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1.
Nature ; 521(7553): 520-4, 2015 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-25807483

RESUMO

Congenital heart disease (CHD) is the most prevalent birth defect, affecting nearly 1% of live births; the incidence of CHD is up to tenfold higher in human fetuses. A genetic contribution is strongly suggested by the association of CHD with chromosome abnormalities and high recurrence risk. Here we report findings from a recessive forward genetic screen in fetal mice, showing that cilia and cilia-transduced cell signalling have important roles in the pathogenesis of CHD. The cilium is an evolutionarily conserved organelle projecting from the cell surface with essential roles in diverse cellular processes. Using echocardiography, we ultrasound scanned 87,355 chemically mutagenized C57BL/6J fetal mice and recovered 218 CHD mouse models. Whole-exome sequencing identified 91 recessive CHD mutations in 61 genes. This included 34 cilia-related genes, 16 genes involved in cilia-transduced cell signalling, and 10 genes regulating vesicular trafficking, a pathway important for ciliogenesis and cell signalling. Surprisingly, many CHD genes encoded interacting proteins, suggesting that an interactome protein network may provide a larger genomic context for CHD pathogenesis. These findings provide novel insights into the potential Mendelian genetic contribution to CHD in the fetal population, a segment of the human population not well studied. We note that the pathways identified show overlap with CHD candidate genes recovered in CHD patients, suggesting that they may have relevance to the more complex genetics of CHD overall. These CHD mouse models and >8,000 incidental mutations have been sperm archived, creating a rich public resource for human disease modelling.


Assuntos
Cílios/patologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Animais , Cílios/diagnóstico por imagem , Cílios/genética , Cílios/fisiologia , Análise Mutacional de DNA , Eletrocardiografia , Exoma/genética , Genes Recessivos , Testes Genéticos , Cardiopatias Congênitas/diagnóstico por imagem , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Transdução de Sinais , Ultrassonografia
2.
Int J Cancer ; 144(11): 2707-2717, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30565669

RESUMO

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.


Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Musculares/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Criança , Conjuntos de Dados como Assunto , Epigênese Genética , Humanos , Neoplasias Musculares/patologia , Músculo Estriado/patologia , Mutação Puntual , Regiões Promotoras Genéticas/genética , Rabdomiossarcoma/patologia , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas ras
3.
J Pathol ; 241(5): 626-637, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28138962

RESUMO

The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalized human myoblasts. Although myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3-FOXO1 and MYCN formed rapidly growing RMS tumours, whereas myoblasts expressing only PAX3-FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3-FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3-FOXO1 expression, and cell lines derived from these recurrent tumours showed transformation in the absence of doxycycline. The doxycycline-independent oncogenicity in these recurrent tumour-derived lines persisted even after PAX3-FOXO1 was inactivated with a CRISPR/Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3-FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour-derived cells without PAX3-FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3-FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3-FOXO1, recurrent tumours can develop by a PAX3-FOXO1-independent mechanism, in which rare cells are postulated to acquire secondary transforming events that were activated or selected by initial PAX3-FOXO1 expression. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.


Assuntos
Carcinogênese/genética , Recidiva Local de Neoplasia/genética , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma/genética , Translocação Genética/genética , Animais , Morte Celular , Diferenciação Celular , Linhagem Celular Tumoral , Doxiciclina/administração & dosagem , Feminino , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Desenvolvimento Muscular , Mioblastos/metabolismo , Mioblastos/patologia , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição PAX3/genética , Fator de Transcrição PAX3/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma/patologia
4.
Hum Mol Genet ; 24(14): 3994-4005, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25877302

RESUMO

Recent studies identified a previously uncharacterized gene C5ORF42 (JBTS17) as a major cause of Joubert syndrome (JBTS), a ciliopathy associated with cerebellar abnormalities and other birth defects. Here we report the first Jbts17 mutant mouse model, Heart Under Glass (Hug), recovered from a forward genetic screen. Exome sequencing identified Hug as a S235P missense mutation in the mouse homolog of JBTS17 (2410089e03rik). Hug mutants exhibit multiple birth defects typical of ciliopathies, including skeletal dysplasia, polydactyly, craniofacial anomalies, kidney cysts and eye defects. Some Hug mutants exhibit congenital heart defects ranging from mild pulmonary stenosis to severe pulmonary atresia. Immunostaining showed JBTS17 is localized in the cilia transition zone. Fibroblasts from Hug mutant mice and a JBTS patient with a JBTS17 mutation showed ciliogenesis defects. Significantly, Hug mutant fibroblasts showed loss of not only JBTS17, but also NPHP1 and CEP290 from the cilia transition zone. Hug mutants exhibited reduced ciliation in the cerebellum. This was associated with reduction in cerebellar foliation. Using a fibroblast wound-healing assay, we showed Hug mutant cells cannot establish cell polarity required for directional cell migration. However, stereocilia patterning was grossly normal in the cochlea, indicating planar cell polarity is not markedly affected. Overall, we showed the JBTS pathophysiology is replicated in the Hug mutant mice harboring a Jbts17 mutation. Our findings demonstrate JBTS17 is a cilia transition zone component that acts upstream of other Joubert syndrome associated transition zone proteins NPHP1 and CEP290, indicating its importance in the pathogenesis of Joubert syndrome.


Assuntos
Doenças Cerebelares/genética , Cerebelo/anormalidades , Proteínas de Membrana/genética , Retina/anormalidades , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Polaridade Celular , Células Cultivadas , Doenças Cerebelares/patologia , Cerebelo/patologia , Cílios , Proteínas do Citoesqueleto , Modelos Animais de Doenças , Anormalidades do Olho/genética , Anormalidades do Olho/patologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Doenças Renais Císticas/genética , Doenças Renais Císticas/patologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Gravidez , Transporte Proteico/genética , Retina/patologia
5.
PLoS Biol ; 11(11): e1001720, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24302887

RESUMO

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.


Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular , Cílios/fisiologia , Proteínas do Citoesqueleto/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Polaridade Celular , Células Cultivadas , Análise Mutacional de DNA , Adesões Focais/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Transporte Proteico , Septinas/metabolismo , Imagem com Lapso de Tempo , Via de Sinalização Wnt , Peixe-Zebra
6.
Mod Pathol ; 28(9): 1214-24, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26226845

RESUMO

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3-FOXO1 or PAX7-FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3-FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3-FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2'-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.


Assuntos
Metilação de DNA/genética , Rabdomiossarcoma/genética , Neoplasias de Tecidos Moles/genética , Análise por Conglomerados , Humanos , Hibridização in Situ Fluorescente , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Fusão Oncogênica , Fatores de Transcrição Box Pareados , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma
7.
Mamm Genome ; 25(3-4): 120-8, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24306492

RESUMO

Mutation mapping in mice can be readily accomplished by genome wide segregation analysis of polymorphic DNA markers. In this study, we showed the efficacy of Ion Torrent next generation sequencing for conducting genome-wide scans to map and identify a mutation causing congenital heart disease in a mouse mutant, Bishu, recovered from a mouse mutagenesis screen. The Bishu mutant line generated in a C57BL/6J (B6) background was intercrossed with another inbred strain, C57BL/10J (B10), and the resulting B6/B10 hybrid offspring were intercrossed to generate mutants used for the mapping analysis. For each mutant sample, a panel of 123 B6/B10 polymorphic SNPs distributed throughout the mouse genome was PCR amplified, bar coded, and then pooled to generate a single library used for Ion Torrent sequencing. Sequencing carried out using the 314 chip yielded >600,000 usable reads. These were aligned and mapped using a custom bioinformatics pipeline. Each SNP was sequenced to a depth >500×, allowing accurate automated calling of the B6/B10 genotypes. This analysis mapped the mutation in Bishu to an interval on the proximal region of mouse chromosome 4. This was confirmed by parallel capillary sequencing of the 123 polymorphic SNPs. Further analysis of genes in the map interval identified a splicing mutation in Dnaic1(c.204+1G>A), an intermediate chain dynein, as the disease causing mutation in Bishu. Overall, our experience shows Ion Torrent amplicon sequencing is high throughput and cost effective for conducting genome-wide mapping analysis and is easily scalable for other high volume genotyping analyses.


Assuntos
Análise Mutacional de DNA/métodos , Modelos Animais de Doenças , Predisposição Genética para Doença/genética , Cardiopatias/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos Mutantes/genética , Animais , Sequência de Bases , Mapeamento Cromossômico/métodos , Cruzamentos Genéticos , Cardiopatias/congênito , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único/genética
8.
Circulation ; 125(18): 2232-42, 2012 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-22499950

RESUMO

BACKGROUND: Patients with congenital heart disease (CHD) and heterotaxy show high postsurgical morbidity/mortality, with some developing respiratory complications. Although this finding is often attributed to the CHD, airway clearance and left-right patterning both require motile cilia function. Thus, airway ciliary dysfunction (CD) similar to that of primary ciliary dyskinesia (PCD) may contribute to increased respiratory complications in heterotaxy patients. METHODS AND RESULTS: We assessed 43 CHD patients with heterotaxy for airway CD. Videomicrocopy was used to examine ciliary motion in nasal tissue, and nasal nitric oxide (nNO) was measured; nNO level is typically low with PCD. Eighteen patients exhibited CD characterized by abnormal ciliary motion and nNO levels below or near the PCD cutoff values. Patients with CD aged >6 years show increased respiratory symptoms similar to those seen in PCD. Sequencing of all 14 known PCD genes in 13 heterotaxy patients with CD, 12 without CD, 10 PCD disease controls, and 13 healthy controls yielded 0.769, 0.417, 1.0, and 0.077 novel variants per patient, respectively. One heterotaxy patient with CD had the PCD causing DNAI1 founder mutation. Another with hyperkinetic ciliary beat had 2 mutations in DNAH11, the only PCD gene known to cause hyperkinetic beat. Among PCD patients, 2 had known PCD causing CCDC39 and CCDC40 mutations. CONCLUSIONS: Our studies show that CHD patients with heterotaxy have substantial risk for CD and increased respiratory disease. Heterotaxy patients with CD were enriched for mutations in PCD genes. Future studies are needed to assess the potential benefit of prescreening and prophylactically treating heterotaxy patients for CD.


Assuntos
Transtornos da Motilidade Ciliar/epidemiologia , Cardiopatias Congênitas/epidemiologia , Síndrome de Heterotaxia/epidemiologia , Anormalidades do Sistema Respiratório/epidemiologia , Adolescente , Adulto , Dineínas do Axonema/genética , Testes Respiratórios , Criança , Pré-Escolar , Transtornos da Motilidade Ciliar/genética , Proteínas do Citoesqueleto , Feminino , Cardiopatias Congênitas/genética , Síndrome de Heterotaxia/genética , Humanos , Lactente , Masculino , Microscopia de Vídeo , Pessoa de Meia-Idade , Mutação , Óxido Nítrico/análise , Prevalência , Proteínas/genética , Anormalidades do Sistema Respiratório/genética , Adulto Jovem
9.
Proc Natl Acad Sci U S A ; 106(9): 3219-24, 2009 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-19218456

RESUMO

Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENU-induced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8(C193R) mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.


Assuntos
Padronização Corporal , Etilnitrosoureia/farmacologia , Proteínas de Membrana/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação/genética , Proteína Nodal/metabolismo , Alinhamento de Sequência , Transdução de Sinais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
10.
Eur J Endocrinol ; 187(1): 185-196, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35861986

RESUMO

Introduction: Recurrent and metastatic pheochromocytoma (PCC) are rare advanced endocrine neoplasms with limited treatment options. Insight into the pathogenic molecular alterations in patients with advanced PCC can provide therapeutic options for precisely targeting dysregulated pathways. Objective: We report the discovery and characterization of a novel BRAF-containing fusion transcript and its downstream molecular alterations in a patient with recurrent PCC with peritoneal seeding (pheochromocytomatosis). Methods: We reviewed the medical record of a patient with pheochromocytomatosis. A comprehensive pan-cancer molecular profiling using next-generation sequencing (NGS) as well as confirmatory real-time-quantitative PCR were performed on surgical specimens. BRAF rearrangement and downstream molecular changes were assayed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Western blot was used to assess the in vitro activation of the mitogen-activated protein kinase (MAPK) signaling pathway and the EMT markers in transfected HEK-293 cells. Results: The NGS analysis of a specimen from a 72-year-old female patient with pheochromocytomatosis showed an in-frame fusion of exon 3 of Glucocorticoid Induced 1 (GLCCI1) to exon 9 of BRAF. The upstream auto-inhibitory domain of BRAF was excluded from the GLCCI1-BRAF fusion; however, the downstream BRAF kinase domain was intact. A BRAF rearrangement was confirmed via a BRAF-specific break-apart FISH assay. Four separate tumor foci harbored GLCCI1-BRAF fusion. IHC demonstrated increased phosphorylated MEK. HEK-293 cells transfected with the GLCCI1-BRAF fusion demonstrated increased phosphorylated MEK as well as higher expression of EMT markers SNAI1 and ZEB1 in vitro. Conclusion: We demonstrate a novel pathogenic gene fusion of GLCCI1 with the oncogenic kinase domain of BRAF, resulting in an activation of the MAPK signaling pathway and EMT markers. Thus, this patient may benefit from clinically available MEK and/or BRAF inhibitors when systemic therapy is indicated. Summary Statement: This report is the first of GLCCI1 fused to BRAF in a human neoplasm and only the second BRAF-containing fusion transcript in PCC. Detailed molecular characterization of PCC can be a valuable tool in managing patients with recurrent PCC and pheochromocytomatosis that represents a significant clinical challenge.


Assuntos
Glucocorticoides , Proteínas Proto-Oncogênicas B-raf , Idoso , Feminino , Células HEK293 , Humanos , Hibridização in Situ Fluorescente/métodos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Recidiva Local de Neoplasia , Proteínas Proto-Oncogênicas B-raf/genética , Transdução de Sinais
11.
Cancer Res ; 81(11): 2930-2942, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33589519

RESUMO

Targeted monotherapies usually fail due to development of resistance by a subgroup of cells that evolve into recurrent tumors. Alveolar rhabdomyosarcoma is an aggressive myogenic soft-tissue cancer that is associated with a characteristic PAX3-FOXO1 gene fusion encoding a novel fusion transcription factor. In our myoblast model of PAX3-FOXO1-induced rhabdomyosarcoma, deinduction of PAX3-FOXO1 simulates a targeted therapy that antagonizes the fusion oncoprotein. This simulated therapy results initially in regression of the primary tumors, but PAX3-FOXO1-independent recurrent tumors eventually form after a delay. We report here that upregulation of FGF8, a direct transcriptional target of PAX3-FOXO1, is a mechanism responsible for PAX3-FOXO1-independent tumor recurrence. As a transcriptional target of PAX3-FOXO1, FGF8 promoted oncogenic activity in PAX3-FOXO1-expressing primary tumors that developed in the myoblast system. In the recurrent tumors forming after PAX3-FOXO1 deinduction, FGF8 expression was necessary and sufficient to induce PAX3-FOXO1-independent tumor growth through an autocrine mechanism. FGF8 was also expressed in human PAX3-FOXO1-expressing rhabdomyosarcoma cell lines and contributed to proliferation and transformation. In a human rhabdomyosarcoma cell line with reduced PAX3-FOXO1 expression, FGF8 upregulation rescued oncogenicity and simulated recurrence after PAX3-FOXO1-targeted therapy. We propose that deregulated expression of a PAX3-FOXO1 transcriptional target can generate resistance to therapy directed against this oncogenic transcription factor and postulate that this resistance mechanism may ultimately be countered by therapeutic approaches that antagonize the corresponding downstream pathways. SIGNIFICANCE: In a model of cancer initiated by a fusion transcription factor, constitutive activation of a downstream transcriptional target leads to fusion oncoprotein-independent recurrences, thereby highlighting a novel progression mechanism and therapeutic target.


Assuntos
Biomarcadores Tumorais/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Regulação Neoplásica da Expressão Gênica , Recidiva Local de Neoplasia/patologia , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Feminino , Fator 8 de Crescimento de Fibroblasto/genética , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/metabolismo , Proteínas de Fusão Oncogênica/genética , Fatores de Transcrição Box Pareados/genética , Rabdomiossarcoma/genética , Rabdomiossarcoma/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Clin Invest ; 131(15)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34166228

RESUMO

The 12q13-q14 chromosomal region is recurrently amplified in 25% of fusion-positive (FP) rhabdomyosarcoma (RMS) cases and is associated with a poor prognosis. To identify amplified oncogenes in FP RMS, we compared the size, gene composition, and expression of 12q13-q14 amplicons in FP RMS with those of other cancer categories (glioblastoma multiforme, lung adenocarcinoma, and liposarcoma) in which 12q13-q14 amplification frequently occurs. We uncovered a 0.2 Mb region that is commonly amplified across these cancers and includes CDK4 and 6 other genes that are overexpressed in amplicon-positive samples. Additionally, we identified a 0.5 Mb segment that is only recurrently amplified in FP RMS and includes 4 genes that are overexpressed in amplicon-positive RMS. Among these genes, only serine hydroxymethyltransferase 2 (SHMT2) was overexpressed at the protein level in an amplicon-positive RMS cell line. SHMT2 knockdown in amplicon-positive RMS cells suppressed growth, transformation, and tumorigenesis, whereas overexpression in amplicon-negative RMS cells promoted these phenotypes. High SHMT2 expression reduced sensitivity of FP RMS cells to SHIN1, a direct SHMT2 inhibitor, but sensitized cells to pemetrexed, an inhibitor of the folate cycle. In conclusion, our study demonstrates that SHMT2 contributes to tumorigenesis in FP RMS and that SHMT2 amplification predicts differential response to drugs targeting this metabolic pathway.


Assuntos
Carcinogênese , Cromossomos Humanos Par 12 , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Glicina Hidroximetiltransferase , Proteínas de Neoplasias , Rabdomiossarcoma , Carcinogênese/genética , Carcinogênese/metabolismo , Cromossomos Humanos Par 12/genética , Cromossomos Humanos Par 12/metabolismo , Feminino , Glicina Hidroximetiltransferase/biossíntese , Glicina Hidroximetiltransferase/genética , Humanos , Masculino , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Rabdomiossarcoma/enzimologia , Rabdomiossarcoma/genética
13.
J Clin Invest ; 117(12): 3742-52, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18037990

RESUMO

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder associated with ciliary defects and situs inversus totalis, the complete mirror image reversal of internal organ situs (positioning). A variable incidence of heterotaxy, or irregular organ situs, also has been reported in PCD patients, but it is not known whether this is elicited by the PCD-causing genetic lesion. We studied a mouse model of PCD with a recessive mutation in Dnahc5, a dynein gene commonly mutated in PCD. Analysis of homozygous mutant embryos from 18 litters yielded 25% with normal organ situs, 35% with situs inversus totalis, and 40% with heterotaxy. Embryos with heterotaxy had complex structural heart defects that included discordant atrioventricular and ventricular outflow situs and atrial/pulmonary isomerisms. Variable combinations of a distinct set of cardiovascular anomalies were observed, including superior-inferior ventricles, great artery alignment defects, and interrupted inferior vena cava with azygos continuation. The surprisingly high incidence of heterotaxy led us to evaluate the diagnosis of PCD. PCD was confirmed by EM, which revealed missing outer dynein arms in the respiratory cilia. Ciliary dyskinesia was observed by videomicroscopy. These findings show that Dnahc5 is required for the specification of left-right asymmetry and suggest that the PCD-causing Dnahc5 mutation may also be associated with heterotaxy.


Assuntos
Transtornos da Motilidade Ciliar/patologia , Dineínas/genética , Cardiopatias Congênitas/ultraestrutura , Mutação , Situs Inversus/ultraestrutura , Animais , Cílios/genética , Cílios/ultraestrutura , Transtornos da Motilidade Ciliar/genética , Transtornos da Motilidade Ciliar/fisiopatologia , Modelos Animais de Doenças , Genes Recessivos , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/patologia , Humanos , Pulmão/fisiopatologia , Pulmão/ultraestrutura , Camundongos , Camundongos Mutantes , Miocárdio/ultraestrutura , Situs Inversus/genética , Situs Inversus/fisiopatologia , Veia Cava Inferior/fisiopatologia , Veia Cava Inferior/ultraestrutura
14.
Gene ; 666: 145-157, 2018 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-29730428

RESUMO

The PAX3 gene encodes a member of the PAX family of transcription factors that is characterized by a highly conserved paired box motif. The PAX3 protein is a transcription factor consisting of an N-terminal DNA binding domain (containing a paired box and homeodomain) and a C-terminal transcriptional activation domain. This protein is expressed during development of skeletal muscle, central nervous system and neural crest derivatives, and regulates expression of target genes that impact on proliferation, survival, differentiation and motility in these lineages. Germline mutations of the murine Pax3 and human PAX3 genes cause deficiencies in these developmental lineages and result in the Splotch phenotype and Waardenburg syndrome, respectively. Somatic genetic rearrangements that juxtapose the PAX3 DNA binding domain to the transcriptional activation domain of other transcription factors deregulate PAX3 function and contribute to the pathogenesis of the soft tissue cancers alveolar rhabdomyosarcoma and biphenotypic sinonasal sarcoma. The wild-type PAX3 protein is also expressed in other cancers related to developmental lineages that normally express this protein and exerts phenotypic effects related to its normal developmental role.


Assuntos
Fator de Transcrição PAX3/genética , Animais , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Fator de Transcrição PAX3/metabolismo , Sarcoma/genética , Síndrome de Waardenburg/genética
15.
Sci Signal ; 11(557)2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30459282

RESUMO

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood with an unmet clinical need for decades. A single oncogenic fusion gene is associated with treatment resistance and a 40 to 45% decrease in overall survival. We previously showed that expression of this PAX3:FOXO1 fusion oncogene in alveolar RMS (aRMS) mediates tolerance to chemotherapy and radiotherapy and that the class I-specific histone deacetylase (HDAC) inhibitor entinostat reduces PAX3:FOXO1 protein abundance. Here, we established the antitumor efficacy of entinostat with chemotherapy in various preclinical cell and mouse models and found that HDAC3 inhibition was the primary mechanism of entinostat-induced suppression of PAX3:FOXO1 abundance. HDAC3 inhibition by entinostat decreased the activity of the chromatin remodeling enzyme SMARCA4, which, in turn, derepressed the microRNA miR-27a. This reexpression of miR-27a led to PAX3:FOXO1 mRNA destabilization and chemotherapy sensitization in aRMS cells in culture and in vivo. Furthermore, a phase 1 clinical trial (ADVL1513) has shown that entinostat is tolerable in children with relapsed or refractory solid tumors and is planned for phase 1B cohort expansion or phase 2 clinical trials. Together, these results implicate an HDAC3-SMARCA4-miR-27a-PAX3:FOXO1 circuit as a driver of chemoresistant aRMS and suggest that targeting this pathway with entinostat may be therapeutically effective in patients.


Assuntos
DNA Helicases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Fatores de Transcrição/metabolismo , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Biologia Computacional , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Feminino , Transferência Ressonante de Energia de Fluorescência , Proteína Forkhead Box O1/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Camundongos , Transplante de Neoplasias , Fator de Transcrição PAX3/metabolismo , Piridinas/farmacologia , Análise de Sequência de RNA , Vincristina/farmacologia
16.
Mol Biol Cell ; 15(11): 4761-74, 2004 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-15342787

RESUMO

Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor alpha (CNTFRalpha) (200 ng/ml), or CNTF-CNTFRalpha. Although CNTF and CNTFRalpha alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFRalpha significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFRalpha treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between -838 to -1693 was deemed necessary for CNTF-CNTFRalpha to induce heightened expression. CNTF-CNTFRalpha did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFRalpha for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling.


Assuntos
Astrócitos/metabolismo , Fator Neurotrófico Ciliar/fisiologia , Conexina 43/biossíntese , Receptor do Fator Neutrófico Ciliar/fisiologia , Regulação para Cima , Animais , Sítios de Ligação , Northern Blotting , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citocinas/metabolismo , Dimerização , Genes Reporter , Proteína Glial Fibrilar Ácida/química , Immunoblotting , Imuno-Histoquímica , Óperon Lac , Camundongos , Modelos Genéticos , Fosforilação , Regiões Promotoras Genéticas , RNA/metabolismo , RNA Mensageiro/metabolismo , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Transfecção , Tirfostinas/farmacologia
17.
Nat Genet ; 49(7): 1152-1159, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28530678

RESUMO

Congenital heart disease (CHD) affects up to 1% of live births. Although a genetic etiology is indicated by an increased recurrence risk, sporadic occurrence suggests that CHD genetics is complex. Here, we show that hypoplastic left heart syndrome (HLHS), a severe CHD, is multigenic and genetically heterogeneous. Using mouse forward genetics, we report what is, to our knowledge, the first isolation of HLHS mutant mice and identification of genes causing HLHS. Mutations from seven HLHS mouse lines showed multigenic enrichment in ten human chromosome regions linked to HLHS. Mutations in Sap130 and Pcdha9, genes not previously associated with CHD, were validated by CRISPR-Cas9 genome editing in mice as being digenic causes of HLHS. We also identified one subject with HLHS with SAP130 and PCDHA13 mutations. Mouse and zebrafish modeling showed that Sap130 mediates left ventricular hypoplasia, whereas Pcdha9 increases penetrance of aortic valve abnormalities, both signature HLHS defects. These findings show that HLHS can arise genetically in a combinatorial fashion, thus providing a new paradigm for the complex genetics of CHD.


Assuntos
Heterogeneidade Genética , Síndrome do Coração Esquerdo Hipoplásico/genética , Sequência de Aminoácidos , Animais , Aorta/embriologia , Sistemas CRISPR-Cas , Mapeamento Cromossômico , Cromossomos Humanos/genética , Modelos Animais de Doenças , Exoma , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Ventrículos do Coração/embriologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Mutação de Sentido Incorreto , Miócitos Cardíacos/patologia , Penetrância , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Obstrução do Fluxo Ventricular Externo/genética , Peixe-Zebra/genética
18.
J Genet ; 95(2): 399-409, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27350685

RESUMO

Mitochondrial DNA control region of Mus terricolor, three aboriginal species M. spretus, M. macedonicus, M. spicilegus; the Asian lineage M. caroli, M. cervicolor, M. cookii; and the two house mice, M. musculus domesticus and M. m. castaneus were analysed to estimate the substitution rate, phylogenetic relationship and the probable time of divergence. Results showed that M. spretus, M. caroli and M. terricolor are highly diverged from each other (caroli/terricolor = 0.146, caroli/spretus = 0.147 and terricolor/spretus = 0.122), whereas M. spretus showed less divergence with two house mice species (0.070 and 0.071). Sequence divergence between M. terricolor and the Palearctic group were found to be ranging from 0.121 to 0.134. Phylogenetic analysis by minimum evolution, neighbour-joining, unweighed pair group method with arithmetic mean and maximum parsimony showed almost similar topology. Two major clusters were found, one included the Asian lineage, M. caroli, M. cookii and M. cervicolor and the other included the house mice M. m. domesticus, M. m. castaneus and the aboriginal mice M. macedonicus and M. spicilegus along with M. spretus, forming the Palearctic clade. M. terricolor was positioned between the Palearctic and Asian clades. Results showed that Palearctic-terricolor and the Asian lineages diverged 5.47 million years ago (Mya), while M. terricolor had split around 4.63 Mya from their ancestor. M. cervicolor, M. cookii and M. caroli diverged between 4.70 and 3.36 Mya, which indicates that M. terricolor and the Asian lineages evolved simultaneously. M. spretus is expected to have diverged nearly 2.9 Mya from their most recent common ancestor.


Assuntos
DNA Mitocondrial/genética , Especiação Genética , Camundongos/genética , Filogenia , Animais , Sequência de Bases , Feminino , Variação Genética , Haplótipos , Masculino , Camundongos/classificação , Mitocôndrias/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie
19.
Biol Open ; 5(3): 323-35, 2016 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-26883626

RESUMO

Planar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development.

20.
Virchows Arch ; 465(2): 233-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24993903

RESUMO

Gene rearrangements involving the Ewing sarcoma breakpoint region 1 (EWSR1) gene are seen in a broad range of sarcomas and some nonmesenchymal neoplasms. Ewing sarcoma is molecularly defined by a fusion of the EWSR1 gene (or rarely the related FUS gene) to a member of the E26 transformation-specific (ETS) family of transcription factors, frequently the EWSR1-FLI1 fusion. More recently, EWSR1 gene fusion to non-ETS family members, including the nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2 (NFATC2) gene, has been reported in a histological variant of Ewing sarcoma. Here, we report a malignant round cell tumor of bone with an EWSR1-NFATC2 fusion gene. This report builds upon the unusual morphological and clinical presentation of bone neoplasms containing an EWSR1-NFATC2 fusion gene.


Assuntos
Neoplasias Ósseas/genética , Proteínas de Ligação a Calmodulina/genética , Fusão Gênica/genética , Fatores de Transcrição NFATC/genética , Proteínas de Ligação a RNA/genética , Sarcoma de Ewing/genética , Adulto , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/terapia , Transplante Ósseo , Terapia Combinada , Curetagem , Tratamento Farmacológico , Rearranjo Gênico/genética , Humanos , Masculino , Proteína EWS de Ligação a RNA , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/terapia , Resultado do Tratamento
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