RESUMO
Binding of transcription factors to mutated DNA sequences is a likely regulator of cancer progression. Noncoding regulatory mutations such as those on the core promoter of the gene encoding human telomerase reverse transcriptase have been shown to affect gene expression in cancer. Using a protein microarray of 667 transcription factor DNA-binding domains and subsequent functional assays, we looked for transcription factors that preferentially bind the mutant hTERT promoter and characterized their downstream effects. One of them, friend leukemia integration 1 (FLI1), which belongs to the E26 transforming-specific family of transcription factors, exhibited particularly strong effects with respect to regulating hTERT expression, while the even better binding ELK3 did not. Depletion of FLI1 decreased expression of the genes for cyclin D1 (CCND1) and E2F transcription factor 2 (E2F2) resulting in a G1/S cell cycle arrest and in consequence a reduction of cell proliferation. FLI1 also affected CMTM7, another gene involved in G1/S transition, although by another process that suggests a balanced regulation of the tumor suppressor gene's activity via opposing regulation processes. FLI1 expression was found upregulated and correlated with an increase in CCND1 expression in pancreatic cancer and brain tumors. In non-neoplastic lung cells, however, FLI1 depletion led to rapid progression through the cell cycle. This coincides with the fact that FLI1 is downregulated in lung tumors. Taken together, our data indicate a cell cycle regulatory hub involving FLI1, hTERT, CCND1 and E2F2 in a tissue- and context-dependent manner.
Assuntos
Neoplasias/genética , Neoplasias/metabolismo , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ciclina D1/biossíntese , Ciclina D1/genética , Ciclina D1/metabolismo , Progressão da Doença , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Neoplasias/patologia , Regiões Promotoras Genéticas , Análise Serial de Proteínas , Proteína Proto-Oncogênica c-fli-1/biossíntese , Telomerase/genética , Telomerase/metabolismoRESUMO
Ewing sarcoma is characterized by the expression of the chimeric EWSR1-FLI1 transcription factor. Proteomic analyses indicate that the decrease of EWSR1-FLI1 expression leads to major changes in effectors of the dynamics of the actin cytoskeleton and the adhesion processes with a shift from cell-to-cell to cell-matrix adhesion. These changes are associated with a dramatic increase of in vivo cell migration and invasion potential. Importantly, EWSR1-FLI1 expression, evaluated by single-cell RT-ddPCR/immunofluorescence analyses, and activity, assessed by expression of EWSR1-FLI1 downstream targets, are heterogeneous in cell lines and in tumours and can fluctuate along time in a fully reversible process between EWSR1-FLI1high states, characterized by highly active cell proliferation, and EWSR1-FLI1low states where cells have a strong propensity to migrate, invade and metastasize. This new model of phenotypic plasticity proposes that the dynamic fluctuation of the expression level of a dominant oncogene is an intrinsic characteristic of its oncogenic potential.
Assuntos
Proteínas de Ligação a Calmodulina/biossíntese , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteínas de Ligação a RNA/biossíntese , Sarcoma de Ewing/metabolismo , Animais , Proteínas de Ligação a Calmodulina/genética , Linhagem Celular Tumoral , Camundongos , Camundongos SCID , Invasividade Neoplásica , Metástase Neoplásica , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Peixe-ZebraRESUMO
M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.
Assuntos
Granulócitos/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Células-Tronco Multipotentes/efeitos dos fármacos , Mielopoese/efeitos dos fármacos , Animais , Butadienos/farmacologia , Linhagem Celular , Ensaio de Unidades Formadoras de Colônias , Ativação Enzimática/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/deficiência , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Mielopoese/fisiologia , Nitrilas/farmacologia , Proteína Quinase C-delta/genética , Proteína Quinase C-delta/fisiologia , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
Hematopoietic lineage commitment is regulated by cytokines and master transcription factors, but it remains unclear how a progenitor cell chooses a lineage in the face of conflicting cues. Through transcript counting in megakaryocyte-erythroid progenitors undergoing erythropoiesis, we show that the expression levels of the pro-erythropoiesis transcription factor EKLF (also known as KLF1) and receptor EpoR are inversely correlated with their pro-megakaryopoiesis counterparts, FLI-1 and TpoR (also known as MPL). Notably, as progenitors commit to the erythrocyte lineage, EpoR is upregulated and TpoR is strongly downregulated, thus boosting the potency of the pro-erythropoiesis cue erythropoietin and effectively eliminating the activity of the pro-megakaryopoiesis cue thrombopoietin. Based on these findings, we propose a new model for exclusive decision making that explicitly incorporates signals from extrinsic cues, and we experimentally confirm a model prediction of temporal changes in transcript noise levels in committing progenitors. Our study suggests that lineage-specific receptor levels can modulate potencies of cues to achieve robust commitment decisions.
Assuntos
Linhagem da Célula/genética , Eritropoese/genética , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição Kruppel-Like/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Receptores da Eritropoetina/biossíntese , Receptores de Trombopoetina/biossíntese , Diferenciação Celular/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Fatores de Transcrição Kruppel-Like/genética , Megacariócitos/citologia , Proteína Proto-Oncogênica c-fli-1/genética , Receptores de Trombopoetina/genética , Trombopoetina/genética , Trombopoetina/metabolismoRESUMO
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.
Assuntos
Fígado/patologia , Sarcoma de Ewing/patologia , Adulto , Biomarcadores Tumorais/metabolismo , Feminino , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/biossíntese , Humanos , Fígado/efeitos dos fármacos , Proteínas Nucleares , Proteínas de Fusão Oncogênica/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína EWS de Ligação a RNA/biossíntese , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/tratamento farmacológico , Fatores de Transcrição/biossíntese , Proteínas de Peixe-ZebraRESUMO
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.
Assuntos
Transformação Celular Neoplásica/metabolismo , Regulação Neoplásica da Expressão Gênica , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Ribonucleoproteínas/metabolismo , Sarcoma de Ewing/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Regulação para Baixo/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K , Humanos , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , RNA Longo não Codificante/genética , RNA Neoplásico/genética , Proteína EWS de Ligação a RNA/biossíntese , Proteína EWS de Ligação a RNA/genética , Ribonucleoproteínas/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Análise de Sequência de RNA , Regulação para Cima/genéticaRESUMO
The friend leukemia insertion site 1 (Fli-1) transcription factor, an Ets family member, is implicated in the pathogenesis of systemic lupus erythematosus in human patients and murine models of lupus. Lupus-prone mice with reduced Fli-1 expression have significantly less nephritis, prolonged survival, and decreased infiltrating inflammatory cells into the kidney. Inflammatory chemokines, including CCL5, are critical for attracting inflammatory cells. In this study, decreased CCL5 mRNA expression was observed in kidneys of lupus-prone NZM2410 mice with reduced Fli-1 expression. CCL5 protein expression was significantly decreased in endothelial cells transfected with Fli-1-specific small interfering RNA compared with controls. Fli-1 binds to endogenous Ets binding sites in the distal region of the CCL5 promoter. Transient transfection assays demonstrate that Fli-1 drives transcription from the CCL5 promoter in a dose-dependent manner. Both Ets1, another Ets family member, and Fli-1 drive transcription from the CCL5 promoter, although Fli-1 transactivation was significantly stronger. Ets1 acts as a dominant-negative transcription factor for Fli-1, indicating that they may have at least one DNA binding site in common. Systematic deletion of DNA binding sites demonstrates the importance of the sites located within a 225-bp region of the promoter. Mutation of the Fli-1 DNA binding domain significantly reduces transactivation of the CCL5 promoter by Fli-1. We identified a novel regulator of transcription for CCL5. These results suggest that Fli-1 is a novel and critical regulator of proinflammatory chemokines and affects the pathogenesis of disease through the regulation of factors that recruit inflammatory cells to sites of inflammation.
Assuntos
Quimiocina CCL5/genética , DNA/química , Células Endoteliais/imunologia , Proteína Proto-Oncogênica c-fli-1/genética , Ativação Transcricional/genética , Células 3T3 , Animais , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Linhagem Celular , Quimiocina CCL5/biossíntese , DNA/imunologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Células Endoteliais/citologia , Inflamação/genética , Inflamação/imunologia , Rim/citologia , Rim/imunologia , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Camundongos , Camundongos Transgênicos , Nefrite/genética , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/imunologia , Interferência de RNA , RNA Mensageiro/biossíntese , RNA Interferente Pequeno , TransfecçãoRESUMO
Distinction between radial growth phase (RGP) and vertical growth phase (VGP) in cutaneous melanomas is prognostically significant. Despite established morphological criteria, molecular markers to separate RGP and VGP have not been well established. The goal of this study was to investigate associations of p16, WT1, and Fli-1 with RGP-to-VGP progression, by immunohistochemistry. The p16 is a tumor suppressor, whereas WT1 and Fli-1 are transcriptional activators. The authors hypothesized that entry into VGP would be associated with decreased p16 and increased WT1 and Fli-1. Paraffin sections from 18 RGP and 15 VGP melanomas were immunostained with well-characterized antibodies to p16, WT1, and Fli-1. Melanoma growth phases were determined using precodified morphological attributes. In RGP melanomas, p16 was expressed in 15 of 18 (83%), WT1 in 17 of 17 (100%), and Fli-1 at least focally in 6 of 18 (33%). The deep dermal component of VGP melanomas stained positively for Fli-1 in 9 of 14 (64%), strongly for WT1 in 10 of 14 (71%), and strongly for p16 in only 2 of 15 (13%). Observed patterns of WT1 immunopositivity did not support the authors' hypothesis; it is not likely to be a good indicator of VGP. On the other hand, Fli-1 staining trended toward more positive deep tumor compartment staining and p16 to weaker staining in the deep compartment. At present, application of histological criteria remains the best method for assignment of growth phase in melanomas; however, p16 and possibly Fli-1 immunostains may serve as useful adjuncts in morphologically indeterminate cases.
Assuntos
Melanoma/patologia , Proteínas de Neoplasias/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Neoplasias Cutâneas/patologia , Proteínas WT1/biossíntese , Adulto , Biomarcadores Tumorais/análise , Inibidor p16 de Quinase Dependente de Ciclina , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/análise , Proteína Proto-Oncogênica c-fli-1/análise , Proteínas WT1/análise , Adulto JovemRESUMO
Diffuse large B-cell lymphoma (DLBCL) is the most common form of human lymphoma. DLBCL is a heterogeneous disease characterized by different genetic lesions. We herein report the functional characterization of a recurrent gain mapping on chromosome 11q24.3, found in 23% of 166 DLBCL cases analyzed. The transcription factors ETS1 and FLI1, located within the 11q24.3 region, had significantly higher expression in clinical samples carrying the gain. Functional studies on cell lines showed that ETS1 and FLI1 cooperate in sustaining DLBCL proliferation and viability and regulate genes involved in germinal center differentiation. Taken together, these data identify the 11q24.3 gain as a recurrent lesion in DLBCL leading to ETS1 and FLI1 deregulated expression, which can contribute to the pathogenesis of this disease.
Assuntos
Cromossomos Humanos Par 11/genética , Linfoma Difuso de Grandes Células B/genética , Proteína Proto-Oncogênica c-ets-1/genética , Proteína Proto-Oncogênica c-fli-1/genética , Western Blotting , Imunoprecipitação da Cromatina , Eletroporação , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Proto-Oncogênica c-ets-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , TransfecçãoRESUMO
Friend leukemia virus integration 1 (Fli1) and erythroid Krüppel-like factor (EKLF) participate under experimental conditions in the differentiation of megakaryocytic and erythroid progenitor in cooperation with other transcription factors, cytokines, cytokine receptors, and microRNAs. Defective erythropoiesis with refractory anemia and effective megakaryopoiesis with normal or increased platelet count is typical for 5q- syndrome. We decided to evaluate the roles of EKLF and Fli1 in the pathogenesis of this syndrome and of another ribosomopathy, Diamond-Blackfan anemia (DBA). Fli1 and EKLF mRNA levels were examined in mononuclear blood and bone marrow cells from patients with 5q- syndrome, low-risk MDS patients with normal chromosome 5, DBA patients, and healthy controls. In 5q- syndrome, high Fli1 mRNA levels in the blood and bone marrow mononuclear cells were found. In DBA, Fli1 expression did not differ from the controls. EKLF mRNA level was significantly decreased in the blood and bone marrow of 5q- syndrome and in all DBA patients. We propose that the elevated Fli1 in 5q- syndrome protects megakaryocytic cells from ribosomal stress contrary to erythroid cells and contributes to effective though dysplastic megakaryopoiesis.
Assuntos
Anemia de Diamond-Blackfan/genética , Anemia Macrocítica/genética , Eritropoese/genética , Fatores de Transcrição Kruppel-Like/fisiologia , Proteína Proto-Oncogênica c-fli-1/fisiologia , Trombopoese/genética , Adolescente , Adulto , Anemia de Diamond-Blackfan/metabolismo , Anemia Macrocítica/metabolismo , Células da Medula Óssea/metabolismo , Criança , Deleção Cromossômica , Cromossomos Humanos Par 5/genética , Cromossomos Humanos Par 5/metabolismo , Ilhas de CpG , Feminino , Humanos , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Leucócitos Mononucleares/metabolismo , Masculino , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/sangue , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/fisiologia , Transcrição Gênica , Adulto JovemRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Neoplasias/farmacologia , Sarcoma de Ewing/metabolismo , Western Blotting , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Fusão Oncogênica/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína EWS de Ligação a RNA/biossíntese , Proteínas Recombinantes/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Neoplasias Ósseas/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sarcoma de Ewing/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Aurora Quinases , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Ciclo Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos SCID , Proteínas de Fusão Oncogênica/biossíntese , Proteínas de Fusão Oncogênica/genética , Piperazinas/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , Interferência de RNA , Proteína EWS de Ligação a RNA/biossíntese , Proteína EWS de Ligação a RNA/genética , Distribuição Aleatória , Sarcoma de Ewing/enzimologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Regiões 3' não Traduzidas , Regulação Neoplásica da Expressão Gênica , Proteínas de Fusão Oncogênica/biossíntese , Regiões Promotoras Genéticas , Proteína Proto-Oncogênica c-fli-1/biossíntese , Precursores de RNA/biossíntese , Estabilidade de RNA , Proteína EWS de Ligação a RNA/biossíntese , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Humanos , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Camundongos , Células NIH 3T3 , Proteínas de Fusão Oncogênica/genética , Proteína Proto-Oncogênica c-fli-1/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Precursores de RNA/genética , Proteína EWS de Ligação a RNA/genética , Transcrição GênicaRESUMO
Elevated homocysteine (Hcys) serum levels represent a risk factor for several chronic pathologies, including cardiovascular disease, atherosclerosis, and chronic renal failure, and affect bone development, quality, and homeostasis. Hcys influences the formation of a stable bone matrix directly through the inhibition of the collagen cross-linking enzyme lysyl oxidase (Lox) and, as we have shown recently, by repressing its mRNA expression. The aim of this study was to investigate the mechanisms involved in this process. Through evaluation of gene arrays, quantitative RT-PCR, immunoblots, and ELISA, we identified a Hcys-dependent stimulation of interleukin 6 (IL-6) and genes involved in IL-6/Janus kinase 2 (JAK2)-dependent signal transduction pathways in pre-osteoblastic MC3T3-E1 cells. Moreover, up-regulation of genes essential for epigenetic DNA methylation (DNA (cytosine-5)-methyltransferases and helicase lymphoid-specific (Hells) was observed. Further investigations demonstrated that Hcys increased via IL-6/JAK2 the expression of Fli1 (Friend leukemia virus integration 1), a transcription factor, which we found essential for IL-6-dependent Dnmt1 stimulation. CpG methylation analysis of CpG-rich Lox proximal promoter revealed an increased CpG methylation status after treatment of the cells with Hcys indicating an epigenetic origin for Hcys-dependent Lox repression. Inhibition of the IL-6/JAK2 pathway or of CpG methylation reversed the repressive effect of Hcys on Lox expression. In conclusion, we demonstrate that Hcys stimulates IL-6 synthesis in osteoblasts, which is known to affect bone metabolism via osteoclasts. Furthermore, IL-6 stimulation results via JAK2, Fli1, and Dnmt1 in down-regulation of Lox expression by epigenetic CpG methylation revealing a new mechanism negatively affecting bone matrix formation.
Assuntos
Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Proteínas da Matriz Extracelular/biossíntese , Homocisteína/farmacologia , Interleucina-6/biossíntese , Proteína-Lisina 6-Oxidase/biossíntese , Proteína Proto-Oncogênica c-fli-1/biossíntese , Animais , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Metilação de DNA/genética , Epigênese Genética/genética , Proteínas da Matriz Extracelular/genética , Homocisteína/metabolismo , Interleucina-6/genética , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Regiões Promotoras Genéticas/genética , Proteína-Lisina 6-Oxidase/genética , Proteína Proto-Oncogênica c-fli-1/genéticaRESUMO
We previously characterized nucleoredoxin (NRX) as a negative regulator of the Wnt signaling pathway through Dishevelled (Dvl). We perform a comprehensive search for other NRX-interacting proteins and identify Flightless-I (Fli-I) as a novel NRX-binding partner. Fli-I binds to NRX and other related proteins, such as Rod-derived cone viability factor (RdCVF), whereas Dvl binds only to NRX. Endogenous NRX and Fli-I in vivo interactions are confirmed. Both NRX and RdCVF link Fli-I with myeloid differentiation primary response gene (88) (MyD88), an important adaptor protein for innate immune response. NRX and RdCVF also potentiate the negative effect of Fli-I upon lipopolysaccharide-induced activation of NF-kappaB through the Toll-like receptor 4/MyD88 pathway. Embryonic fibroblasts derived from NRX gene-targeted mice show aberrant NF-kappaB activation upon lipopolysaccharide stimulation. These results suggest that the NRX subfamily of proteins forms a link between MyD88 and Fli-I to mediate negative regulation of the Toll-like receptor 4/MyD88 pathway.
Assuntos
Regulação da Expressão Gênica , Proteínas Nucleares/fisiologia , Oxirredutases/fisiologia , Proteína Proto-Oncogênica c-fli-1/biossíntese , Receptor 4 Toll-Like/metabolismo , Animais , Células COS , Chlorocebus aethiops , Fibroblastos/metabolismo , Humanos , Imunidade Inata , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Transgênicos , NF-kappa B/metabolismo , Células NIH 3T3 , Proteínas Nucleares/química , Oxirredutases/química , Ligação Proteica , Transdução de SinaisRESUMO
The transcription factor Fli-1 is implicated in the pathogenesis of both murine and human lupus. Decreased expression of Fli-1 in heterozygous (Fli-1(+/-)) Murphy Roths Large (MRL)/lpr mice resulted in significantly lower kidney pathological scores and markedly increased survival. In this study, bone marrow (BM) transplantation was used to investigate the role of decreased expression of Fli-1 in haematopoietic versus non-haematopoietic cell lineages in autoimmune disease development. Wild-type (WT) MRL/lpr that received BM from Fli-1(+/-) MRL/lpr mice had statistically significantly lower autoantibodies, less proteinuria, reduced renal disease and prolonged survival compared to WT MRL/lpr mice that received BM from WT MRL/lpr mice. Although not statistically significant, Fli-1(+/-) MRL/lpr mice that received BM from WT MRL/lpr mice also had lower autoantibodies and improved survival compared to WT MRL/lpr mice that received BM from WT MRL/lpr mice. Our data indicate that expression of Fli-1 in haematopoietic cell lineages has a significant effect on disease development in MRL/lpr mice.
Assuntos
Transplante de Medula Óssea , Lúpus Eritematoso Sistêmico/terapia , Proteína Proto-Oncogênica c-fli-1/fisiologia , Animais , Autoanticorpos/biossíntese , Autoanticorpos/sangue , Linhagem da Célula , Progressão da Doença , Feminino , Células-Tronco Hematopoéticas/metabolismo , Heterozigoto , Rim/patologia , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Nefrite Lúpica/genética , Nefrite Lúpica/imunologia , Nefrite Lúpica/patologia , Nefrite Lúpica/terapia , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/deficiência , Proteína Proto-Oncogênica c-fli-1/genética , Quimera por Radiação , Organismos Livres de Patógenos EspecíficosRESUMO
BACKGROUND: Despite improvements in the treatment of patients with Ewing family tumours (EFT) during the past decades, the prognosis for patients with advanced disease is still unsatisfying. New treatment strategies have to be developed. MATERIALS AND METHODS: A hypoxanthine/aminopterin/thymidine (HAT)-sensitive EFT cell line was developed by repetitive treatment of the EFT cell line SK-N-MC with 8'-azaguanine (8AG). By using DNA microarrays, the gene expression profile of this cell line was characterized. Immunostimulatory activity was assessed by mixed lymphocyte/tumour cell culture (MLTC). Artificial fusion of tumour cells and dendritic cells was visualized by flow cytometry. RESULTS: After selection of 8AG-resistant cells, a cell line with high sensitivity for treatment with HAT was obtained. Expression of the X chromosome inactivation specific transcript XIST was higher in HAT-sensitive cells. Nevertheless, HAT-sensitive cells retained the EFT-associated gene expression profile. Moreover, in the presence of HAT, it was possible to use these cells without irradiation as stimulatory cells in MLTC or as fusion partner for dendritic cells. CONCLUSION: HAT-sensitive EFT cells might be an interesting tool for the development of new immunotherapeutic approaches for the treatment of EFT.
Assuntos
Aminopterina/farmacologia , Hipoxantina/farmacologia , Imunoterapia Ativa/métodos , Sarcoma de Ewing/imunologia , Timidina/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/imunologia , Azaguanina/farmacologia , Proteínas de Ligação a Calmodulina/biossíntese , Proteínas de Ligação a Calmodulina/imunologia , Vacinas Anticâncer/imunologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Antígeno HLA-A2/imunologia , Antígeno HLA-A2/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/imunologia , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/imunologia , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/biossíntese , Proteínas de Ligação a RNA/imunologia , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologiaRESUMO
Ewing's sarcoma (ES)/peripheral neuroectodermal tumor (PNET) are malignant neoplasms affecting children and young adults. We performed a study to typify the histological diversity and evaluate antibodies that may offer diagnostic/prognostic support. In total, 415 cases of genetically confirmed paraffin-embedded ES/PNET were analyzed on whole sections and in tissue microarrays. This study confirms the structural heterogeneity of ES/PNET, distinguishing three major subtypes: conventional ES (280 cases); PNET (53 cases); and atypical ES/PNET (80), including large cells, vascular-like patterns, spindle pattern, and adamantinoma-like configuration. All cases presented positivity for at least three of the four tested antibodies (CD99, FLI1, HNK1, and CAV1). CAV1 appeared as a diagnostic immunomarker of ES/PNET being positive in CD99-negative cases. Hence, the immunohistochemical analysis confirmed the diagnostic value of all four antibodies, which together cover more than 99% of the tumors, independently of the histological variety. The univariate analysis for survival revealed atypical ES as the only histological parameter apparently associated with less favorable clinical outcome, particularly in the subgroup of patients treated with surgery. In conclusion, the diagnosis of atypical ES is a challenge for the pathologist and needs support from molecular techniques to perform an optimal differential diagnosis with other small round cell tumors.
Assuntos
Anticorpos , Biomarcadores Tumorais/análise , Neoplasias Ósseas/patologia , Tumores Neuroectodérmicos Primitivos Periféricos/patologia , Sarcoma de Ewing/patologia , Antígeno 12E7 , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/biossíntese , Neoplasias Ósseas/metabolismo , Antígenos CD57/biossíntese , Caveolina 1/biossíntese , Moléculas de Adesão Celular/biossíntese , Criança , Pré-Escolar , Progressão da Doença , Feminino , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Lactente , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Tumores Neuroectodérmicos Primitivos Periféricos/metabolismo , Prognóstico , Proteína Proto-Oncogênica c-fli-1/biossíntese , Sarcoma de Ewing/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Neoplasias Renais/diagnóstico , Tumores Neuroectodérmicos Primitivos/diagnóstico , Tumor de Wilms/diagnóstico , Antígeno 12E7 , Antígenos CD/biossíntese , Antígenos CD/genética , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/genética , Criança , Pré-Escolar , Cromossomos Humanos Par 11/genética , Cromossomos Humanos Par 11/metabolismo , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 22/metabolismo , Diagnóstico Diferencial , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Tumores Neuroectodérmicos Primitivos/genética , Tumores Neuroectodérmicos Primitivos/metabolismo , Tumores Neuroectodérmicos Primitivos/patologia , Proteína Proto-Oncogênica c-fli-1/biossíntese , Proteína Proto-Oncogênica c-fli-1/genética , Proteína EWS de Ligação a RNA/biossíntese , Proteína EWS de Ligação a RNA/genética , Translocação Genética/genética , Tumor de Wilms/genética , Tumor de Wilms/metabolismo , Tumor de Wilms/patologiaRESUMO
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.