RESUMEN
We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.
Asunto(s)
Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Encéfalo/embriología , Encéfalo/metabolismo , Supervivencia Celular , Niño , Discapacidades del Desarrollo , Femenino , Humanos , Cinesinas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación Missense , Transporte de Proteínas , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
Deep-sequencing reveals extensive variation in the sequence of endogenously expressed microRNAs (termed 'isomiRs') in human cell lines and tissues, especially in relation to the 3' end. From the immunoprecipitation of the microRNA-binding protein Argonaute and the sequencing of associated small RNAs, we observe extensive 3'-isomiR variation, including for miR-222 where the majority of endogenously expressed miR-222 is extended by 1-5 nt compared to the canonical sequence. We demonstrate this 3' heterogeneity has dramatic implications for the phenotype of miR-222 transfected cells, with longer isoforms promoting apoptosis in a size (but not 3' sequence)-dependent manner. The transfection of longer miR-222 isomiRs did not induce an interferon response, but did downregulate the expression of many components of the pro-survival PI3K-AKT pathway including PIK3R3, a regulatory subunit whose knockdown phenocopied the expression of longer 222 isoforms in terms of apoptosis and the inhibition of other PI3K-AKT genes. As this work demonstrates the capacity for 3' isomiRs to mediate differential functions, we contend more attention needs to be given to 3' variance given the prevalence of this class of isomiR.
Asunto(s)
Apoptosis/genética , Proliferación Celular/genética , Regulación de la Expresión Génica , MicroARNs/genética , Isoformas de ARN/genética , Línea Celular , Línea Celular Tumoral , Humanos , Immunoblotting , Células MCF-7 , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
PURPOSE: The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. METHODS: We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. RESULTS: There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. CONCLUSIONS: This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.
Asunto(s)
Genes de Neurofibromatosis 1 , Neurofibromatosis 1/diagnóstico , Neurofibromatosis 1/genética , Adulto , Anciano , Biomarcadores de Tumor , Análisis Mutacional de ADN , Femenino , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana Edad , Mutación , Clasificación del Tumor , Estadificación de Neoplasias , Neurofibromatosis 1/epidemiología , Neurofibromatosis 1/mortalidadRESUMEN
A series of azobenzene-containing peptidic boronate esters was prepared and the activity of the thermally adapted states (TAS), enriched in trans isomer, and the photostationary states (PSS), enriched in cis isomer, for each compound were evaluated against ß5 and ß1 proteasome subunits. Compounds with a sterically demanding phenyl-substituted azobenzene at P2 (4c), and a less sterically demanding unsubstituted azobenzene at the N-terminus (5a), showed the greatest difference in activity between the two states. In both cases, the more active trans-enriched TAS had activity comparable to bortezomib and delanzomib. Furthermore, cis-enriched 4c inhibited tumor growth in both breast and colorectal carcinoma cell lines. Significantly, the initial trans-enriched TAS of 4c was not cytotoxic against the non-malignant MCF-10A cells.
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Antineoplásicos/química , Antineoplásicos/farmacología , Compuestos Azo/química , Compuestos Azo/farmacología , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Isomerismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Procesos Fotoquímicos , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas F-Box/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Fase G2 , Humanos , Unión Proteica , Proteolisis , Proteínas Supresoras de Tumor/genética , UbiquitinaciónRESUMEN
Mutations in ANKRD11 have recently been reported to cause KBG syndrome, an autosomal dominant condition characterized by intellectual disability (ID), behavioral problems, and macrodontia. To understand the pathogenic mechanism that relates ANKRD11 mutations with the phenotype of KBG syndrome, we studied the cellular characteristics of wild-type ANKRD11 and the effects of mutations in humans and mice. We show that the abundance of wild-type ANKRD11 is tightly regulated during the cell cycle, and that the ANKRD11 C-terminus is required for the degradation of the protein. Analysis of 11 pathogenic ANKRD11 variants in humans, including six reported in this study, and one reported in the Ankrd11 (Yod/+) mouse, shows that all mutations affect the C-terminal regions and that the mutant proteins accumulate aberrantly. In silico analysis shows the presence of D-box sequences that are signals for proteasome degradation. We suggest that ANKRD11 C-terminus plays an important role in regulating the abundance of the protein, and a disturbance of the protein abundance due to the mutations leads to KBG syndrome.
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Anomalías Múltiples , Enfermedades del Desarrollo Óseo , Ciclo Celular/genética , Proteínas de Unión al ADN , Facies , Discapacidad Intelectual , Mutación , Proteolisis , Proteínas Represoras , Anomalías Dentarias , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Animales , Enfermedades del Desarrollo Óseo/genética , Enfermedades del Desarrollo Óseo/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Masculino , Ratones , Ratones Mutantes , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Anomalías Dentarias/genética , Anomalías Dentarias/metabolismoRESUMEN
Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole-containing macrocyclic protease inhibitors pre-organized into a ß-strand conformation and an evaluation of their activity against a panel of proteases. Acyclic azido-alkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpainâ II, cathepsinâ L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent macrocyclic inhibitors of cathepsinâ S were identified. These adopt a well-defined ß-strand geometry as shown by NMR spectroscopy, X-ray analysis, and molecular docking studies.
Asunto(s)
Compuestos Macrocíclicos/síntesis química , Péptidos/química , Inhibidores de Proteasas/síntesis química , Triazoles/síntesis química , Calpaína/antagonistas & inhibidores , Catepsina L/antagonistas & inhibidores , Química Clic , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Peptidomiméticos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Triazoles/química , Triazoles/farmacologíaRESUMEN
BACKGROUND: Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. METHODS: A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. RESULTS: Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. CONCLUSIONS: The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Azacitidina/análogos & derivados , Mama/citología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Proteínas Luminiscentes/metabolismo , Nitrorreductasas/metabolismo , Antineoplásicos/farmacología , Azacitidina/farmacología , Proteínas Portadoras/genética , Células Cultivadas , Citomegalovirus/genética , Metilación de ADN , Decitabina , Epigénesis Genética , Regulación de la Expresión Génica , Genes Reporteros , Vectores Genéticos , Humanos , Ácidos Hidroxámicos/farmacología , Proteínas Luminiscentes/genética , Nitrorreductasas/genética , Plásmidos , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Liposomas Unilamelares , Vorinostat , Proteína Fluorescente RojaRESUMEN
A significant proportion of transcription factors encoded by the human genome are classical C(2) H(2) zinc finger proteins that regulate gene expression by directly interacting with their cognate DNA binding motifs. We previously showed that one such C(2) H(2) zinc finger DNA binding protein, ZNF652 (zinc finger protein 652), specifically and functionally interacts with CBFA2T3 to repress transcription of genes involved in breast oncogenesis. To identify potential targets by which ZNF652 exerts its putative tumour suppressive function, its promoter-specific cistrome was mapped by ChIP-chip. De novo motif scanning of the ZNF652 binding sites identified a novel ZNF652 recognition motif that closely resembles the previously characterised in vitro binding site, being a 10 nucleotide core of that 13 nucleotide sequence. Genes with ZNF652 binding sites function in diverse cellular pathways, and many are involved in cancer development and progression. Characterisation of the in vivo ZNF652 DNA binding motif and identification of potential ZNF652 target genes are key steps towards elucidating the function(s) of this transcription factor in the normal and malignant breast cell.
Asunto(s)
Neoplasias de la Mama/genética , Mapeo Cromosómico/métodos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas/genética , Sitios de Unión/genética , Línea Celular Tumoral , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Humanos , Hidroliasas/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The p53 tumour suppressor plays a pivotal role in the prevention of oncogenic transformation. Cancers frequently evade the potent antitumour surveillance mechanisms of p53 through mutation of the TP53 gene, with approximately 50% of all human malignancies expressing dysfunctional, mutated p53 proteins. Interestingly, genetic lesions in the TP53 gene are only observed in 10% of Ewing Sarcomas, with the majority of these sarcomas expressing a functional wild-type p53. In addition, the p53 downstream signaling pathways and DNA-damage cell cycle checkpoints remain functionally intact in these sarcomas. This paper summarizes recent insights into the functional capabilities and regulation of p53 in Ewing Sarcoma, with a particular focus on the cross-talk between p53 and the EWS-FLI1 gene rearrangement frequently associated with this disease. The development of several activators of p53 is discussed, with recent evidence demonstrating the potential of small molecule p53 activators as a promising systemic therapeutic approach for the treatment of Ewing Sarcomas with wild-type p53.
RESUMEN
We report on the role of PAMAM dendrimer concentration and generation (G2, G4, G6) on cell growth and cytotoxicity in HEK293T and HeLa cell lines and make comparisons with dendrimer-induced leakage from liposomes to probe the mechanisms in action. Specifically, we observed a striking transition from cell growth enhancement to a reduction in cell viability at a critical PAMAM dendrimer concentration, that is, approximately 500 nM. Confocal microscopy studies show evidence of a transition from cell membrane adhesion to cell internalization and cell nucleus interaction at equivalent dendrimer concentrations. A dendrimer concentration window of 500-700 nM was identified for effective cell internalization without significant cytotoxicity. Though liposome leakage correlated with cytotoxicity, no quantitative agreement was observed, that is, cells are 100 times (based on surface coverage) more resistant to dendrimers than liposomes. These findings have significant implications in the design of effective drug/gene delivery vehicles based on dendrimers.
Asunto(s)
Materiales Biocompatibles/toxicidad , Proliferación Celular/efectos de los fármacos , Citotoxinas/toxicidad , Dendrímeros/toxicidad , Materiales Biocompatibles/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Citotoxinas/metabolismo , Dendrímeros/metabolismo , Células HeLa , Humanos , Liposomas/metabolismoRESUMEN
Human chromosome 16 features one of the highest levels of segmentally duplicated sequence among the human autosomes. We report here the 78,884,754 base pairs of finished chromosome 16 sequence, representing over 99.9% of its euchromatin. Manual annotation revealed 880 protein-coding genes confirmed by 1,670 aligned transcripts, 19 transfer RNA genes, 341 pseudogenes and three RNA pseudogenes. These genes include metallothionein, cadherin and iroquois gene families, as well as the disease genes for polycystic kidney disease and acute myelomonocytic leukaemia. Several large-scale structural polymorphisms spanning hundreds of kilobase pairs were identified and result in gene content differences among humans. Whereas the segmental duplications of chromosome 16 are enriched in the relatively gene-poor pericentromere of the p arm, some are involved in recent gene duplication and conversion events that are likely to have had an impact on the evolution of primates and human disease susceptibility.
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Cromosomas Humanos Par 16/genética , Duplicación de Gen , Mapeo Físico de Cromosoma , Animales , Genes/genética , Genómica , Heterocromatina/genética , Humanos , Datos de Secuencia Molecular , Polimorfismo Genético/genética , Análisis de Secuencia de ADN , Sintenía/genéticaRESUMEN
In vitro studies have suggested proteasome inhibitors could be effective in triple-negative breast cancer (TNBC). We found that bortezomib and carfilzomib induce proteotoxic stress and apoptosis via the unfolded protein response (UPR) in TNBC cell lines, with sensitivity correlated with expression of immuno-(PSMB8/9/10) but not constitutive-(PSMB5/6/7) proteasome subunits. Equally, the transcriptomes of i-proteasome-high human TNBCs are enriched with UPR gene sets, and the genomic copy number landscape reflects positive selection pressure favoring i-proteasome activity, but in the setting of adjuvant treatment, this is actually associated with favorable prognosis. Tumor expression of PSMB8 protein (ß5i) is associated with levels of MHC-I, interferon-γ-inducible proteasome activator PA28ß, and the densities of stromal antigen-presenting cells and lymphocytes (TILs). Crucially, TILs were protective among TNBCs that maintain high ß5i but did not stratify survival amongst ß5i-low TNBCs. Moreover, ß5i expression was lower in brain metastases than in patient-matched primary breast tumors (n = 34; P = 0.007), suggesting that suppression contributes to immune evasion and metastatic progression. Hence, inhibiting proteasome activity could be counterproductive in the adjuvant treatment setting because it potentiates anti-TNBC immunity.
Asunto(s)
Metabolismo Energético , Evasión Inmune , Complejo de la Endopetidasa Proteasomal/metabolismo , Neoplasias de la Mama Triple Negativas/etiología , Neoplasias de la Mama Triple Negativas/metabolismo , Bortezomib/farmacología , Variaciones en el Número de Copia de ADN , Susceptibilidad a Enfermedades , Resistencia a Antineoplásicos , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Evasión Inmune/genética , Estimación de Kaplan-Meier , Pronóstico , Inhibidores de Proteasoma/farmacología , Transcriptoma , Neoplasias de la Mama Triple Negativas/mortalidad , Neoplasias de la Mama Triple Negativas/patología , Respuesta de Proteína DesplegadaRESUMEN
Active vitamin D (1,25(OH)2D) has been shown to regulate numerous cell processes in mammary cells. Degradation of 1,25(OH)2D is initiated by the mitochondrial enzyme, 25-hydroxyvitamin D 24-hydroxylase (CYP24 A1), and provides local control of 1,25(OH)2D bioactivity. Several reports of the association between elevated CYP24 A1 activity and breast cancer incidence, suggest that CYP24 A1 may be a target for therapeutic intervention. Whether CYP24 A1 activity within the mammary epithelium regulates 1,25(OH)2D levels and mammary gland development is yet to shown. We have used a conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium to demonstrate reduced terminal end bud number, ductal outgrowth and branching during puberty and alveologenesis at early pregnancy, by inhibiting proliferation but not apoptosis in both basal and luminal MECs. In vitro study showed increased sensitivity of luminal MECs to lower levels of 1,25(OH)2D with the ablation of Cyp24a1 activity. In summary, Cyp24a1 within MECs plays an important role in modulating postnatal and pregnancy-associated mammary gland development which provides support for inhibiting CYP24 A1 as a potential approach to activating the vitamin D pathway in breast cancer prevention and therapy.
Asunto(s)
Eliminación de Gen , Glándulas Mamarias Animales/metabolismo , Vitamina D3 24-Hidroxilasa/genética , Vitamina D/metabolismo , Animales , Proliferación Celular , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/crecimiento & desarrollo , Glándulas Mamarias Animales/ultraestructura , Ratones , Ratones Endogámicos C57BL , Maduración Sexual , Vitamina D/análogos & derivados , Vitamina D3 24-Hidroxilasa/metabolismoRESUMEN
The biologically active form of vitamin D3 (1,25(OH)2D) regulates epithelial cell differentiation, proliferation, and apoptosis, lending weight to clinical evidence linking vitamin D3 insufficiency to breast cancer incidence and mortality. Local dysregulation of vitamin D3 metabolism has been identified in patients with breast cancer, implying that disruption of 1,25(OH)2D signaling may contribute to breast cancer development in an autocrine or paracrine manner. Mouse mammary glands express the critical enzymes responsible for 1,25(OH)2D synthesis (Cyp2r1 and Cyp27b1), degradation (Cyp24a1), as well as the vitamin D3 receptor (Vdr), and genetically modified mouse models have revealed a great deal about the role of vitamin D3 in cancer initiation and progression. Ablation of Vdr or Cyp27b1 in murine models of mammary cancer reduces the anti-tumor effects of vitamin D3, while elevation of Cyp24a1 levels increases degradation of 1,25(OH)2D, leading to diminished anti-tumor effects. This review discusses the recent transgenic mouse models of vitamin D3 metabolism and the Vdr signaling network, and how these contribute to mammary gland development, and cancer tumorigenesis and progression. Collectively, these mouse models have helped clarify mechanisms of action of vitamin D3 signaling and suggest that activation or restoration of the vitamin D3 regulated pathway is a potential approach for human breast cancer prevention.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Colecalciferol/metabolismo , Modelos Animales de Enfermedad , Vitaminas/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones TransgénicosRESUMEN
Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 -1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 µmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902-16. ©2018 AACR.
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Neoplasias Óseas/tratamiento farmacológico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Sarcoma de Ewing/tratamiento farmacológico , Adolescente , Apoptosis/efectos de los fármacos , Apoptosis/genética , Neoplasias Óseas/enzimología , Neoplasias Óseas/genética , Línea Celular Tumoral , Niño , Estrés del Retículo Endoplásmico/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Interferencia de ARN , Sarcoma de Ewing/enzimología , Sarcoma de Ewing/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The transcriptional repressor CBFA2T3 is a putative breast tumor suppressor. To define the role of CBFA2T3, we used a segment of this protein as bait in a yeast two-hybrid screen and identified a novel uncharacterized protein, ZNF652. In general, primary tumors and cancer cell lines showed lower expression of ZNF652 than normal tissues. Together with the location of this gene on the long arm of chromosome 17q, a region of frequent loss of heterozygosity in cancer, these results suggest a possible role of ZNF652 in tumorigenesis. In silico analysis of this protein revealed that it contains multiple classic zinc finger domains that are predicted to bind DNA. Coimmunoprecipitation assays showed that ZNF652 strongly interacts with CBFA2T3 and this interaction occurs through the COOH-terminal 109 amino acids of ZNF652. In contrast, there was a weak interaction of ZNF652 with CBFA2T1 and CBFA2T2, the other two members of this ETO family. Transcriptional reporter assays further confirmed the strength and selectivity of the ZNF652-CBFA2T3 interaction. The transcriptional repression of growth factor independent-1 (GFI-1), a previously characterized ETO effector zinc finger protein, was shown to be enhanced by CBFA2T1, but to a lesser extent by CBFA2T2 and CBFA2T3. We therefore suggest that each of the various gene effector zinc finger proteins may specifically interact with one or more of the ETO proteins to generate a defined range of transcriptional repressor complexes.
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Neoplasias de la Mama/genética , Proteínas de Unión al ADN/genética , Fosfoproteínas/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Proteínas Supresoras de Tumor/genética , Dedos de Zinc/fisiología , Secuencia de Aminoácidos , Animales , Neoplasias de la Mama/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Supresores de Tumor , Humanos , Ratones , Datos de Secuencia Molecular , Fosfoproteínas/biosíntesis , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteína 1 Compañera de Translocación de RUNX1 , Conejos , Ratas , Proteínas Represoras/biosíntesis , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Supresoras de Tumor/biosíntesis , Proteínas Supresoras de Tumor/metabolismo , Técnicas del Sistema de Dos Híbridos , Dedos de Zinc/genéticaRESUMEN
A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumor cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. FBXO31 has properties consistent with a tumor suppressor, because ectopic expression of FBXO31 in two breast cancer cell lines inhibited colony growth on plastic and inhibited cell proliferation in the MCF-7 cell line. In addition, compared with the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumor cell lines and primary tumors. FBXO31 was cell cycle regulated in the breast cell lines MCF-10A and SKBR3 with maximal expression from late G(2) to early G(1) phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G(1) phase of the cell cycle. FBXO31 contains an F-box domain and is associated with the proteins Skp1, Roc-1, and Cullin-1, suggesting that FBXO31 is a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumor suppressor by generating SCF(FBXO31) complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation.
Asunto(s)
Neoplasias de la Mama/genética , Senescencia Celular/genética , Cromosomas Humanos Par 16 , Proteínas F-Box/genética , Genes Supresores de Tumor , Proteínas Supresoras de Tumor/genética , Northern Blotting , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Cromosomas Artificiales Bacterianos , Ensayo de Unidades Formadoras de Colonias , Proteínas Cullin/metabolismo , Proteínas F-Box/metabolismo , Femenino , Fase G1 , Fase G2 , Humanos , Inmunoprecipitación , Riñón/metabolismo , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/metabolismoRESUMEN
p53, a transcription factor that participates in multiple cellular functions, is considered the most important tumor suppressor. Previous evidence suggests that post-transcriptional deregulation of p53 by microRNAs contributes to tumorigenesis, tumor progression and therapeutic resistance. In the present study, we found that the microRNA miR-766 was aberrantly expressed in breast cancer, and that over-expression of miR-766 caused accumulation of wild-type p53 protein in multiple cancer cell lines. Supporting its role in the p53 signalling pathway, miR-766 decreased cell proliferation and colony formation in several cancer cell lines, and cell cycle analyses revealed that miR-766 causes G2 arrest. At a mechanistic level, we demonstrate that miR-766 enhances p53 signalling by directly targeting MDM4, an oncogene and negative regulator of p53. Analysis of clinical genomic data from multiple cancer types supports the relevance of miR-766 in p53 signalling. Collectively, our study demonstrates that miR-766 can function as a novel tumor suppressor by enhancing p53 signalling.
Asunto(s)
Puntos de Control de la Fase G2 del Ciclo Celular/genética , MicroARNs/genética , Proteínas Nucleares/genética , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Proteína p53 Supresora de Tumor/metabolismo , Regiones no Traducidas 3' , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Transducción de Señal , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Numerous cytogenetic and molecular studies of breast cancer have identified frequent loss of heterozygosity (LOH) of the long arm of human chromosome 16. On the basis of these data, the likely locations of breast cancer tumor suppressor genes are bands 16q22.1 and 16q24.3. We have mapped the CBFA2T3 (MTG16) gene, previously cloned as a fusion partner of the AML1 protein from a rare (16;21) leukemia translocation, to the 16q24.3 breast cancer LOH region. The expression of CBFA2T3 was significantly reduced in a number of breast cancer cell lines and in primary breast tumors, including early ductal carcinomas in situ, when compared with nontransformed breast epithelial cell lines and normal breast tissue. Reintroduction of CBFA2T3 into different breast tumor derived cell lines with decreased expression of this gene reduced colony growth on plastic and in soft agar. CBFA2T3 was shown to function as a transcriptional repressor when tethered to the GAL4 DNA-binding domain in a reporter gene assay and, therefore, has the potential to be a transcriptional repressor in normal breast epithelial cells. Taken together, these findings suggest that CBFA2T3 is a likely candidate for the breast cancer tumor suppressor gene that is the target for the frequent 16q24 LOH in breast neoplasms.