolismo , Masculino , Mutação , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Complexo Repressor Polycomb 1/antagonistas & inibidores , Complexo Repressor Polycomb 1/genéticaRESUMO
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.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Tumor Rabdoide/genética , Proteína SMARCB1/genética , Teratoma/genética , Animais , Azepinas/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Linhagem Celular Tumoral , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Senescência Celular/efeitos dos fármacos , Senescência Celular/genética , Cromatina/genética , Cromatina/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Proteínas Proto-Oncogênicas c-myc/genética , Tumor Rabdoide/patologia , Teratoma/patologia , Triazóis/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
RESUMO
Kinase inhibitors are effective cancer therapies, but tumors frequently develop resistance. Current strategies to circumvent resistance target the same or parallel pathways. We report here that targeting a completely different process, autophagy, can overcome multiple BRAF inhibitor resistance mechanisms in brain tumors. BRAFV600Emutations occur in many pediatric brain tumors. We previously reported that these tumors are autophagy-dependent and a patient was successfully treated with the autophagy inhibitor chloroquine after failure of the BRAFV600E inhibitor vemurafenib, suggesting autophagy inhibition overcame the kinase inhibitor resistance. We tested this hypothesis in vemurafenib-resistant brain tumors. Genetic and pharmacological autophagy inhibition overcame molecularly distinct resistance mechanisms, inhibited tumor cell growth, and increased cell death. Patients with resistance had favorable clinical responses when chloroquine was added to vemurafenib. This provides a fundamentally different strategy to circumvent multiple mechanisms of kinase inhibitor resistance that could be rapidly tested in clinical trials in patients with BRAFV600E brain tumors.
Assuntos
Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Cloroquina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Indóis/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Sulfonamidas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Resultado do Tratamento , VemurafenibRESUMO
We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , Cromatina/genética , Epigenômica/métodos , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Tumor Rabdoide/genética , Proteína SMARCB1/genética , Teratoma/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Metilação de DNA , Dasatinibe/farmacologia , Dasatinibe/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Humanos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Tumor Rabdoide/tratamento farmacológico , Teratoma/tratamento farmacológicoRESUMO
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.
Assuntos
Biomarcadores Tumorais/análise , Neoplasias Cerebelares/patologia , Quinase 1 do Ponto de Checagem/biossíntese , Meduloblastoma/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Benzodiazepinonas/farmacologia , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Cerebelares/enzimologia , Neoplasias Cerebelares/mortalidade , Cisplatino/farmacologia , Intervalo Livre de Doença , Genes myc , Humanos , Estimativa de Kaplan-Meier , Meduloblastoma/enzimologia , Meduloblastoma/mortalidade , Prognóstico , Pirazóis/farmacologia , Tiofenos/farmacologia , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Ependymoma (EPN) in childhood is a brain tumor with substantial mortality. Inflammatory response has been identified as a molecular signature of high-risk Group A EPN. To better understand the biology of this phenotype and aid therapeutic development, transcriptomic data from Group A and B EPN patient tumor samples, and additional malignant and normal brain data, were analyzed to identify the mechanism underlying EPN Group A inflammation. Enrichment of IL6 and STAT3 pathway genes were found to distinguish Group A EPN from Group B EPN and other brain tumors, implicating an IL6 activation of STAT3 mechanism. EPN tumor cell growth was shown to be dependent on STAT3 activity, as demonstrated using shRNA knockdown and pharmacologic inhibition of STAT3 that blocked proliferation and induced apoptosis. The inflammatory factors secreted by EPN tumor cells were shown to reprogram myeloid cells, and this paracrine effect was characterized by a significant increase in pSTAT3 and IL8 secretion. Myeloid polarization was shown to be dependent on tumor secretion of IL6, and these effects could be reversed using IL6-neutralizing antibody or IL6 receptor-targeted therapeutic antibody tocilizumab. Polarized myeloid cell production of IL8 drove unpolarized myeloid cells to upregulate CD163 and to produce a number of proinflammatory cytokines. Collectively, these findings indicate that constitutive IL6/STAT3 pathway activation is important in driving tumor growth and inflammatory cross-talk with myeloid cells within the Group A EPN microenvironment. Effective design of Group A-targeted therapy for children with EPN may require reversal of this potentially immunosuppressive and protumor pathway.
Assuntos
Ependimoma/metabolismo , Ependimoma/patologia , Interleucina-6/metabolismo , Fenótipo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/biossíntese , Ependimoma/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Células Mieloides/metabolismo , FosforilaçãoRESUMO
INTRODUCTION: Pediatric adamantinomatous craniopharyngioma (ACP) is a histologically benign but clinically aggressive brain tumor that arises from the sellar/suprasellar region. Despite a high survival rate with current surgical and radiation therapy (75-95 % at 10 years), ACP is associated with debilitating visual, endocrine, neurocognitive and psychological morbidity, resulting in excheptionally poor quality of life for survivors. Identification of an effective pharmacological therapy could drastically decrease morbidity and improve long term outcomes for children with ACP. RESULTS: Using mRNA microarray gene expression analysis of 15 ACP patient samples, we have found several pharmaceutical targets that are significantly and consistently overexpressed in our panel of ACP relative to other pediatric brain tumors, pituitary tumors, normal pituitary and normal brain tissue. Among the most highly expressed are several targets of the kinase inhibitor dasatinib - LCK, EPHA2 and SRC; EGFR pathway targets - AREG, EGFR and ERBB3; and other potentially actionable cancer targets - SHH, MMP9 and MMP12. We confirm by western blot that a subset of these targets is highly expressed in ACP primary tumor samples. CONCLUSIONS: We report here the first published transcriptome for ACP and the identification of targets for rational therapy. Experimental drugs targeting each of these gene products are currently being tested clinically and pre-clinically for the treatment of other tumor types. This study provides a rationale for further pre-clinical and clinical studies of novel pharmacological treatments for ACP. Development of mouse and cell culture models for ACP will further enable the translation of these targets from the lab to the clinic, potentially ushering in a new era in the treatment of ACP.
Assuntos
Craniofaringioma/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Família de Proteínas EGF/metabolismo , Receptores ErbB/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Qualidade de Vida/psicologia , RNA Mensageiro/metabolismo , Receptor EphA2/metabolismo , Receptor ErbB-3/metabolismo , Quinases da Família src/metabolismo , Adolescente , Anfirregulina , Criança , Pré-Escolar , Craniofaringioma/tratamento farmacológico , Craniofaringioma/genética , Família de Proteínas EGF/genética , Receptores ErbB/genética , Feminino , Expressão Gênica , Humanos , Lactente , Recém-Nascido , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Masculino , Análise em Microsséries/métodos , Receptor EphA2/genética , Receptor ErbB-3/genética , Regulação para Cima , Quinases da Família src/genéticaRESUMO
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.
Assuntos
Proliferação de Células , Neoplasias Cerebelares/prevenção & controle , Meduloblastoma/prevenção & controle , Células-Tronco Neoplásicas/patologia , Proteínas Nucleares/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Apoptose , Azepinas/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Ciclo Celular , Proteínas de Ciclo Celular , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/mortalidade , Neoplasias Cerebelares/patologia , Criança , Perfilação da Expressão Gênica , Humanos , Meduloblastoma/genética , Meduloblastoma/mortalidade , Meduloblastoma/patologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Triazóis/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: Autophagy inhibition is a potential therapeutic strategy in cancer, but it is unknown which tumors will benefit. The BRAF(V600E) mutation has been identified as important in pediatric central nervous system (CNS) tumors and is known to affect autophagy in other tumor types. We evaluated CNS tumor cells with BRAF(V600E) and found that mutant (but not wild-type) cells display high rates of induced autophagy, are sensitive to pharmacologic and genetic autophagy inhibition, and display synergy when the clinically used autophagy inhibitor chloroquine was combined with the RAF inhibitor vemurafenib or standard chemotherapeutics. Importantly, we also demonstrate that chloroquine can improve vemurafenib sensitivity in a resistant ex vivo primary culture and provide the first demonstration in a patient harboring the V600E mutation treated with vemurafenib that the addition of chloroquine can improve clinical outcomes. These findings suggest that CNS tumors with BRAF(V600E) are autophagy-dependent and should be targeted with autophagy inhibition in combination with other therapeutic strategies. SIGNIFICANCE: Autophagy inhibition may improve cancer therapy, but it is unclear which tumors will benefit. We found that BRAF mutations cause brain tumor cells to depend on autophagy and display selective chemosensitization with autophagy inhibition. We present a pediatric case in which deliberate autophagy inhibition halted tumor growth and overcame acquired BRAF-inhibition resistance.
Assuntos
Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Sistema Nervoso Central/efeitos dos fármacos , Cloroquina/farmacologia , Indóis/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Sulfonamidas/farmacologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sistema Nervoso Central/patologia , Criança , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Mutação , VemurafenibRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Meduloblastoma/genética , Terapia de Alvo Molecular , Proteínas Nucleares/biossíntese , Proteínas Tirosina Quinases/biossíntese , Apoptose/efeitos dos fármacos , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Pré-Escolar , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano , Genômica , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Proteínas Nucleares/genética , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/genética , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , PirimidinonasRESUMO
Better understanding of ependymoma (EPN) biology at relapse is needed to improve therapy at this critical event. Convincing data exist defining transcriptionally distinct posterior fossa (PF) sub-groups A and B at diagnosis. The clinical and biological consequence of these sub-groups at recurrence has not yet been defined. Genome and transcriptome microarray profiles and clinical variables of matched primary and first recurrent PF EPN pairs were used to identify biologically distinct patterns of progression between EPN sub-groups at recurrence. Key findings were validated by histology and immune function assays. Transcriptomic profiles were partially conserved at recurrence. However, 4 of 14 paired samples changed sub-groups at recurrence, and significant sub-group-specific transcriptomic changes between primary and recurrent tumors were identified, which were predominantly immune-related. Further examination revealed that Group A primary tumors harbor an immune gene signature and cellular functionality consistent with an immunosuppressive phenotype associated with tissue remodeling and wound healing. Conversely, Group B tumors develop an adaptive, antigen-specific immune response signature and increased T-cell infiltration at recurrence. Clinical distinctions between sub-groups become more apparent after first recurrence. Group A tumors were more often sub-totally resected and had a significantly shorter time to subsequent progression and worse overall survival. Minimal tumor-specific genomic changes were observed for either PF Groups A or B at recurrence. Molecular sub-groups of PF EPN convey distinct immunobiologic signatures at diagnosis and recurrence, providing potential biologic rationale to their disparate clinical outcomes. Immunotherapeutic approaches may be warranted, particularly in Group A PF EPN.
Assuntos
Ependimoma/diagnóstico , Ependimoma/imunologia , Neoplasias Infratentoriais/diagnóstico , Neoplasias Infratentoriais/imunologia , Recidiva Local de Neoplasia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Estudos de Coortes , Citocinas/metabolismo , Ependimoma/genética , Ependimoma/cirurgia , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Infratentoriais/genética , Neoplasias Infratentoriais/cirurgia , Masculino , Análise em Microsséries , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Prognóstico , Transcriptoma , Adulto JovemRESUMO
Brainstem gangliogliomas (GGs), often cannot be resected, have a much poorer prognosis than those located in more common supratentorial sites and may benefit from novel therapeutic approaches. Therapeutically targetable BRAF c.1799T>A (p.V600E) (BRAF(V600E) ) mutations are harbored in roughly 50% of collective GGs taken from all anatomical sites. Large numbers of pediatric brainstem GGs, however, have not been specifically assessed and anatomic-and age-restricted assessment of genetic and biological factors are becoming increasingly important. Pediatric brainstem GGs (n = 13), non-brainstem GGs (n = 11) and brainstem pilocytic astrocytomas (PAs) (n = 8) were screened by standard Sanger DNA sequencing of BRAF exon 15. Five of 13 (38%) pediatric GG harbored a definitive BRAF(V600E) mutation, with two others exhibiting an equivocal result by this method. BRAF(V600E) was also seen in five of 11 (45%) non-brainstem GGs and one of eight (13%) brainstem PAs. VE1 immunostaining for BRAF(V600E) showed concordance with sequencing in nine of nine brainstem GGs including the two cases equivocal by Sanger. The equivocal brainstem GGs were subsequently shown to harbor BRAF(V600E) using a novel, more sensitive, RNA-sequencing approach, yielding a final BRAF(V600E) mutation frequency of 54% (seven of 13) in brainstem GGs. BRAF(V600E) -targeted therapeutics should be a consideration for the high percentage of pediatric brainstem GGs refractory to conventional therapies.
Assuntos
Neoplasias do Tronco Encefálico/genética , Ganglioglioma/genética , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Neoplasias do Tronco Encefálico/patologia , Criança , Pré-Escolar , Éxons , Feminino , Ganglioglioma/patologia , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Estudos Retrospectivos , Adulto JovemRESUMO
BACKGROUND: Although, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy. METHODS: Overall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients' clinical characteristics and pathological measures. RESULTS: In several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied. CONCLUSIONS: Deeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.
Assuntos
Neoplasias do Sistema Nervoso Central/genética , MicroRNAs/biossíntese , Neoplasias Embrionárias de Células Germinativas/genética , Adolescente , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias do Sistema Nervoso Central/metabolismo , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Criança , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Masculino , Meduloblastoma/genética , Meduloblastoma/metabolismo , MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/metabolismo , Valor Preditivo dos Testes , Prognóstico , Teratoma/genética , Teratoma/metabolismo , Resultado do TratamentoRESUMO
Despite increasing evidence that antitumor immune control exists in the pediatric brain, these findings have yet to be exploited successfully in the clinic. A barrier to development of immunotherapeutic strategies in pediatric brain tumors is that the immunophenotype of these tumors' microenvironment has not been defined. To address this, the current study used multicolor FACS of disaggregated tumor to systematically characterize the frequency and phenotype of infiltrating immune cells in the most common pediatric brain tumor types. The initial study cohort consisted of 7 pilocytic astrocytoma (PA), 19 ependymoma (EPN), 5 glioblastoma (GBM), 6 medulloblastoma (MED), and 5 nontumor brain (NT) control samples obtained from epilepsy surgery. Immune cell types analyzed included both myeloid and T cell lineages and respective markers of activated or suppressed functional phenotypes. Immune parameters that distinguished each of the tumor types were identified. PA and EPN demonstrated significantly higher infiltrating myeloid and lymphoid cells compared with GBM, MED, or NT. Additionally, PA and EPN conveyed a comparatively activated/classically activated myeloid cell-skewed functional phenotype denoted in particular by HLA-DR and CD64 expression. In contrast, GBM and MED contained progressively fewer infiltrating leukocytes and more muted functional phenotypes similar to that of NT. These findings were recapitulated using whole tumor expression of corresponding immune marker genes in a large gene expression microarray cohort of pediatric brain tumors. The results of this cross-tumor comparative analysis demonstrate that different pediatric brain tumor types exhibit distinct immunophenotypes, implying that specific immunotherapeutic approaches may be most effective for each tumor type.
Assuntos
Neoplasias Encefálicas/classificação , Neoplasias Encefálicas/imunologia , Imunofenotipagem , Células Mieloides/imunologia , Linfócitos T/imunologia , Adolescente , Astrocitoma/imunologia , Encéfalo/imunologia , Neoplasias Encefálicas/genética , Criança , Estudos de Coortes , Ependimoma/imunologia , Epilepsia/imunologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glioblastoma/imunologia , Antígenos HLA-DR/metabolismo , Humanos , Meduloblastoma/imunologia , Receptores de IgG/metabolismo , Microambiente TumoralRESUMO
BRAF V600E mutation has been identified in up to 2/3 of pleomorphic xanthoastrocytomas (PXAs), World Health Organization grade II, as well as in varying percentages of PXAs with anaplastic features (PXA-A), gangliogliomas, extracerebellar pilocytic astrocytomas, and, rarely, giant cell glioblastoma multiforme (GC-GBMs). GC-GBMs and epithelioid GBMs (E-GBMs) can be histologically challenging to distinguish from PXA-A. We undertook this study specifically to address whether these 2 tumor types also showed the mutation. We tested our originally reported cohort of 8 E-GBMs and 2 rhabdoid GBMs (R-GBM) as well as 5 new E-GBMs (1 pediatric, 4 adult) and 9 GC-GBMs (2 pediatric, 7 adult) (n=24) for BRAF V600E mutational status. Twenty-one of 24 had sufficient material for IDH-1 immunostaining, which is usually absent in PXAs, PXA-As, and primary GBMs but present in secondary GBMs. Patients ranged in age from 4 to 67 years. BRAF V600E mutation was identified in 7/13 of E-GBMs, including 3 of our original cases; patients with mutation were aged 10 to 50 years. None of the 9 GC-GBMs or 2 R-GBMs manifested this mutation, including pediatric patients. The sole secondary E-GBM was the single case manifesting positive IDH-1 immunoreactivity. A high percentage of E-GBMs manifest BRAF V600E mutation, paralleling PXAs. All R-GBMs and GC-GBMs were negative, although larger multi-institutional cohorts will have to be tested to extend this result. BRAF V600E mutational analyses should be performed on E-GBMs, particularly in all pediatric and young-aged adults, given the potential for BRAF inhibitor therapy in this subset of GBM patients.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Glioblastoma/genética , Glioblastoma/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Microdissecção , Pessoa de Meia-Idade , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Prognostic factors in pilocytic astrocytomas (PAs) and pilomyxoid astrocytomas (PMAs) include extent of resection, location, and age, but no molecular markers have been established. Insulin-like growth factor 2 mRNA binding protein 3 (IMP3, IGF2BP3) is predictive of an unfavorable prognosis in other tumors, including high-grade astrocytomas, but its role in PA/PMA is unknown. This study aimed to determine the expression and prognostic value of IMP3 in pediatric PA/PMAs. Insulin-like growth factor 2 mRNA binding protein 3 protein expression was examined by immunohistochemistry in 77 pediatric PAs (n = 70) and PMAs (n = 7) and scored on a subjective scale. Strong diffuse staining for IMP3 was observed in 31% (24 of 77) of tumors and associated with a shorter progression-free survival (hazard ratio, 2.63; p = 0.008). This cohort confirmed previously identified prognostic factors, including extent of resection, age, and tumor location. Currently, only clinical factors are weighed to stratify risk for patients and to identify those who should receive further therapy. Multivariate analyses identified IMP3 expression as an independent prognostic factor when combined with high-/low-risk stratification (hazard ratio, 2.45; p = 0.016). High IMP3, as assessed by immunohistochemistry, has potential use as an additional predictor of poor prognosis in pediatric PA/PMAs and warrants evaluation in larger cohorts.
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Astrocitoma/metabolismo , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Ligação a RNA/genética , Adolescente , Astrocitoma/genética , Biomarcadores Tumorais/biossíntese , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Prognóstico , Proteínas de Ligação a RNA/biossínteseRESUMO
BACKGROUND: Rhabdoid tumors (RTs) are aggressive tumors of early childhood that occur most often in brain (AT/RTs) or kidney (KRTs). Regardless of location, they are characterized by loss of functional SMARCB1 protein, a component of the SWI/SNF chromatin remodeling complex. The aim of this study was to determine genes and biological process dysregulated in common to both AT/RTs and KRTs. PROCEDURE: Gene expression for AT/RTs was compared to that of other brain tumors and normal brain using microarray data from our lab. Similar analysis was performed for KRTs and other kidney tumors and normal kidney using data from GEO. Dysregulated genes common to both analyses were analyzed for functional significance. RESULTS: Unsupervised hierarchical clustering of RTs identified three major subsets: two comprised of AT/RTs, and one of KRTs. Compared to other tumors, 1,187, 663, and 539 genes were dysregulated in each subset, respectively. Only 14 dysregulated genes were common to all three subsets. Compared to normal tissue, 5,209, 4,275, and 2,841 genes were dysregulated in each subset, with an overlap of 610 dysregulated genes. Among these genes, processes associated with cell proliferation, MYC activation, and epigenetic dysregulation were common to all three RT subsets. CONCLUSIONS: The low overlap of dysregulated genes in AT/RTs and KRTs suggests that factors in addition to SMARCB1 loss play a role in determining subsequent gene expression. Drugs which target cell cycle or epigenetic genes may be useful in all RTs. Additionally, targeted therapies tailored to specific RT subset molecular profiles should be considered.
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Neoplasias Encefálicas/genética , Epigênese Genética/genética , Neoplasias Renais/genética , Tumor Rabdoide/genética , Transcriptoma , Neoplasias Encefálicas/patologia , Ciclo Celular/genética , Análise por Conglomerados , Humanos , Neoplasias Renais/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Tumor Rabdoide/patologiaRESUMO
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 Cerebelares/patologia , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica/fisiologia , Meduloblastoma/patologia , Tolerância a Radiação/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/efeitos da radiação , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Pré-Escolar , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Meduloblastoma/tratamento farmacológico , Camundongos , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/fisiologia , Piperazinas/farmacologia , Piridinas/farmacologia , Interferência de RNA/fisiologia , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/efeitos da radiação , Radiação Ionizante , Tubulina (Proteína)/metabolismoRESUMO
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.