RESUMEN
Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
Asunto(s)
ARN , Ubiquitina-Proteína Ligasas , Humanos , ARN/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Formaldehído/toxicidad , Aldehídos/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
SOX9 is a master transcription factor that regulates development and stem cell programs. However, its potential oncogenic activity and regulatory mechanisms that control SOX9 protein stability are poorly understood. Here, we show that SOX9 is a substrate of FBW7, a tumor suppressor, and a SCF (SKP1/CUL1/F-box)-type ubiquitin ligase. FBW7 recognizes a conserved degron surrounding threonine 236 (T236) in SOX9 that is phosphorylated by GSK3 kinase and consequently degraded by SCFFBW7α Failure to degrade SOX9 promotes migration, metastasis, and treatment resistance in medulloblastoma, one of the most common childhood brain tumors. FBW7 is either mutated or downregulated in medulloblastoma, and in cases where FBW7 mRNA levels are low, SOX9 protein is significantly elevated and this phenotype is associated with metastasis at diagnosis and poor patient outcome. Transcriptional profiling of medulloblastoma cells expressing a degradation-resistant SOX9 mutant reveals activation of pro-metastatic genes and genes linked to cisplatin resistance. Finally, we show that pharmacological inhibition of PI3K/AKT/mTOR pathway activity destabilizes SOX9 in a GSK3/FBW7-dependent manner, rendering medulloblastoma cells sensitive to cytostatic treatment.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Meduloblastoma/metabolismo , Factor de Transcripción SOX9/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Compuestos de Anilina/farmacología , Animales , Benzamidas , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular , Cromonas/farmacología , Cisplatino/farmacología , Proteínas F-Box/genética , Proteína 7 que Contiene Repeticiones F-Box-WD , Glucógeno Sintasa Quinasa 3/metabolismo , Células HEK293 , Humanos , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Ratones Desnudos , Morfolinas/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , Pirimidinas/farmacología , Pirroles/farmacología , Factor de Transcripción SOX9/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Smokers have an elevated risk of cardiovascular disease but the origin(s) of this increased risk are incompletely defined. Considerable evidence supports an accumulation of the oxidant-generating enzyme MPO (myeloperoxidase) in the inflamed artery wall, and smokers have high levels of SCN(-), a preferred MPO substrate, with this resulting in HOSCN (hypothiocyanous acid) formation. We hypothesized that this thiol-specific oxidant may target the Zn(2+)-thiol cluster of eNOS (endothelial nitric oxide synthase), resulting in enzyme dysfunction and reduced formation of the critical signalling molecule NOâ¢. Decreased NO⢠bioavailability is an early and critical event in atherogenesis, and HOSCN-mediated damage to eNOS may contribute to smoking-associated disease. In the present study it is shown that exposure of isolated eNOS to HOSCN or MPO/H2O2/SCN(-) decreased active dimeric eNOS levels, and increased inactive monomer and Zn(2+) release, compared with controls, HOCl (hypochlorous acid)- or MPO/H2O2/Cl(-)-treated samples. eNOS activity was increasingly compromised by MPO/H2O2/Cl(-) with increasing SCN(-) concentrations. Exposure of HCAEC (human coronary artery endothelial cell) lysates to pre-formed HOSCN, or MPO/H2O2/Cl(-) with increasing SCN(-), increased eNOS monomerization and Zn(2+) release, and decreased activity. Intact HCAECs exposed to HOCl and HOSCN had decreased eNOS activity and NO2(-)/NO3(-) formation (products of NO⢠decomposition), and increased free Zn(2+). Exposure of isolated rat aortic rings to HOSCN resulted in thiol loss, and decreased eNOS activity and cGMP levels. Overall these data indicate that high SCN(-) levels, as seen in smokers, can increase HOSCN formation and enhance eNOS dysfunction in human endothelial cells, with this potentially contributing to increased atherogenesis in smokers.
Asunto(s)
Óxido Nítrico Sintasa de Tipo III/fisiología , Oxidantes/toxicidad , Fumar , Tiocianatos/toxicidad , Animales , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Humanos , Ácido Hipocloroso/farmacología , Masculino , Oxidantes/metabolismo , Peroxidasa/metabolismo , Multimerización de Proteína/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
BRAF inhibitors (BRAFi) selectively target oncogenic BRAFV600E/K and are effective in 80% of advanced cutaneous malignant melanoma cases carrying the V600 mutation. However, the development of drug resistance limits their clinical efficacy. Better characterization of the underlying molecular processes is needed to further improve treatments. We previously demonstrated that transcription of PTEN is negatively regulated by the PTEN pseudogene antisense RNA, PTENP1-AS, and here we investigated the impact of this transcript on clinical outcome and BRAFi resistance in melanoma. We observed that increased expression levels of PTENP1-AS in BRAFi resistant cells associated with enrichment of EZH2 and H3K27me3 at the PTEN promoter, consequently reducing the expression levels of PTEN. Further, we showed that targeting of the PTENP1-AS transcript sensitized resistant cells to BRAFi treatment and that high expression of PTENP1-AS in stage III melanoma correlated with poor survival. Collectively, the data presented here show that PTENP1-AS is a promising target for re-sensitizing cells to BRAFi and also a possible prognostic marker for clinical outcome in stage III melanoma.
Asunto(s)
Melanoma , Proteínas Proto-Oncogénicas B-raf , Neoplasias Cutáneas , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal/efectos de los fármacos , Vemurafenib/farmacología , Melanoma Cutáneo MalignoRESUMEN
Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): 'PTEN low' BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while 'PTEN high' BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Ciclo Celular/genética , Proteína Quinasa Activada por ADN/genética , Fosfohidrolasa PTEN/genética , Proteínas Tirosina Quinasas/genética , Pirazoles/farmacología , Pirimidinonas/farmacología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteína Quinasa Activada por ADN/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Fosfohidrolasa PTEN/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ProteomaRESUMEN
Epithelial-myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial-myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial-myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC.
Asunto(s)
Biomarcadores de Tumor/antagonistas & inhibidores , Everolimus/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Mutación , Mioepitelioma/tratamiento farmacológico , Neoplasias de las Glándulas Salivales/tratamiento farmacológico , Adulto , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/genética , Carcinoma/tratamiento farmacológico , Carcinoma/genética , Carcinoma/patología , Análisis Mutacional de ADN , Femenino , Perfilación de la Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Mioepitelioma/genética , Mioepitelioma/patología , Pronóstico , Neoplasias de las Glándulas Salivales/genética , Neoplasias de las Glándulas Salivales/patología , Células Tumorales CultivadasRESUMEN
Cancer cells with stem-like properties are believed to contribute to treatment resistance, dissemination, and recurrence. SOX9 controls stem cell plasticity and its deregulation may provide a basis for tumor progression. Here, we summarize our findings of targeted SOX9 destruction by SCFFBW7 (Skp1/Cul1/F-box) in medulloblastoma and its potential for therapeutic intervention.
RESUMEN
Singlet oxygen ((1)O(2)) is a reactive oxygen species generated during photo-oxidation, inflammation, and via peroxidase-catalyzed reactions (e.g., myeloperoxidase and eosinophil peroxidase). (1)O(2) oxidizes the free amino acids Trp, Tyr, His, Cys, and Met, and those species present on peptides/proteins, with this resulting in modulation of protein structure and function. Impairment of the activity of antioxidant enzymes may be of relevance to the oxidative stress observed in a number of pathologies involving either light exposure or inflammation. In this study, the effects of (1)O(2) on glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activity, including the mechanisms of their inactivation, were investigated. Exposure of GPx or TrxR, either as purified proteins or in cell lysates, to Rose Bengal and visible light (an established source of (1)O(2)) resulted in significant, photolysis time-dependent reductions in enzyme activity (10-40%, P<0.05). More extensive inhibition (ca. 2-fold) was detected when the reactions were carried out in D(2)O, consistent with the intermediacy of (1)O(2). No additional inhibition was detected after the cessation of photolysis, eliminating a role for photo-products. Methionine, which reacts rapidly with (1)O(2) (k~10(7)M(-1) s(-1))(,) significantly reduced photo-inactivation at large molar excesses, presumably by acting as an alternative target. Reductants (NaBH(4), DTT, GSH, or NADPH) added after the cessation of (1)O(2) formation were unable to reverse enzyme inactivation, consistent with irreversible enzyme oxidation. Formation of nonreducible protein aggregates and/or fragments was detected for both photo-oxidized GPx and TrxR by SDS-PAGE. An oxidant concentration-dependent increase in protein carbonyls was detected with TrxR but not GPx. These studies thus demonstrate that the antioxidant enzymes GPx and TrxR can be irreversibly inactivated by (1)O(2).
Asunto(s)
Glutatión Peroxidasa/química , Oxidantes Fotoquímicos/química , Rosa Bengala/química , Reductasa de Tiorredoxina-Disulfuro/química , Animales , Borohidruros/química , Bovinos , Extractos Celulares/química , Línea Celular , Ditiotreitol/química , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/efectos de la radiación , Depuradores de Radicales Libres/química , Glutatión/química , Peróxido de Hidrógeno/química , Metionina/química , Ratones , Oxidación-Reducción , Fotólisis , Carbonilación Proteica/efectos de los fármacos , Carbonilación Proteica/efectos de la radiación , Ratas , Sustancias Reductoras/química , Oxígeno Singlete/químicaRESUMEN
Selenium is a critical trace element, with deficiency associated with numerous diseases including cardiovascular disease, diabetes, and cancer. Selenomethionine (SeMet; a selenium analogue of the amino acid methionine, Met) is a major form of organic selenium and an important dietary source of selenium for selenoprotein synthesis in vivo. As selenium compounds can be readily oxidized and reduced, and selenocysteine residues play a critical role in the catalytic activity of the key protective enzymes glutathione peroxidase and thioredoxin reductase, we investigated the ability of SeMet (and its sulfur analogue, Met) to scavenge hydroperoxides present on amino acids, peptides, and proteins, which are key intermediates in protein oxidation. We show that SeMet, but not Met, can remove these species both stoichiometrically and catalytically in the presence of glutathione (GSH) or a thioredoxin reductase (TrxR)/thioredoxin (Trx)/NADPH system. Reaction of the hydroperoxide with SeMet results in selenoxide formation as detected by HPLC. Recycling of the selenoxide back to SeMet occurs rapidly with GSH, TrxR/NADPH, or a complete TrxR/Trx/NADPH reducing system, with this resulting in an enhanced rate of peroxide removal. In the complete TrxR/Trx/NADPH system loss of peroxide is essentially stoichiometric with NADPH consumption, indicative of a highly efficient system. Similar reactions do not occur with Met under these conditions. Studies using murine macrophage-like J774A.1 cells demonstrate a greater peroxide-removing capacity in cells supplemented with SeMet, compared to nonsupplemented controls. Overall, these findings demonstrate that SeMet may play an important role in the catalytic removal of damaging peptide and protein oxidation products.