RESUMO
WWOX-related epileptic encephalopathy (WOREE) syndrome caused by human germline bi-allelic mutations in WWOX is a neurodevelopmental disorder characterized by intractable epilepsy, severe developmental delay, ataxia and premature death at the age of 2-4 years. The underlying mechanisms of WWOX actions are poorly understood. In the current study, we show that specific neuronal deletion of murine Wwox produces phenotypes typical of the Wwox-null mutation leading to brain hyperexcitability, intractable epilepsy, ataxia and postnatal lethality. A significant decrease in transcript levels of genes involved in myelination was observed in mouse cortex and hippocampus. Wwox-mutant mice exhibited reduced maturation of oligodendrocytes, reduced myelinated axons and impaired axonal conductivity. Brain hyperexcitability and hypomyelination were also revealed in human brain organoids with a WWOX deletion. These findings provide cellular and molecular evidence for myelination defects and hyperexcitability in the WOREE syndrome linked to neuronal function of WWOX.
Assuntos
Epilepsia/genética , Deleção de Genes , Bainha de Mielina/genética , Neurônios/fisiologia , Oxidorredutase com Domínios WW/deficiência , Oxidorredutase com Domínios WW/genética , Animais , Encéfalo/patologia , Técnicas de Cocultura , Epilepsia/patologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Bainha de Mielina/patologia , Neurônios/patologia , Organoides , Oxidorredutase com Domínios WW/antagonistas & inibidoresRESUMO
BACKGROUND: Tumor suppressor WWOX physically binds p53 and TIAF1 and together induces apoptosis and tumor suppression. To understand the molecular action, here we investigated the formation of WWOX/TIAF1/p53 triad and its regulation of cancer cell migration, anchorage-independent growth, SMAD promoter activation, apoptosis, and potential role in neurodegeneration. METHODS: Time-lapse microscopy was used to measure the extent of cell migration. Protein/protein interactions were determined by co-immunoprecipitation, FRET microscopy, and yeast two-hybrid analysis. The WWOX/TIAF1/p53 triad-mediated cancer suppression was determined by measuring the extent of cell migration, anchorage-independent growth, SMAD promoter activation, and apoptosis. p53-deficient lung cancer cell growth in nude mice was carried out to assess the tumor suppressor function of ectopic p53 and/or WWOX. RESULTS: Wwox-deficient MEF cells exhibited constitutive Smad3 and p38 activation and migrated individually and much faster than wild type cells. TGF-ß increased the migration of wild type MEF cells, but significantly suppressed Wwox knockout cell migration. While each of the triad proteins is responsive to TGF-ß stimulation, ectopically expressed triad proteins suppressed cancer cell migration, anchorage-independent growth, and SMAD promoter activation, as well as caused apoptosis. The effects are due in part to TIAF1 polymerization and its retention of p53 and WWOX in the cytoplasm. p53 and TIAF1 were effective in suppressing anchorage-independent growth, and WWOX ineffective. p53 and TIAF1 blocked WWOX or Smad4-regulated SMAD promoter activation. WWOX suppressed lung cancer NCI-H1299 growth and inhibited splenomegaly by inflammatory immune response, and p53 blocked the event in nude mice. The p53/WWOX-cancer mice exhibited BACE upregulation, APP degradation, tau tangle formation, and amyloid ß generation in the brain and lung. CONCLUSION: The WWOX/TIAF1/p53 triad is potent in cancer suppression by blocking cancer cell migration, anchorage-independent growth and SMAD promoter activation, and causing apoptosis. Yet, p53 may functionally antagonize with WWOX. p53 blocks WWOX inhibition of inflammatory immune response induced by cancer, and this leads to protein aggregation in the brain as seen in the Alzheimer's disease and other neurodegeneration.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias da Mama/terapia , Neoplasias Pulmonares/terapia , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Camundongos Nus , Proteínas Nucleares/antagonistas & inibidores , Agregados Proteicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/deficiência , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/deficiência , Oxidorredutase com Domínios WW/antagonistas & inibidores , Oxidorredutase com Domínios WW/deficiênciaRESUMO
WW domain-containing oxidoreductase (Wwox) is a putative tumor suppressor. Several germline mutations of Wwox have been associated with infant neurological disorders characterized by epilepsy, growth retardation, and early death. Less is known, however, about the pathological link between Wwox mutations and these disorders or the physiological role of Wwox in brain development. In this study, we examined age-related expression and histological localization of Wwox in forebrains as well as the effects of loss of function mutations in the Wwox gene in the immature cortex of a rat model of lethal dwarfism with epilepsy (lde/lde). Immunostaining revealed that Wwox is expressed in neurons, astrocytes, and oligodendrocytes. lde/lde cortices were characterized by a reduction in neurite growth without a reduced number of neurons, severe reduction in myelination with a reduced number of mature oligodendrocytes, and a reduction in cell populations of astrocytes and microglia. These results indicate that Wwox is essential for normal development of neurons and glial cells in the cerebral cortex.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Antiporters/deficiência , Córtex Cerebral/metabolismo , Nanismo/genética , Epilepsia/genética , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/genética , Doenças Mitocondriais/genética , Neurogênese/genética , Transtornos Psicomotores/genética , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Proteína da Polipose Adenomatosa do Colo/genética , Proteína da Polipose Adenomatosa do Colo/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Animais , Antiporters/genética , Antiporters/metabolismo , Astrócitos/metabolismo , Astrócitos/patologia , Contagem de Células , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/patologia , Modelos Animais de Doenças , Nanismo/metabolismo , Nanismo/patologia , Epilepsia/metabolismo , Epilepsia/patologia , Regulação da Expressão Gênica no Desenvolvimento , Mutação em Linhagem Germinativa , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/metabolismo , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central/patologia , Masculino , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/patologia , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Oligodendroglia/metabolismo , Oligodendroglia/patologia , Prosencéfalo/crescimento & desenvolvimento , Prosencéfalo/metabolismo , Prosencéfalo/patologia , Transtornos Psicomotores/metabolismo , Transtornos Psicomotores/patologia , Ratos , Ratos Transgênicos , Transdução de Sinais , Proteínas Supressoras de Tumor/deficiência , Oxidorredutase com Domínios WW/deficiênciaRESUMO
OBJECTIVE: WWOX, a well-established tumor suppressor, is frequently lost in cancer and plays important roles in DNA damage response and cellular metabolism. METHODS: We re-analyzed several genome-wide association studies (GWAS) using the Type 2 Diabetes Knowledge Portal website to uncover WWOX's association with metabolic syndrome (MetS). Using several engineered mouse models, we studied the effect of somatic WWOX loss on glucose homeostasis. RESULTS: Several WWOX variants were found to be strongly associated with MetS disorders. In mouse models, somatic ablation of Wwox in skeletal muscle (WwoxΔSKM) results in weight gain, glucose intolerance, and insulin resistance. Furthermore, WwoxΔSKM mice display reduced amounts of slow-twitch fibers, decreased mitochondrial quantity and activity, and lower glucose oxidation levels. Mechanistically, we found that WWOX physically interacts with the cellular energy sensor AMP-activated protein kinase (AMPK) and that its loss is associated with impaired activation of AMPK, and with significant accumulation of the hypoxia inducible factor 1 alpha (HIF1α) in SKM. CONCLUSIONS: Our studies uncover an unforeseen role of the tumor suppressor WWOX in whole-body glucose homeostasis and highlight the intimate relationship between cancer progression and metabolic disorders, particularly obesity and type-2 diabetes. SUBJECT AREAS: Genetics, Metabolic Syndrome, Diabetes.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Oxidorredutase com Domínios WW/deficiência , Oxidorredutase com Domínios WW/metabolismo , Animais , Metabolismo dos Carboidratos , Engenharia Genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Oxidorredutase com Domínios WW/genéticaRESUMO
Inactivation of WW domain-containing oxidoreductase (WWOX), the gene product of the common fragile site FRA16D, is a common event in breast cancer and is associated with worse prognosis of triple-negative breast cancer (TNBC) and basal-like breast cancer (BLBC). Despite recent progress, the role of WWOX in driving breast carcinogenesis remains unknown. Here we report that ablation of Wwox in mammary tumor-susceptible mice results in increased tumorigenesis, and that the resultant tumors resemble human BLBC. Interestingly, copy number loss of Trp53 and downregulation of its transcript levels were observed in the Wwox knockout tumors. Moreover, tumors isolated from Wwox and Trp53 mutant mice were indistinguishable histologically and transcriptionally. Finally, we find that deletion of TP53 and WWOX co-occurred and is associated with poor survival of breast cancer patients. Altogether, our data uncover an essential role for WWOX as a bona fide breast cancer tumor suppressor through the maintenance of p53 stability.
Assuntos
Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/metabolismo , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Células Epiteliais/citologia , Feminino , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Humanos , Células MCF-7 , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Knockout , Mutação , Taxa de Sobrevida , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/deficiência , Oxidorredutase com Domínios WW/genéticaRESUMO
Liver cancer is one of the most lethal malignancies with very poor prognosis once diagnosed. The most common form of liver cancer is hepatocellular carcinoma (HCC). The WW domain-containing oxidoreductase (WWOX) is a large gene that is often perturbed in a wide variety of tumors, including HCC. WWOX has been shown to act as a tumor suppressor modulating cellular metabolism via regulating hypoxia-inducible factor 1α (HIF-1α) levels and function. Given that WWOX is commonly inactivated in HCC, we set to determine whether specific targeted deletion of murine Wwox affects liver biology and HCC development. WWOX liver-specific knockout mice (Wwox ΔHep ) showed more potent liver regeneration potential and enhanced proliferation as compared with their control littermates. Moreover, WWOX deficiency in hepatocytes combined with diethylnitrosamine treatment increased the tumor burden, which was associated with increased HIF1α levels and target gene transactivation. Inhibition of HIF1α by systemic treatment with digoxin significantly delayed HCC formation. Our work suggests that WWOX inactivation has a central role in promoting HCC through rewiring of cellular metabolism and modulating proliferation.
Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Hepáticas/genética , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/genética , Animais , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células , Dieta Hiperlipídica/efeitos adversos , Dietilnitrosamina/farmacologia , Digoxina/farmacologia , Modelos Animais de Doenças , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Prognóstico , Transdução de Sinais , Carga Tumoral/efeitos dos fármacos , Proteínas Supressoras de Tumor/deficiência , Oxidorredutase com Domínios WW/deficiênciaRESUMO
The human WWOX (WW domain-containing oxidoreductase) gene, originally known as a tumor suppressor gene, has been shown to be important for brain function and development. In recent years, mutations in WWOX have been associated with a wide phenotypic spectrum of autosomal recessively inherited neurodevelopmental disorders. Whole exome sequencing was completed followed by Sanger sequencing to verify segregation of the identified variants. Functional WWOX analysis was performed in fibroblasts of one patient. Transcription and translation were assessed by quantitative real-time PCR and Western blotting. We report two related patients who presented with early epilepsy refractory to treatment, progressive microcephaly, profound developmental delay, and brain MRI abnormalities. Additionally, one of the patients showed bilateral optic atrophy. Whole exome sequencing revealed homozygosity for a novel missense variant affecting the evolutionary conserved amino acid Gln230 in the catalytic short-chain dehydrogenase/reductase (SDR) domain of WWOX in both girls. Functional studies showed normal levels of WWOX transcripts but absence of WWOX protein. To our knowledge, our patients are the first individuals presenting the more severe end of the phenotypic spectrum of WWOX deficiency, although they were only affected by a single missense variant of WWOX. This could be explained by the functional data indicating an impaired translation or premature degradation of the WWOX protein.
Assuntos
Deficiências do Desenvolvimento/genética , Mutação de Sentido Incorreto , Espasmos Infantis/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Oxidorredutase com Domínios WW/deficiência , Oxidorredutase com Domínios WW/genética , Afeganistão , Idade de Início , Células Cultivadas , Criança , Consanguinidade , Deficiências do Desenvolvimento/complicações , Epilepsia/complicações , Epilepsia/genética , Família , Feminino , Células HEK293 , Humanos , Recém-Nascido , Linhagem , Domínios Proteicos/genética , Estabilidade de RNA/genética , Índice de Gravidade de Doença , Espasmos Infantis/complicações , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Oxidorredutase com Domínios WW/química , Oxidorredutase com Domínios WW/metabolismoRESUMO
Expression of Fhit and Wwox protein is frequently lost or reduced in many human cancers. In this report, we provide data that further characterizes the molecular consequences of Fhit loss in the initiation of DNA double-strand breaks (DSBs), and of Wwox loss in altered repair of DSBs. We show that loss of Fhit initiates mild genome instability in early passage mouse kidney cells, confirming that DNA damage associated with Fhit-deficiency is not limited to cancer cells. We also demonstrate that the cause of Fhit-deficient DSBs: thymidine deficiency-induced replication stress, can be resolved with thymidine supplementation in early passage mouse kidney cells before extensive genome instability occurs. As for consequences of Wwox loss in cancer, we show in a small panel of breast cancer cells and mouse embryonic fibroblasts that Wwox expression predicts response to radiation and mitomycin C, all agents that cause DSBs. In addition, loss of Wwox significantly reduced progression free survival in a cohort of ovarian cancer patients treated with platin-based chemotherapies. Finally, stratification of a cohort of squamous lung cancers by Fhit expression reveals that Wwox expression is significantly reduced in the low Fhit-expressing group, suggesting that loss of Fhit is quickly succeeded by loss of Wwox. We propose that Fhit and Wwox loss work synergistically in cancer progression and that DNA damage caused by Fhit could be targeted early in cancer initiation for prevention, while DNA damage caused by Wwox loss could be targeted later in cancer progression, particularly in cancers that develop resistance to genotoxic therapies.