RESUMO
Cell transformation is clearly linked to epigenetic changes. However, the role of the histone-modifying enzymes in this process is still poorly understood. In this study, we investigated the contribution of the histone acetyltransferase (HAT) enzymes to Ras-mediated transformation. Our results demonstrated that lysine acetyltransferase 5, also known as Tip60, facilitates histone acetylation of bulk chromatin in Ras-transformed cells. As a consequence, global H4 acetylation (H4K8ac and H4K12ac) increases in Ras-transformed cells, rendering a more decompacted chromatin than in parental cells. Furthermore, low levels of CREB-binding protein (CBP) lead to hypoacetylation of retinoblastoma 1 (Rb1) and cyclin-dependent kinase inhibitor 1B (Cdkn1b or p27Kip1) tumour suppressor gene promoters to facilitate Ras-mediated transformation. In agreement with these data, overexpression of Cbp counteracts Ras transforming capability in a HAT-dependent manner. Altogether our results indicate that CBP and Tip60 coordinate histone acetylation at both local and global levels to facilitate Ras-induced transformation.
Assuntos
Proteína de Ligação a CREB/metabolismo , Transformação Celular Neoplásica/genética , Genes ras , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Transativadores/metabolismo , Acetilação , Animais , Proteína de Ligação a CREB/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Histona Acetiltransferases/genética , Lisina Acetiltransferase 5 , Camundongos , Células NIH 3T3/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Transativadores/genéticaRESUMO
Active chromatin remodelling is integral to the DNA damage response in eukaryotes, as damage sensors, signalling molecules and repair enzymes gain access to lesions. A variety of nucleosome remodelling complexes is known to promote different stages of DNA repair. The nucleosome sliding factors CHRAC/ACF of Drosophila are involved in chromatin organization during development. Involvement of corresponding hACF1-containing mammalian nucleosome sliding factors in replication, transcription and very recently also non-homologous end-joining of DNA breaks have been suggested. We now found that hACF1-containing factors are more generally involved in the DNA damage response. hACF1 depletion increases apoptosis, sensitivity to radiation and compromises the G2/M arrest that is activated in response to UV- and X-rays. In the absence of hACF1, γH2AX and CHK2ph signals are diminished. hACF1 and its ATPase partner SNF2H rapidly accumulate at sites of laser-induced DNA damage. hACF1 is also required for a tight checkpoint that is induced upon replication fork collapse. ACF1-depleted cells that are challenged with aphidicolin enter mitosis despite persistence of lesions and accumulate breaks in metaphase chromosomes. hACF1-containing remodellers emerge as global facilitators of the cellular response to a variety of different types of DNA damage.
Assuntos
Dano ao DNA , Reparo do DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Pontos de Checagem da Fase M do Ciclo Celular , Fatores de Transcrição/fisiologia , Afidicolina/toxicidade , Apoptose , Linhagem Celular , Proteínas Cromossômicas não Histona , Fragilidade Cromossômica , Humanos , Interferência de RNA , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genéticaRESUMO
Ewing Sarcoma (EWS) is an aggressive bone and soft tissue tumor that mainly affects children, adolescents, and young adults. The standard therapy, including chemotherapy, surgery, and radiotherapy, has substantially improved the survival of EWS patients with localized disease. Unfortunately, this multimodal treatment remains elusive in clinics for those patients with recurrent or metastatic disease who have an unfavorable prognosis. Consistently, there is an urgent need to find new strategies for patients that fail to respond to standard therapies. In this regard, in the last decade, treatments targeting epigenetic dependencies in tumor cells and the immune system have emerged into the clinical scenario. Additionally, recent advances in nanomedicine provide novel delivery drug systems, which may address challenges such as side effects and toxicity. Therefore, therapeutic strategies stemming from epigenetics, immunology, and nanomedicine yield promising alternatives for treating these patients. In this review, we highlight the most relevant EWS preclinical and clinical studies in epigenetics, immunotherapy, and nanotherapy conducted in the last five years.
RESUMO
Ewing sarcoma (EwS) is a human malignant tumor typically driven by the Ewing sarcoma-Friend leukemia integration (EWS-FLI) fusion protein. A paucity of genetically modified animal models, partially owed to the high toxicity of EWS-FLI, hinders research on EwS. Here, we report a spontaneous mutant variant, EWS-FLI1FS, that circumvents the toxicity issue in Drosophila. Through proteomic and genomic analyses, we show that human EWS-FLI1FS interacts with the Drosophila homologues of EWS-FLI human protein partners, including core subunits of chromatin remodeling complexes, the transcription machinery, and the spliceosome; brings about a massive dysregulation of transcription that affects a significant fraction of known targets of EWS-FLI in human cells; and modulates splicing. We also show that EWS-FLI1FS performs in Drosophila the two major neomorphic activities that it is known to have in human cells: activation of transcription from GGAA microsatellites and out competition of ETS transcription factors. We conclude that EWS-FLI1FS reproduces in Drosophila the known oncogenic activities of EWS-FLI that drive EwS tumorigenesis in humans. These results open up an unprecedented opportunity to investigate EWS-FLI's oncogenic pathways in vivo in a genetically tractable organism.
RESUMO
Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor with high susceptibility to metastasize. The underlying molecular mechanisms leading to EWS metastases remain poorly understood. Epigenetic changes have been implicated in EWS tumor growth and progression. Linking epigenetics and metastases may provide insight into novel molecular targets in EWS and improve its treatment. Here, we evaluated the effects of a selective G9a histone methyltransferase inhibitor (BIX01294) on EWS metastatic process. Our results showed that overexpression of G9a in tumors from EWS patients correlates with poor prognosis. Moreover, we observe a significantly higher expression of G9a in metastatic EWS tumor as compared to either primary or recurrent tumor. Using functional assays, we demonstrate that pharmacological G9a inhibition using BIX01294 disrupts several metastatic steps in vitro, such as migration, invasion, adhesion, colony formation and vasculogenic mimicry. Moreover, BIX01294 reduces tumor growth and metastases in two spontaneous metastases mouse models. We further identified the sialidase NEU1 as a direct target and effector of G9a in the metastatic process in EWS. NEU1 overexpression impairs migration, invasion and clonogenic capacity of EWS cell lines. Overall, G9a inhibition impairs metastases in vitro and in vivo through the overexpression of NEU1. G9a has strong potential as a prognostic marker and may be a promising therapeutic target for EWS patients.
Assuntos
Sarcoma de Ewing , Animais , Linhagem Celular Tumoral , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Histona Metiltransferases/metabolismo , Humanos , Camundongos , Recidiva Local de Neoplasia , Neuraminidase/metabolismo , Neuraminidase/uso terapêutico , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologiaRESUMO
Ewing sarcoma (EWS) is an aggressive bone and soft tissue tumor of children and young adults in which the principal driver is a fusion gene, EWSR1-FLI1. Although the essential role of EWSR1-FLI1 protein in the regulation of oncogenesis, survival, and tumor progression processes has been described in-depth, little is known about the regulation of chimeric fusion-gene expression. Here, we demonstrate that the active nuclear HDAC6 in EWS modulates the acetylation status of specificity protein 1 (SP1), consequently regulating the SP1/P300 activator complex binding to EWSR1 and EWSR1-FLI1 promoters. Selective inhibition of HDAC6 impairs binding of the activator complex SP1/P300, thereby inducing EWSR1-FLI1 downregulation and significantly reducing its oncogenic functions. In addition, sensitivity of EWS cell lines to HDAC6 inhibition is higher than other tumor or non-tumor cell lines. High expression of HDAC6 in primary EWS tumor samples from patients correlates with a poor prognosis in two independent series accounting 279 patients. Notably, a combination treatment of a selective HDAC6 and doxorubicin (a DNA damage agent used as a standard therapy of EWS patients) dramatically inhibits tumor growth in two EWS murine xenograft models. These results could lead to suitable and promising therapeutic alternatives for patients with EWS.
Assuntos
Proteína Proto-Oncogênica c-fli-1 , Sarcoma de Ewing , Acetilação , Carcinogênese , Desacetilase 6 de Histona , Humanos , Regiões Promotoras GenéticasRESUMO
The presence of the chimeric EWSR1-FLI1 oncoprotein is the main and initiating event defining Ewing sarcoma (ES). The dysregulation of epigenomic and proteomic homeostasis induced by the oncoprotein contributes to a wide variety of events involved in oncogenesis and tumor progression. Attempts at studying the effects of EWSR1-FLI1 in non-tumor cells to understand the mechanisms underlying sarcomagenesis have been unsuccessful to date, as ectopic expression of EWSR1-FLI1 blocks cell cycle progression and induces apoptosis in the tested cell lines. Therefore, it is essential to find a permissive cell type for EWSR1-FLI1 expression that allows its endogenous molecular functions to be studied. Here we have demonstrated that HeLa cell lines are permissive to EWSR1-FLI1 ectopic expression, and that our model substantially recapitulates the endogenous activity of the EWSR1-FLI1 fusion protein. This model could contribute to better understanding ES sarcomagenesis by helping to understand the molecular mechanisms induced by the EWSR1-FLI1 oncoprotein.
Assuntos
Carcinogênese/genética , Expressão Ectópica do Gene , Proteínas de Fusão Oncogênica/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Sítios de Ligação , DNA/metabolismo , Células HeLa , Humanos , Proteínas de Fusão Oncogênica/metabolismoRESUMO
Ewing sarcoma (EwS) is an aggressive tumor that affects adolescents and young adults. EwS is defined by a chromosomal translocation, EWSR1-FLI1 being the most common, that causes genome reprogramming through remodeling of enhancers. Here, we describe an unexpected function of RING1B, which is highly expressed in EwS. While retaining its repressive activity at Polycomb developmental regulated genes, RING1B colocalizes with EWSR1-FLI1 at active enhancers. We demonstrate that RING1B is necessary for the expression of key EWSR1-FLI1 targets by facilitating oncogene recruitment to their enhancers. Knockdown of RING1B impairs growth of tumor xenografts and expression of genes regulated by EWSR1-FLI1 bound enhancers. Pharmacological inhibition of AURKB with AZD1152 increases H2Aub levels causing down-regulation of RING1B/EWSR1-FLI1 common targets. Our findings demonstrate that RING1B is a critical modulator of EWSR1-FLI1-induced chromatin remodeling, and its inhibition is a potential therapeutic strategy for the treatment of these tumors.
Assuntos
Cromatina , Sarcoma de Ewing , Adolescente , Carcinogênese , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Cromatina/genética , Montagem e Desmontagem da Cromatina , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Fusão Oncogênica/genética , Proteína EWS de Ligação a RNA/genética , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Adulto JovemRESUMO
The CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300 acetyltransferases function as transcriptional co-activators and play critical roles in cell differentiation and proliferation. Accumulating evidence shows that alterations of the CBP/p300 protein levels are linked to human tumours. In the present study, we show that the levels of the CBP/p300 co-activators are decreased dramatically by continuous PDGF (platelet-derived growth factor) and Ras signalling pathway activation in NIH 3T3 fibroblasts. This effect occurs by reducing the expression levels of the CBP/p300 genes. In addition, CBP and p300 are degraded by the 26 S proteasome pathway leading to an overall decrease in the levels of the CBP/p300 proteins. Furthermore, we provide evidence that Mdm2 (murine double minute 2), in the presence of active H-Ras or N-Ras, induces CBP/p300 degradation in NIH 3T3 cells. These findings support a novel mechanism for modulating other signalling transduction pathways that require these common co-activators.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Proteínas ras/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Histona Acetiltransferases/genética , Camundongos , Células NIH 3T3 , Fator de Crescimento Derivado de Plaquetas/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Fatores de Transcrição/genética , Ubiquitina/metabolismo , Valina/genética , Valina/metabolismo , Fatores de Transcrição de p300-CBP , Proteínas ras/genéticaRESUMO
Ewing sarcoma (ES) is an aggressive tumor defined by EWSR1 gene fusions that behave as an oncogene. Here we demonstrate that RING1B is highly expressed in primary ES tumors, and its expression is independent of the fusion oncogene. RING1B-depleted ES cells display an expression profile enriched in genes functionally involved in hematological development but RING1B depletion does not induce cellular differentiation. In ES cells, RING1B directly binds the SCN8A sodium channel promoter and its depletion results in enhanced Nav1.6 expression and function. The signaling pathway most significantly modulated by RING1B is NF-κB. RING1B depletion results in enhanced p105/p50 expression, which sensitizes ES cells to apoptosis by FGFR/SHP2/STAT3 blockade. Reduced NaV1.6 function protects ES cells from apoptotic cell death by maintaining low NF-κB levels. Our findings identify RING1B as a trait of the cell-of-origin and provide a potential targetable vulnerability.
Assuntos
Neoplasias Ósseas/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.6/metabolismo , NF-kappa B/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Sarcoma de Ewing/metabolismo , Transdução de Sinais , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Humanos , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
The global inhibition of transcription at the mitotic phase of the cell cycle occurs together with the general displacement of transcription factors from the mitotic chromatin. Nevertheless, the DNase- and potassium permanganate-hypersensitive sites are maintained on potentially active promoters during mitosis, helping to mark active genes at this stage of the cell cycle. Our study focuses on the role of histone acetylation and H3 (Lys-4) methylation in the maintenance of the competency of these active genes during mitosis. To this end we have analyzed histone modifications across the promoters and coding regions of constitutively active, inducible, and inactive genes in mitotic arrested cells. Our results show that basal histone modifications are maintained during mitosis at promoters and coding regions of the active and inducible RNA polymerase II-transcribed genes. In addition we have demonstrated that, together with H3 acetylation and H3 (Lys-4) methylation, H4 (Lys-12) acetylation at the coding regions contributes to the formation of a stable mark on active genes at this stage of the cell cycle. Finally, analysis of cyclin B1 gene activation during mitosis revealed that the former occurs with a strong increase of H3 (Lys-4) trimethylation but not H3 or H4 acetylation, suggesting that histone methyltransferases are active during this stage. These data demonstrate a critical role of histone acetylation and H3 (Lys-4) methylation during mitosis in marking and activating genes during the mitotic stage of the cell cycle.
Assuntos
Histonas/química , Mitose , Acetilação , Ciclo Celular , Núcleo Celular/metabolismo , Divisão do Núcleo Celular , Cromatina/química , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Ciclina B/metabolismo , Ciclina B1 , Técnica Indireta de Fluorescência para Anticorpo , Regulação da Expressão Gênica , Células HeLa , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Immunoblotting , Interfase , Lisina/química , Metilação , Nucleossomos/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Proteínas Metiltransferases , Processamento de Proteína Pós-Traducional , RNA Polimerase II/metabolismo , Temperatura , Transcrição Gênica , Ativação TranscricionalRESUMO
The existence of different patterns of chemical modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) of the histone tails led, some years ago, to the histone code hypothesis. According to this hypothesis, these modifications would provide binding sites for proteins that can change the chromatin state to either active or repressed. Interestingly, some protein domains present in histone-modifying enzymes are known to interact with these covalent marks in the histone tails. This was first shown for the bromodomain, which was found to interact selectively with acetylated lysines at the histone tails. More recently, it has been described that the chromodomain can be targeted to methylation marks in histone N-terminal domains. Finally, the interaction between the SANT domain and histones is also well documented. Overall, experimental evidence suggests that these domains could be involved in the recruitment of histone-modifying enzymes to discrete chromosomal locations, and/or in the regulation their enzymatic activity. Within this context, we review the distribution of bromodomains, chromodomains and SANT domains among chromatin-modifying enzymes and discuss how they can contribute to the translation of the histone code.