RESUMO
Capillary malformation-arteriovenous malformation (CM-AVM) is caused by germline RASA1 and EPHB4 alterations. RASA1 intralesional second hits have also been reported. Here we report RASA1 constitutional mosaicism, defined here as the presence of a mosaic variant in all cell types of an individual, in two patients with CM-AVM. High-throughput sequencing was used to search for RASA1 pathogenic variants in blood samples from two unrelated patients with CM-AVM. An affected tissue sample from one of the patients was also analyzed. Both patients showed different nonsense RASA1 variants in mosaic, ranging from 7% to 21.5%, in blood samples and in the corresponding affected tissue sample from one of the patients. In conclusion, we report for the first time the presence of RASA1 constitutional mosaicism in CM-AVM. Constitutional mosaicism has implications for accurate molecular diagnosis and recurrence risk and helps to explain the great phenotypic variability in CM-AVM.
Assuntos
Malformações Arteriovenosas/diagnóstico , Malformações Arteriovenosas/genética , Capilares/anormalidades , Estudos de Associação Genética , Predisposição Genética para Doença , Mosaicismo , Mutação , Mancha Vinho do Porto/diagnóstico , Mancha Vinho do Porto/genética , Proteína p120 Ativadora de GTPase/genética , Alelos , Substituição de Aminoácidos , Angiografia por Tomografia Computadorizada , Feminino , Estudos de Associação Genética/métodos , Genótipo , Humanos , MasculinoRESUMO
Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.
Assuntos
Classe I de Fosfatidilinositol 3-Quinases , Linfangioleiomiomatose , Sistema Linfático , Mutação de Sentido Incorreto , Sirolimo/administração & dosagem , Adolescente , Adulto , Substituição de Aminoácidos , Criança , Pré-Escolar , Classe I de Fosfatidilinositol 3-Quinases/genética , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Humanos , Linfangioleiomiomatose/diagnóstico por imagem , Linfangioleiomiomatose/tratamento farmacológico , Linfangioleiomiomatose/enzimologia , Linfangioleiomiomatose/genética , Sistema Linfático/anormalidades , Sistema Linfático/diagnóstico por imagem , Sistema Linfático/enzimologia , Masculino , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Cyclooxygenase (COX) catalyzes the first step in prostanoid biosynthesis and exists as two isoforms. COX-1 is a constitutive enzyme involved in physiological processes, whereas COX-2 is induced by a variety of stimuli. MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. Although it is known that COX-2 expression is regulated by miRNAs, there are no data regarding COX-2 involvement in miRNA regulation. Considering our previous results showing that COX-2 expression in hepatocytes protects against insulin resistance, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs implicated in insulin signaling in liver cells. Our results provide evidence of the molecular basis for a novel function of COX-2 in miRNA processing. COX-2 represses miRNA 23b (miR-23b), miR-146b, and miR-183 expression in liver cells by increasing the level of DEAD-box helicase p68 (DDX5) through phosphatidylinositol 3-kinase (PI3K)/p300 signaling and by modulating the enzymatic function of the Drosha (RNase type III) complex through its physical association with DDX5. The decrease of miR-183 expression promotes protection against insulin resistance by increasing insulin receptor substrate 1 (IRS1) levels. These results indicate that the modulation of miRNA processing by COX-2 is a key event in insulin signaling in liver and has potential clinical implications for the management of various hepatic dysfunctions.
Assuntos
Ciclo-Oxigenase 2/genética , RNA Helicases DEAD-box/genética , Fígado/metabolismo , MicroRNAs/genética , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/metabolismo , RNA Helicases DEAD-box/metabolismo , Regulação da Expressão Gênica , Hepatócitos/metabolismo , Humanos , Proteínas Substratos do Receptor de Insulina/metabolismo , Resistência à Insulina/genética , Camundongos Transgênicos , MicroRNAs/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease III/metabolismoRESUMO
Type 2 diabetes has a complex pathology that involves a chronic inflammatory state. Emerging evidence suggests a link between the innate immune system receptor NOD1 (nucleotide-binding and oligomerization domain 1) and the pathogenesis of diabetes, in monocytes and hepatic and adipose tissues. The aim of the present study was to assess the role of NOD1 in the progression of diabetic cardiomyopathy. We have measured NOD1 protein in cardiac tissue from Type 2 diabetic (db) mice. Heart and isolated cardiomyocytes from db mice revealed a significant increase in NOD1, together with an up-regulation of nuclear factor κB (NF-κB) and increased apoptosis. Heart tissue also exhibited an enhanced expression of pro-inflammatory cytokines. Selective NOD1 activation with C12-γ-D-glutamyl-m-diaminopimelic acid (iEDAP) resulted in an increased NF-κB activation and apoptosis, demonstrating the involvement of NOD1 both in wild-type and db mice. Moreover, HL-1 cardiomyocytes exposed to elevated concentrations of glucose plus palmitate displayed an enhanced NF-κB activity and apoptotic profile, which was prevented by silencing of NOD1 expression. To address this issue in human pathology, NOD1 expression was evaluated in myocardium obtained from patients with Type 2 diabetes (T2DMH) and from normoglycaemic individuals without cardiovascular histories (NH). We have found that NOD1 was expressed in both NH and T2DMH; however, NOD1 expression was significantly pronounced in T2DMH. Furthermore, both the pro-inflammatory cytokine tumour necrosis factor α (TNF-α) and the apoptosis mediator caspase-3 were up-regulated in T2DMH samples. Taken together, our results define an active role for NOD1 in the heightened inflammatory environment associated with both experimental and human diabetic cardiac disease.
Assuntos
Cardiomiopatias Diabéticas/metabolismo , Miocárdio/metabolismo , Proteína Adaptadora de Sinalização NOD1/metabolismo , Animais , Apoptose , Linhagem Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Cardiomiopatias Diabéticas/genética , Cardiomiopatias Diabéticas/patologia , Progressão da Doença , Glucose/farmacologia , Humanos , Camundongos , NF-kappa B/metabolismo , Palmitatos/farmacologia , Regulação para CimaRESUMO
Cyclooxygenase-2 (COX-2), a rate limiting step in arachidonic acid cascade, plays a key role in the biosynthesis of prostaglandin E2 (PGE2) upon inflammatory stimuli, growth factors, hormones and other cellular stresses. Overproduction of PGE2 stimulates proliferation of various cancer cells, confers resistance to apoptosis and favors metastasis and angiogenesis. The steady-state level of PGE2 is maintained by interplay between the biosynthetic pathway including COX and PGE2 synthases and the catabolic pathways involving nicotinamide adenine dinucleotide (NAD(+))-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 15-PGDH is a crucial enzyme responsible for the biological inactivation of PGE2. Adult hepatocytes fail to induce COX-2 expression regardless of the pro-inflammatory factors used. COX-2 is induced in hepatocytes after partial hepatectomy (PH), in animal models of cirrhosis, in human hepatoma cell lines, in human HCC and after HBV and HCV infection. However, no data are available regarding 15-PGDH expression in HCC. Our results show that 15-PGDH is downregulated in human hepatoma cells with a high COX-2 expression, in chemical and genetic murine models of HCC and in human HCC biopsies. Moreover, 15-PGDH expression is suppressed by EGF (epidermal growth factor) and HGF (hepatocyte growth factor) mainly involving PI3K (phosphatidylinositol-3-kinase), ERK (extracellular signal-regulated kinase) and p38MAPK (mitogen-activated protein kinase) activation. Conversely, ectopic expression of 15-PGDH induces apoptosis in hepatoma cells and decreases the growth of hepatoma cells in nude mice whereas the silencing of 15-PGDH increases the tumor formation. These data suggest a potential therapeutic application of 15-PGDH in HCC.
Assuntos
Carcinoma Hepatocelular/enzimologia , Hidroxiprostaglandina Desidrogenases/metabolismo , Neoplasias Hepáticas/enzimologia , Adulto , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Biópsia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Ciclo-Oxigenase 2/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Oxirredutases Intramoleculares/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Prostaglandina-E Sintases , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Ewing sarcoma is the second most common bone malignancy in children and young adults. It is driven by oncogenic fusion proteins (i.e. EWS/FLI1) acting as aberrant transcription factors that upregulate and downregulate target genes, leading to cellular transformation. Thus, identificating these target genes and understanding their contribution to Ewing sarcoma tumorigenesis are key for the development of new therapeutic strategies. In this study we show that lysyl oxidase (LOX), an enzyme involved in maintaining structural integrity of the extracellular matrix, is downregulated by the EWS/FLI1 oncoprotein and in consequence it is not expressed in Ewing sarcoma cells and primary tumors. Using a doxycycline inducible system to restore LOX expression in an Ewing sarcoma derived cell line, we showed that LOX displays tumor suppressor activities. Interestingly, we showed that the tumor suppressor activity resides in the propeptide domain of LOX (LOX-PP), an N-terminal domain produced by proteolytic cleavage during the physiological processing of LOX. Expression of LOX-PP reduced cell proliferation, cell migration, anchorage-independent growth in soft agar and formation of tumors in immunodeficient mice. By contrast, the C-terminal domain of LOX, which contains the enzymatic activity, had the opposite effects, corroborating that the tumor suppressor activity of LOX is mediated exclusively by its propeptide domain. Finally, we showed that LOX-PP inhibits ERK/MAPK signalling pathway, and that many pathways involved in cell cycle progression were significantly deregulated by LOX-PP, providing a mechanistic explanation to the cell proliferation inhibition observed upon LOX-PP expression. In summary, our observations indicate that deregulation of the LOX gene participates in Ewing sarcoma development and identify LOX-PP as a new therapeutic target for one of the most aggressive paediatric malignancies. These findings suggest that therapeutic strategies based on the administration of LOX propeptide or functional analogues could be useful for the treatment of this devastating paediatric cancer.
Assuntos
Regulação para Baixo , Proteínas de Fusão Oncogênica/metabolismo , Proteína-Lisina 6-Oxidase/química , Proteína-Lisina 6-Oxidase/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/patologia , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Proteínas de Fusão Oncogênica/química , Estrutura Terciária de Proteína , Proteína-Lisina 6-Oxidase/metabolismo , Proteína Proto-Oncogênica c-fli-1/química , Proteína EWS de Ligação a RNA/química , Proteínas Supressoras de Tumor/metabolismoRESUMO
Cyclooxygenase-2 (COX-2) has been associated with cell growth regulation, tissue remodeling and carcinogenesis. Overexpression of COX-2 in hepatocytes constitutes an ideal condition to evaluate the role of prostaglandins (PGs) in liver pathogenesis. The effect of COX-2-dependent PGs in genetic hepatocarcinogenesis has been investigated in triple c-myc/transforming growth factor α (TGF-α) transgenic mice that express human COX-2 in hepatocytes on a B6CBAxCD1xB6DBA2 background. Analysis of the contribution of COX-2-dependent PGs to the development of hepatocarcinogenesis, evaluated in this model, suggested a minor role of COX-2-dependent prostaglandins to liver oncogenesis as indicated by liver histopathology, morphometric analysis and specific markers of tumor progression. This allows concluding that COX-2 is insufficient for modifying the hepatocarcinogenesis course mediated by c-myc/TGF-α.
Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/patologia , Ciclo-Oxigenase 2/genética , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas c-myc/genética , Fator de Crescimento Transformador alfa/genética , Animais , Carcinoma Hepatocelular/genética , Progressão da Doença , Feminino , Expressão Gênica , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Transgênicos , Transdução de Sinais/genéticaRESUMO
Tumours of the Ewing family, which comprise Ewing's sarcoma and peripheral primitive neuroectodermal tumours, are highly aggressive and mostly affect children and adolescents. They are characterized by chromosomal translocations leading to the generation of fusion proteins between EWS (or very rarely FUS) and members of the E-twenty-six (ETS) family of transcription factors that are capable of transforming cells. EWS/FLI1, the most frequent fusion, is thought to cause transformation through activation or repression of specific target genes. We present evidence demonstrating that the Wnt inhibitor and beta-catenin/T-cell factor (TCF)-responsive gene DICKKOPF-1 (DKK-1) is a transcriptional target of EWS/FLI1, which can inhibit both basal and beta-catenin-induced transactivation of the DKK-1 promoter. Moreover, our data indicate that EWS/FLI1 has a more general effect on beta-catenin/TCF-mediated transcription since it can block transactivation of a consensus beta-catenin/TCF reporter construct. Consistently, Ewing tumour cells expressing different EWS/ETS translocations cannot engage beta-catenin/TCF-dependent transcription, whereas silencing of EWS/FLI1 restores beta-catenin responsiveness in A673 and RD-ES Ewing tumour cells. Accordingly, gene set enrichment analysis shows that beta-catenin/TCF target genes are significantly enriched among genes downregulated by EWS/FLI1 in the Ewing cell line A673. Mechanistically, the inhibitory effect of EWS/FLI1 can be overcome by a constitutively active TCF4 protein (TCF4-VP16). Moreover, EWS/FLI1 binds lymphoid enhancer factor 1, a TCF family member, and interferes with its binding to beta-catenin, which could explain its negative effect on beta-catenin/TCF-mediated transcription. Our results show that EWS/FLI1 inhibits both DKK-1 expression as well as beta-catenin/TCF-dependent transcription, which could contribute to progression of tumours of the Ewing family.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Fator 1 de Ligação ao Facilitador Linfoide/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Fusão Oncogênica/fisiologia , Fator 1 de Transcrição de Linfócitos T/antagonistas & inibidores , Fatores de Transcrição/fisiologia , beta Catenina/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/fisiologia , Linhagem Celular Tumoral/metabolismo , Transformação Celular Neoplásica/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HeLa/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Família Multigênica , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Proteína Proto-Oncogênica c-fli-1 , Proteína EWS de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoma de Ewing/patologia , Fator de Transcrição 4 , Fatores de Transcrição/genética , Transcrição Gênica , Transgenes , Proteínas Wnt/fisiologiaRESUMO
The Ewing family of tumors is a group of highly malignant tumors that mainly arise in bone and most often affect children and young adults in the first two decades of life. Despite the use of multimodal therapy, the long-term disease-free survival rate of patients with Ewing tumors is still disappointingly low, making the discovery of innovative therapeutic strategies all the more necessary. We have recently shown that cholecystokinin (CCK), a neuroendocrine peptide, involved in many biological functions, including cell growth and proliferation, is a relevant target of the EWS/FLI1 oncoprotein characteristic of Ewing tumors. CCK silencing inhibits cell proliferation and tumor growth in vivo, suggesting that CCK acts as an autocrine growth factor for Ewing cells. Here, we analyzed the impact of two CCK receptor antagonists, devazepide (a CCK1-R antagonist) and L365 260 (a CCK2-R antagonist), on the growth of Ewing tumor cells. Devazepide (10 micromol/l) inhibited cell growth of four different Ewing tumor cells in vitro (range 85-88%), whereas the effect of the CCK2-R antagonist on cell growth was negligible. In a mouse tumor xenograft model, devazepide reduced tumor growth by 40%. Flow cytometry experiments showed that devazepide, but not L365 260, induced apoptosis of Ewing tumor cells. In summary, devazepide induces cell death of Ewing tumor cells, suggesting that it could represent a new therapeutic approach in the management of Ewing's tumor patients.
Assuntos
Apoptose/efeitos dos fármacos , Devazepida/farmacologia , Antagonistas de Hormônios/farmacologia , Sarcoma de Ewing/tratamento farmacológico , Animais , Benzodiazepinonas/farmacologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Compostos de Fenilureia/farmacologia , Receptores da Colecistocinina/antagonistas & inibidores , Sarcoma de Ewing/fisiopatologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Tumors of the Ewing family are characterized by chromosomal translocations that yield chimeric transcription factors, such as EWS/FLI1, which regulate the expression of specific genes that contribute to the malignant phenotype. In the present study, we show that cholecystokinin (CCK) is a new target of the EWS/FLI1 oncoprotein and assess its functional role in Ewing tumor pathogenesis. EXPERIMENTAL DESIGN: Relevant EWS/FLI1 targets were identified using a combination of cell systems with inducible EWS/FLI1 expression, Ewing tumors and cell lines, microarrays, and RNA interference with doxycycline-inducible small hairpin RNA (shRNA) vectors. A doxycycline-inducible CCK-shRNA vector was stably transfected in A673 and SK-PN-DW Ewing cell lines to assess the role of CCK in cell proliferation and tumor growth. RESULTS: Microarray analysis revealed that CCK was up-regulated by EWS/FLI1 in HeLa cells. CCK was overexpressed in Ewing tumors as compared with other pediatric malignancies such as rhabdomyosarcoma and neuroblastoma, with levels close to those detected in normal tissues expressing the highest levels of CCK. Furthermore, EWS/FLI1 knockdown in A673 and SK-PN-DW Ewing cells using two different doxycycline-inducible EWS/FLI1-specific shRNA vectors down-regulated CCK mRNA expression and diminished the levels of secreted CCK, showing that CCK is a EWS/FLI1 specific target gene in Ewing cells. A doxycycline-inducible CCK-specific shRNA vector successfully down-regulated CCK expression, reduced the levels of secreted CCK in Ewing cell lines, and inhibited cell growth and proliferation in vitro and in vivo. Finally, we show that Ewing cell lines and tumors express CCK receptors and that the growth inhibition produced by CCK silencing can be rescued by culturing the cells with medium containing CCK. CONCLUSIONS: Our data support the hypothesis that CCK acts as an autocrine growth factor stimulating the proliferation of Ewing cells and suggest that therapies targeting CCK could be promising in the treatment of Ewing tumors.