RESUMEN
Multiple myeloma (MM) is cytogenetically, genetically and molecularly heterogenous even among subclones in one patient, therefore, it is essential to identify both frequent and patient-specific drivers of molecular abnormality. Following previous molecular investigations, we in this study investigated the expression patterns and function of the Ewing sarcoma breakpoint region 1 (EWSR1) gene in MM. The EWSR1 transcriptional level in CD138-positive myeloma cells was higher in 36.4% of monoclonal gammopathy of undetermined significance, in 67.4% of MM patients compared with normal plasma cells, and significantly higher in ten human myeloma-derived cell lines (HMCLs) examined. EWSR1 gene knockdown caused growth inhibition with an increase of apoptotic cells in NCI-H929 and KMS-12-BM cells. Gene expression profiling using microarray analysis suggested EWSR1 gene knockdown caused transcriptional modulation of several genes associated with processes such as cell proliferation, cell motility, cell metabolism, and gene expression. Of particular, EWSR1 gene knockdown caused upregulation of let-7c and downregulation of its known targets K-RAS and AKT. Finally, our analysis using community database suggested that high EWSR1 expression positively associates with poor prognosis and advanced disease stage in MM. These findings suggest that EWSR1 overexpression is a pro-oncogenic molecular abnormality that may participate in MM progression.
Asunto(s)
MicroARNs/biosíntesis , Mieloma Múltiple/metabolismo , Proteínas de Neoplasias/biosíntesis , Proteína EWS de Unión a ARN/biosíntesis , Apoptosis/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Evolución Clonal , Progresión de la Enfermedad , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Estimación de Kaplan-Meier , MicroARNs/genética , Gammopatía Monoclonal de Relevancia Indeterminada/genética , Gammopatía Monoclonal de Relevancia Indeterminada/metabolismo , Gammopatía Monoclonal de Relevancia Indeterminada/patología , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Proteínas de Neoplasias/genética , Supervivencia sin Progresión , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Proteína EWS de Unión a ARN/genética , Transcripción GenéticaRESUMEN
Brain development involves proliferation, migration and specification of neural progenitor cells, culminating in neuronal circuit formation. Mounting evidence indicates that improper regulation of RNA binding proteins (RBPs), including members of the FET (FUS, EWS, TAF15) family, results in defective cortical development and/or neurodegenerative disorders. However, in spite of their physiological relevance, the precise pattern of FET protein expression in developing neurons is largely unknown. Herein, we found that FUS, EWS and TAF15 expression is differentially regulated during brain development, both in time and in space. In particular, our study identifies a fine-tuned regulation of FUS and EWS during neuronal differentiation, whereas TAF15 appears to be more constitutively expressed. Mechanistically FUS and EWS protein expression is regulated at the post-transcriptional level during neuron differentiation and brain development. Moreover, we identified miR-141 as a key regulator of these FET proteins that modulate their expression levels in differentiating neuronal cells. Thus, our studies uncover a novel link between post-transcriptional regulation of FET proteins expression and neurogenesis.
Asunto(s)
MicroARNs/metabolismo , Neuronas/fisiología , Procesamiento Postranscripcional del ARN , Proteína EWS de Unión a ARN/biosíntesis , Proteína FUS de Unión a ARN/biosíntesis , Animales , Encéfalo/citología , Encéfalo/embriología , Encéfalo/metabolismo , Diferenciación Celular/fisiología , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , Neurogénesis/fisiología , Neuronas/citología , Neuronas/metabolismo , Procesamiento Proteico-Postraduccional , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Proteína FUS de Unión a ARN/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores Asociados con la Proteína de Unión a TATA/biosíntesis , Factores Asociados con la Proteína de Unión a TATA/genética , Factores Asociados con la Proteína de Unión a TATA/metabolismoRESUMEN
BACKGROUND: Ewing sarcoma is a rare tumor that occurs commonly in the long bones of children or adolescents that can also arise in soft tissues including the extremities, retroperitoneum, chest wall, and rarely in the liver as primary sites. We report a case of Ewing sarcoma arising primarily in the liver and, to our knowledge, this is the fourth reported case of Ewing sarcoma occurring in the liver. CASE PRESENTATION: A 27-year-old Japanese woman was admitted with sudden onset right upper abdominal pain. Clinical examination revealed a multilocular cystic mass consisting of thickened, irregular septa and nodal walls in the right hepatic lobe. Ultrasound-guided aspiration biopsy of the liver mass showed clusters of small atypical round cells and the clinical preoperative diagnosis was mucinous cystadenoma of the liver. The patient underwent an extended right hepatectomy and histopathological findings revealed sheet-like proliferation of small- to medium-sized round cells. Tumor cells were positive for periodic acid-Schiff reaction and immunoreactive for glycoprotein C99 and gene NKX2.2, as well as the neuroendocrine markers, CD56 and synaptophysin. EWS-FLI-1 fusion transcript type 1 was detected by reverse transcriptase polymerase chain reaction. Pathological and molecular analysis confirmed the diagnosis of Ewing sarcoma arising primarily in the liver and the patient received adjuvant systemic chemotherapy with vincristine, doxorubicin, and cyclophosphamide, alternating with ifosfamide and etoposide. We found no evidence of recurrence 15 months after completing chemotherapy. CONCLUSION: We present an extremely rare case of Ewing sarcoma arising primarily in the liver. To our knowledge, this is the fourth reported case of Ewing sarcoma occurring in the liver, and the first case with a multilocular cystic liver mass. Imaging examinations of the other three reported cases showed solid tumors and a diffuse enlarged liver without mass lesion. Clinicians should consider the possibility of Ewing sarcoma in young patients with a multilocular cystic mass with thick and/or irregular cyst walls in the liver.
Asunto(s)
Hígado/patología , Sarcoma de Ewing/patología , Adulto , Biomarcadores de Tumor/metabolismo , Femenino , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/biosíntesis , Humanos , Hígado/efectos de los fármacos , Proteínas Nucleares , Proteínas de Fusión Oncogénica/biosíntesis , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteína EWS de Unión a ARN/biosíntesis , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/tratamiento farmacológico , Factores de Transcripción/biosíntesis , Proteínas de Pez CebraRESUMEN
Chromosomal translocation that results in fusion of the genes encoding RNA-binding protein EWS and transcription factor FLI1 (EWS-FLI1) is pathognomonic for Ewing sarcoma. EWS-FLI1 alters gene expression through mechanisms that are not completely understood. We performed RNA sequencing (RNAseq) analysis on primary pediatric human mesenchymal progenitor cells (pMPCs) expressing EWS-FLI1 in order to identify gene targets of this oncoprotein. We determined that long noncoding RNA-277 (Ewing sarcoma-associated transcript 1 [EWSAT1]) is upregulated by EWS-FLI1 in pMPCs. Inhibition of EWSAT1 expression diminished the ability of Ewing sarcoma cell lines to proliferate and form colonies in soft agar, whereas EWSAT1 inhibition had no effect on other cell types tested. Expression of EWS-FLI1 and EWSAT1 repressed gene expression, and a substantial fraction of targets that were repressed by EWS-FLI1 were also repressed by EWSAT1. Analysis of RNAseq data from primary human Ewing sarcoma further supported a role for EWSAT1 in mediating gene repression. We identified heterogeneous nuclear ribonucleoprotein (HNRNPK) as an RNA-binding protein that interacts with EWSAT1 and found a marked overlap in HNRNPK-repressed genes and those repressed by EWS-FLI1 and EWSAT1, suggesting that HNRNPK participates in EWSAT1-mediated gene repression. Together, our data reveal that EWSAT1 is a downstream target of EWS-FLI1 that facilitates the development of Ewing sarcoma via the repression of target genes.
Asunto(s)
Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica , ARN Largo no Codificante/biosíntesis , ARN Neoplásico/biosíntesis , Ribonucleoproteínas/metabolismo , Sarcoma de Ewing/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Regulación hacia Abajo/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K , Humanos , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteína Proto-Oncogénica c-fli-1/genética , ARN Largo no Codificante/genética , ARN Neoplásico/genética , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Ribonucleoproteínas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Análisis de Secuencia de ARN , Regulación hacia Arriba/genéticaRESUMEN
Bladder cancer-associated protein (BLCAP) is a novel candidate tumor suppressor gene identified from human bladder carcinoma and highly associated with the invasion of bladder cancer. We previously reported that it also plays a key role in the tumorigenesis and metastasis of human osteosarcoma. In the present study, we constructed a recombinant encoding BLCAP cDNA. Overexpression of BLCAP resulted in growth inhibition and induced apoptosis of human TC-135 Ewing's sarcoma cells in vitro. We further investigated the caspase-3/7 activity and expressions of the fusion transcription factor Ewing's sarcoma protein-friend leukemia virus integration 1 (EWS-FLI1) and the apoptosis regulator B-cell lymphoma 2 (BCL-2). Cell apoptosis was accompanied by the down-regulated expression of EWS-FLI1 and BCL-2. Our present results suggest that BLCAP may play a role not only in regulating cell proliferation but also in coordinating apoptosis through the down-regulation of BCL-2 and EWS-FLI1 in human Ewing's sarcoma cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Neoplasias/farmacología , Sarcoma de Ewing/metabolismo , Western Blotting , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Fusión Oncogénica/biosíntesis , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteína EWS de Unión a ARN/biosíntesis , Proteínas Recombinantes/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option.
Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sarcoma de Ewing/tratamiento farmacológico , Animales , Apoptosis/efectos de los fármacos , Aurora Quinasas , Neoplasias Óseas/enzimología , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Ciclo Celular/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Sinergismo Farmacológico , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones SCID , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , Piperazinas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteína Proto-Oncogénica c-fli-1/genética , Interferencia de ARN , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Distribución Aleatoria , Sarcoma de Ewing/enzimología , Sarcoma de Ewing/genética , Sarcoma de Ewing/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ewing family tumors are characterized by a translocation between the RNA binding protein EWS and one of five ETS transcription factors, most commonly FLI1. The fusion protein produced by the translocation has been thought to act as an aberrant transcription factor, leading to changes in gene expression and cellular transformation. In this study, we investigated the specific processes EWS/FLI1 utilizes to alter gene expression. Using both heterologous NIH 3T3 and human Ewing Family Tumor cell lines, we have demonstrated by quantitative pre-mRNA analysis that EWS/FLI1 repressed the expression of previously validated direct target genes at the level of transcript synthesis. ChIP experiments showed that EWS/FLI1 decreases the amount of Pol II at the promoter of down-regulated genes in both murine and human model systems. However, in down-regulated target genes, there was a significant disparity between the modulation of cognate mRNA and pre-mRNAs, suggesting that these genes could also be regulated at a posttranscriptional level. Confirming this, we found that EWS/FLI1 decreased the transcript half-life of insulin-like growth factor binding protein 3, a down-regulated direct target gene in human tumor-derived Ewing's sarcoma cell lines. Additionally, we have shown through reexpression experiments that full EWS/FLI1-mediated transcriptional repression requires intact EWS and ETS domains. Together these data demonstrate that EWS/FLI1 can dictate steady-state target gene expression by modulating both transcript synthesis and degradation.
Asunto(s)
Regiones no Traducidas 3' , Regulación Neoplásica de la Expresión Génica , Proteínas de Fusión Oncogénica/biosíntesis , Regiones Promotoras Genéticas , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Precursores del ARN/biosíntesis , Estabilidad del ARN , Proteína EWS de Unión a ARN/biosíntesis , Animales , Línea Celular Tumoral , Regulación hacia Abajo/genética , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Ratones , Células 3T3 NIH , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Precursores del ARN/genética , Proteína EWS de Unión a ARN/genética , Transcripción GenéticaRESUMEN
POU5F1 is a transcription factor essential for the self-renewal activity and pluripotency of embryonic stem cells and germ cells. We have previously reported that POU5F1 is fused to EWSR1 in a case of undifferentiated sarcoma with chromosomal translocation t(6;22)(p21;q12). In addition, the EWS-POU5F1 chimeras have been recently identified in human neoplasms of the skin and salivary glands. To clarify the roles of the EWS-POU5F1 chimera in tumorigenesis and tumor cell maintenance, we used small-interfering RNA-mediated gene silencing. Knockdown of EWS-POU5F1 in the t(6;22) sarcoma-derived GBS6 cell line resulted in a significant decrease of cell proliferation because of G1 cell cycle arrest associated with p27(Kip1) up-regulation. Moreover, senescence-like morphological changes accompanied by actin polymerization were observed. In contrast, EWS-POU5F1 down-regulation markedly increased the cell migration and invasion as well as activation of metalloproteinase 2 and metalloproteinase 14. The results indicate that the proliferative activity of cancer cells and cell motility are discrete processes in multistep carcinogenesis. These findings reveal the functional role of the sarcoma-related chimeric protein as well as POU5F1 in the development and progression of human neoplasms.
Asunto(s)
Factor 3 de Transcripción de Unión a Octámeros/biosíntesis , Factor 3 de Transcripción de Unión a Octámeros/genética , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Sarcoma/patología , Línea Celular Tumoral , Movimiento Celular , Senescencia Celular , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Citometría de Flujo/métodos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Invasividad Neoplásica , Análisis de Secuencia por Matrices de Oligonucleótidos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Sarcoma/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
This paper presents the results of an analysis the chimeric genes FUS/CHOP and EWS/CHOP in patients diagnosed as having liposarcoma in order to make a differential diagnosis in both soft tissue tumors and various variants of liposarcoma. Liposarcomas were found in 5 of 7 cases of primary tumors: 4 chimeric transcripts of the FUS/CHOP type (5-2), a variant of alternative splicing of the FUS/CHOP type (5-2) with depletion in 14 p.n. anda rare variant of the EWS/CHOP type (7-2). Fluorescence in situ hybridization (FISH) confirmed translocations in the tumor samples with the chimeric genes being detected. Reverse transcription-polymerase chain reaction and FISH revealed no chimeric genes specific to myxoid sarcoma in a group of patients with other variants of liposarcoma. Thus, the findings support the strict specificity of the chimeric genes FUS/CHOP and EWS/CHOP for myxoid liposarcoma and the expression of these genes in most tumors of this type.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Liposarcoma/metabolismo , Liposarcoma/patología , Proteínas de Fusión Oncogénica/biosíntesis , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Proteína EWS de Unión a ARN/biosíntesis , Proteína FUS de Unión a ARN/biosíntesis , Factor de Transcripción CHOP/biosíntesis , Adulto , Anciano , Empalme Alternativo , Diagnóstico Diferencial , Femenino , Humanos , Liposarcoma/diagnóstico , Masculino , Persona de Mediana EdadRESUMEN
There may be a number of tumors made up by small round blue cells in the kidneys of children. One of them is primitive neuroectodermal tumor (PNET). The differences in therapeutic approaches determine the need to establish an accurate diagnosis. The differential diagnosis of PNET and the blastemal component of Wilms tumor can be difficult due to the similar histological pattern. There is a need for a close analysis of morphological manifestations, by keeping in mind the age of patients, and supplementary studies. A strong CD99 membrane expression and nuclear FLI1 expression in tumor cells are the signs of PNET. Reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization can determine PNET-specific translocations [t(11;22)(q24;q12), by involving the EWS gene.
Asunto(s)
Neoplasias Renales/diagnóstico , Tumores Neuroectodérmicos Primitivos/diagnóstico , Tumor de Wilms/diagnóstico , Antígeno 12E7 , Antígenos CD/biosíntesis , Antígenos CD/genética , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Niño , Preescolar , Cromosomas Humanos Par 11/genética , Cromosomas Humanos Par 11/metabolismo , Cromosomas Humanos Par 22/genética , Cromosomas Humanos Par 22/metabolismo , Diagnóstico Diferencial , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Masculino , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patología , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Translocación Genética/genética , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Tumor de Wilms/patologíaRESUMEN
The most frequently occurring chromosomal translocation that gives rise to the Ewing's sarcoma family of tumors (ESFT) is the chimeric fusion gene EWS-FLI1 that encodes an oncogenic protein composed of the N terminus of EWS and the C terminus of FLI1. Although the genetic basis of ESFT is fairly well understood, its putative cellular origin remains to be determined. Previous work has proposed that neural crest progenitor cells may be the causative cell type responsible for ESFT. However, surprisingly little is known about the expression pattern or role of either wild-type EWS or wild-type FLI1 in this cell population during early embryonic development. Using the developing chick embryo as a model system, we identified EWS expression in emigrating and migratory neural crest stem cells, whereas FLI1 transcripts were found to be absent in these populations and were restricted to developing endothelial cells. By ectopically expressing EWS-FLI1 or wild-type FLI1 in the developing embryo, we have been able to study the cellular transformations that ensue in the context of an in vivo model system. Our results reveal that misexpression of the chimeric EWS-FLI1 fusion gene, or wild-type FLI1, in the developing neural crest stem cell population leads to significant aberrations in neural crest development. An intriguing possibility is that misexpression of the EWS-FLI1 oncogene in neural crest-derived stem cells may be an initiating event in ESFT genesis.
Asunto(s)
Células Madre Embrionarias/fisiología , Células Endoteliales/citología , Cresta Neural/fisiología , Proteínas de Fusión Oncogénica/fisiología , Factores de Transcripción/fisiología , Animales , Neoplasias Óseas/embriología , Neoplasias Óseas/metabolismo , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Embrión de Pollo , Células Madre Embrionarias/citología , Células Endoteliales/fisiología , Cresta Neural/citología , Cresta Neural/embriología , Proteínas de Fusión Oncogénica/biosíntesis , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/embriología , Sarcoma de Ewing/metabolismo , Factores de Transcripción/biosíntesisRESUMEN
Although much is known about the oncogenic functions of chimeric Ewing sarcoma (EWS) fusion proteins that result from chromosomal translocations, the cellular role of the normal EWS protein is not well characterized. We have previously identified a WD domain-containing protein, serine-threonine kinase receptor-associated protein (STRAP), which inhibits transforming growth factor beta (TGF-beta) signaling through interaction with receptors and Smad7 and promotes growth and enhances tumorigenicity. Here, we report the interaction between STRAP and EWS using matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry. Although STRAP is localized in both cytoplasm and nucleus, nuclear STRAP colocalizes and associates specifically with EWS in the nucleus through its NH(2) and COOH termini. We have found that normal EWS protein is up-regulated in human cancers, which correlates with the up-regulation of STRAP in 71% of colorectal cancers and 54% of lung cancers, suggesting a cooperative role of these two proteins in human cancers. TGF-beta has no effect on STRAP and EWS interaction. However, EWS, like STRAP, attenuates TGF-beta-dependent transcription. STRAP inhibits EWS-dependent p300-mediated transactivation of EWS target genes, such as ApoCIII and c-fos, in a TGF-beta-independent manner. Interestingly, we have shown that STRAP blocks the interaction between EWS and p300, whereas the complex formation between STRAP and EWS is not affected by p300. These results suggest that STRAP inhibits the transactivation function of EWS by displacing p300 from the functional transcriptional complex. Thus, this study provides a novel TGF-beta-independent function of STRAP and describes a mechanism by which STRAP regulates the function of oncogenic EWS protein.
Asunto(s)
Proteínas de Neoplasias/metabolismo , Proteína EWS de Unión a ARN/metabolismo , Animales , Línea Celular , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Proteína p300 Asociada a E1A/metabolismo , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Células 3T3 NIH , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Unión Proteica , Proteómica , Proteína EWS de Unión a ARN/antagonistas & inhibidores , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Proteínas de Unión al ARN , Fracciones Subcelulares/metabolismo , TransfecciónRESUMEN
Ewing sarcoma is the second most common malignant pediatric bone tumor. Over 80% of Ewing sarcoma contain the oncogene EWS/FLI-1, which encodes the EWS/FLI-1 oncoprotein, a hybrid transcription factor comprised of NH2-terminal sequences from the RNA-binding protein EWS and the DNA-binding and COOH-terminal regions of the Ets transcription factor FLI-1. Although numerous genes are dysregulated by EWS/FLI-1, advances in Ewing sarcoma cancer biology have been hindered by the lack of an animal model because of EWS/FLI-1-mediated cytotoxicity. In this study, we have developed conditions for the isolation and propagation of murine primary bone-derived cells (mPBDC) that stably express EWS/FLI-1. Early-passage EWS/FLI-1 mPBDCs were immortalized in culture but inefficient at tumor induction, whereas later-passage cells formed sarcomatous tumors in immunocompetent syngeneic mice. Murine EWS/FLI-1 tumors contained morphologically primitive cells that lacked definitive lineage markers. Molecular characterization of murine EWS/FLI-1 tumors revealed that some but not all had acquired a novel, clonal in-frame p53 mutation associated with a constitutive loss of p21 expression. Despite indications that secondary events facilitated EWS/FLI-1 mPBDC tumorigenesis, cells remained highly dependent on EWS/FLI-1 for efficient transformation in clonogenic assays. This Ewing sarcoma animal model will be a useful tool for dissecting the molecular pathogenesis of Ewing sarcoma and provides rationale for the broader use of organ-specific progenitor cell populations for the study of human sarcoma.
Asunto(s)
Neoplasias Óseas/metabolismo , Proteínas de Fusión Oncogénica/biosíntesis , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteína EWS de Unión a ARN/biosíntesis , Sarcoma de Ewing/metabolismo , Secuencia de Aminoácidos , Animales , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Ciclo Celular/fisiología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Modelos Animales de Enfermedad , Expresión Génica , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Proteínas de Fusión Oncogénica/genética , Proteína Proto-Oncogénica c-fli-1/genética , Proteína EWS de Unión a ARN/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologíaRESUMEN
The EWS/ETS fusion proteins associated with Ewings family tumors (EFTs) are thought to promote oncogenesis by acting as aberrant transcription factors. Uridine phosphorylase is a gene that is up-regulated by structurally distinct EWS/ETS fusions. Ectopic expression of uridine phosphorylase was able to support anchorage-independent cell growth, indicating that it plays an active role in the oncogenic process. Transcriptional up-regulation of uridine phosphorylase is shown to be mediated in a DNA binding-dependent manner, and reporter gene assays demonstrated that EWS/FLI1 and RAS mediate activation through a single activator protein 1/ETS site located in the uridine phosphorylase promoter. Chromatin immunoprecipitation assays reveal that EWS/FLI1 directly associates with the uridine phosphorylase promoter in vivo. Up-regulation of uridine phosphorylase by EWS/FLI1 sensitizes cells to growth inhibition by the pyrimidine analogue, 5'-deoxy-5'fluorouridine, both in tissue culture and in vivo model systems.
Asunto(s)
Proteínas Proto-Oncogénicas/fisiología , Proteína EWS de Unión a ARN/fisiología , Proteínas Recombinantes de Fusión/fisiología , Factores de Transcripción/fisiología , Uridina Fosforilasa/fisiología , Células 3T3 , Animales , ADN/metabolismo , Floxuridina/farmacología , Humanos , Ratones , Proteínas de Fusión Oncogénica/biosíntesis , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/fisiología , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Proteína Proto-Oncogénica c-fli-1 , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-ets , Proteína EWS de Unión a ARN/biosíntesis , Proteína EWS de Unión a ARN/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Transcripción Genética , Transfección , Regulación hacia Arriba , Uridina Fosforilasa/biosíntesis , Uridina Fosforilasa/genética , Proteínas ras/fisiologíaRESUMEN
Nuclear receptors represent a large family of transcription factors involved in development, differentiation, homeostasis, and cancer. In recent years, a growing number of cofactors has been discovered that participate in the regulation of the transcriptional activity of these proteins. We present in this study the identification of a cofactor, the homeotic protein Six3, which differentially regulates the transcriptional activity of the orphan nuclear receptor NOR-1 (NR4A3). NOR-1 is normally involved in the balance between cell proliferation and cell death, and is implicated in oncogenesis as part of the EWS/NOR-1 fusion protein found in human extraskeletal myxoid chondrosarcoma (EMC) tumors. Reverse transcription-PCR analyses indicate that EMC tumors expressing the EWS/NOR-1 mRNA also express mRNAs encoding NOR-1 and Six3. Glutathione S-transferase fusion protein assays show that Six3 binds in vitro the DNA-binding domain of NOR-1 and the EWS domain of EWS/NOR-1 and that the homeodomain of Six3 is required for these interactions. Mammalian two-hybrid experiments, using immortalized human chondrocytes as a model, indicate that Six3 also interacts with NOR-1 and EWS/NOR-1 in vivo. Cotransfection experiments show that Six3 stimulates the transcriptional activity of NOR-1, whereas it represses that of EWS/NOR-1. Considering the highly specific expression pattern of Six3, our finding that it is expressed in EMC suggests that it plays a pivotal role in the development of these tumors. We propose that Six3 maintains a transcriptional balance between the activities of NOR-1 and EWS/NOR-1, the net effect being to deregulate the expression of specific target genes and push the equilibrium toward uncontrolled cell proliferation.