RESUMO
BACKGROUND: Myxoid liposarcoma (MLS) displays a distinctive tumor microenvironment and is characterized by the FUS::DDIT3 fusion oncogene, however, the precise functional contributions of these two elements remain enigmatic in tumor development. METHODS: To study the cell-free microenvironment in MLS, we developed an experimental model system based on decellularized patient-derived xenograft tumors. We characterized the cell-free scaffold using mass spectrometry. Subsequently, scaffolds were repopulated using sarcoma cells with or without FUS::DDIT3 expression that were analyzed with histology and RNA sequencing. RESULTS: Characterization of cell-free MLS scaffolds revealed intact structure and a large variation of protein types remaining after decellularization. We demonstrated an optimal culture time of 3 weeks and showed that FUS::DDIT3 expression decreased cell proliferation and scaffold invasiveness. The cell-free MLS microenvironment and FUS::DDIT3 expression both induced biological processes related to cell-to-cell and cell-to-extracellular matrix interactions, as well as chromatin remodeling, immune response, and metabolism. Data indicated that FUS::DDIT3 expression more than the microenvironment determined the pre-adipocytic phenotype that is typical for MLS. CONCLUSIONS: Our experimental approach opens new means to study the tumor microenvironment in detail and our findings suggest that FUS::DDIT3-expressing tumor cells can create their own extracellular niche.
Assuntos
Lipossarcoma Mixoide , Proteínas de Fusão Oncogênica , Proteína FUS de Ligação a RNA , Microambiente Tumoral , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Proliferação de Células , Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Lipossarcoma Mixoide/patologia , Lipossarcoma Mixoide/metabolismo , Lipossarcoma Mixoide/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Alicerces Teciduais/química , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
DDIT3 is a tightly regulated basic leucine zipper (bZIP) transcription factor and key regulator in cellular stress responses. It is involved in a variety of pathological conditions and may cause cell cycle block and apoptosis. It is also implicated in differentiation of some specialized cell types and as an oncogene in several types of cancer. DDIT3 was originally believed to act as a dominant-negative inhibitor by forming heterodimers with other bZIP transcription factors, preventing their DNA binding and transactivating functions. DDIT3 has, however, been reported to bind DNA and regulate target genes. Here, we employed ChIP sequencing combined with microarray-based expression analysis to identify direct binding motifs and target genes of DDIT3. The results reveal DDIT3 binding to motifs similar to other bZIP transcription factors, known to form heterodimers with DDIT3. Binding to a class III satellite DNA repeat sequence was also detected. DDIT3 acted as a DNA-binding transcription factor and bound mainly to the promotor region of regulated genes. ChIP sequencing analysis of histone H3K27 methylation and acetylation showed a strong overlap between H3K27-acetylated marks and DDIT3 binding. These results support a role for DDIT3 as a transcriptional regulator of H3K27ac-marked genes in transcriptionally active chromatin.
Assuntos
Genômica , Fatores de Transcrição , Sítios de Ligação , Fatores de Transcrição/genética , Fatores de Transcrição de Zíper de Leucina Básica , DNARESUMO
Members of the human FET family of RNA-binding proteins, comprising FUS, EWSR1, and TAF15, are ubiquitously expressed and engage at several levels of gene regulation. Many sarcomas and leukemias are characterized by the expression of fusion oncogenes with FET genes as 5' partners and alternative transcription factor-coding genes as 3' partners. Here, we report that the N terminus of normal FET proteins and their oncogenic fusion counterparts interact with the SWI/SNF chromatin remodeling complex. In contrast to normal FET proteins, increased fractions of FET oncoproteins bind SWI/SNF, indicating a deregulated and enhanced interaction in cancer. Forced expression of FET oncogenes caused changes of global H3K27 trimethylation levels, accompanied by altered gene expression patterns suggesting a shift in the antagonistic balance between SWI/SNF and repressive polycomb group complexes. Thus, deregulation of SWI/SNF activity could provide a unifying pathogenic mechanism for the large group of tumors caused by FET fusion oncoproteins. These results may help to develop common strategies for therapy.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Linhagem Celular Tumoral , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Metilação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/genética , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Myxoid liposarcoma (MLS) shows extensive intratumoural heterogeneity with distinct subpopulations of tumour cells. Despite improved survival of MLS patients, existing therapies have shortcomings as they fail to target all tumour cells. The nature of chemotherapy-resistant cells in MLS remains unknown. Here, we show that MLS cell lines contained subpopulations of cells that can form spheres, efflux Hoechst dye and resist doxorubicin, all properties attributed to cancer stem cells (CSCs). By single-cell gene expression, western blot, phospho-kinase array, immunoprecipitation, immunohistochemistry, flow cytometry and microarray analysis we showed that a subset of MLS cells expressed JAK-STAT genes with active signalling. JAK1/2 inhibition via ruxolitinib decreased, while stimulation with LIF increased, phosphorylation of STAT3 and the number of cells with CSC properties indicating that JAK-STAT signalling controlled the number of cells with CSC features. We also show that phosphorylated STAT3 interacted with the SWI/SNF complex. We conclude that MLS contains JAK-STAT-regulated subpopulations of cells with CSC features. Combined doxorubicin and ruxolitinib treatment targeted both proliferating cells as well as cells with CSC features, providing new means to circumvent chemotherapy resistance in treatment of MLS patients.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Lipossarcoma Mixoide/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Janus Quinases/metabolismo , Lipossarcoma Mixoide/tratamento farmacológico , Células-Tronco Neoplásicas/efeitos dos fármacos , Nitrilas , Fosforilação , Pirazóis/farmacologia , Pirimidinas , Fatores de Transcrição STAT/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismoRESUMO
FUS-DDIT3 belongs to the FET (FUS, EWSR1, and TAF15) family of fusion oncogenes, which collectively are considered to be key players in tumor development. Even though over 90% of all myxoid liposarcomas (MLS) have a FUS-DDIT3 gene fusion, there is limited understanding of the signaling pathways that regulate its expression. In order to study cell proliferation and FUS-DDIT3 regulation at mRNA and protein levels, we first developed a direct cell lysis approach that allows DNA, mRNA, and protein to be analyzed in the same sample using quantitative PCR, reverse transcription quantitative qPCR and proximity ligation assay, respectively. We screened 70 well-characterized kinase inhibitors and determined their effects on cell proliferation and expression of FUS-DDIT3 and FUS at both mRNA and protein levels in the MLS 402-91 cell line, where twelve selected inhibitors were evaluated further in two additional MLS cell lines. Both FUS-DDIT3 and FUS mRNA expression correlated with cell proliferation and both transcripts were co-regulated in most conditions, indicating that the common 5' FUS promotor is important in transcriptional regulation. In contrast, FUS-DDIT3 and FUS protein levels displayed more cell line dependent expression. Furthermore, most JAK inhibitors caused FUS-DDIT3 downregulation at both mRNA and protein levels. In conclusion, defining factors that regulate FUS-DDIT3 expression opens new means to understand MLS development at the molecular level.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lipossarcoma Mixoide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Linhagem Celular Tumoral , DNA/análise , DNA/genética , DNA/metabolismo , Humanos , Lipossarcoma Mixoide/genética , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Fusion oncogenes are among the most common types of oncogene in human cancers. The gene rearrangements result in new combinations of regulatory elements and functional protein domains. Here we studied a subgroup of sarcomas and leukaemias characterized by the FET (FUS, EWSR1, TAF15) family of fusion oncogenes, including FUS-DDIT3 in myxoid liposarcoma (MLS). We investigated the regulatory mechanisms, expression levels and effects of FUS-DDIT3 in detail. FUS-DDIT3 showed a lower expression than normal FUS at both the mRNA and protein levels, and single-cell analysis revealed a lack of correlation between FUS-DDIT3 and FUS expression. FUS-DDIT3 transcription was regulated by the FUS promotor, while its mRNA stability depended on the DDIT3 sequence. FUS-DDIT3 protein stability was regulated by protein interactions through the FUS part, rather than the leucine zipper containing DDIT3 part. In addition, in vitro as well as in vivo FUS-DDIT3 protein expression data displayed highly variable expression levels between individual MLS cells. Combined mRNA and protein analyses at the single-cell level showed that FUS-DDIT3 protein expression was inversely correlated to the expression of cell proliferation-associated genes. We concluded that FUS-DDIT3 is uniquely regulated at the transcriptional as well as the post-translational level and that its expression level is important for MLS tumour development. The FET fusion oncogenes are potentially powerful drug targets and detailed knowledge about their regulation and functions may help in the development of novel treatments.
Assuntos
Biomarcadores Tumorais/metabolismo , Proliferação de Células , Lipossarcoma Mixoide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Meia-Vida , Humanos , Lipossarcoma Mixoide/genética , Lipossarcoma Mixoide/patologia , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Tempo , Transcrição Gênica , TransfecçãoRESUMO
Myxoid liposarcoma has the pathognomonic fusion oncogene FUS-DDIT3 encoding a chimeric transcription factor. Metastatic risk is higher with an increased round cell component and has been linked to aberrations involving the IGFR/PI3K/AKT pathway. These molecular insights have yet to translate to targeted therapies, and the lack of experimental models is a major hindrance. We describe the initial in-depth characterization of a new cell line (DL-221) and establishment of a mouse xenograft model. The cell line DL-221 was derived from a metastatic pleural lesion showing myxoid and round cell histology. This newly established cell line was characterized for phenotypic properties and molecular cytogenetic profile, using PCR, COBRA-FISH, and western blot. Next-generation whole-exome sequencing was performed to further characterize the cell line and the parent tumor. NOD-SCID-IL2R gamma knockout mice were xenograft hosts. DL-221 cells grew an adhering monolayer and COBRA-FISH showed an aneuploid karyotype with t(12;16)(q13;p11) and several other rearrangements; RT-PCR demonstrated a FUS-DDIT3 fusion transcript type 1. Both the cell line and the original tumor harbored a TP53 compound heterozygous mutation in exon 4 and 7, and were wild-type for PIK3CA. Moreover, among the 1254 variants called by whole-exome sequencing, there was 77% concordance between the cell line and parent tumor. The recently described hotspot mutation in the TERT promoter region in myxoid liposarcomas was also found at C228T in DL-221. Xenografts suitable for additional preclinical studies were successfully established in mice after subcutaneous injection. The established DL-221 cell line is the only published available myxoid liposarcoma cell line that underwent spontaneous immortalization, without requiring SV40 transformation. The cell line and its xenograft model are unique and helpful tools to study the biology and novel potential-targeted treatment approaches for myxoid liposarcoma.
Assuntos
Lipossarcoma Mixoide/genética , Proteínas de Fusão Oncogênica/genética , Translocação Genética , Animais , Linhagem Celular Tumoral , Análise Mutacional de DNA , Feminino , Xenoenxertos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Lipossarcoma Mixoide/patologia , Lipossarcoma Mixoide/secundário , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologia , Neoplasias Pleurais/secundárioRESUMO
Liposarcomas (LS) are the most common malignant mesenchymal tumors, with an overall long-term mortality rate of 60%. LS comprise three major subtypes, i.e., well-differentiated/dedifferentiated liposarcoma (WDLS/DDLS), myxoid/round cell liposarcoma (MLS) and pleomorphic liposarcoma (PLS). Aiming at the preclinical identification of novel therapeutic options, we here investigate the functional significance of SRC in primary human LS and in LS-derived cell lines. Immunohistochemical and Western blot analyses reveal relevant levels of activated p-(Tyr416)-SRC in LS of the different subtypes with particular activation in MLS and PLS. Dysregulation of the SRC modifiers CSK and PTP1B was excluded as major reason for the activation of the kinase. Consistent siRNA-mediated knockdown of SRC or inhibition by the SRC inhibitor Dasatinib led to decreased proliferation of LS cell lines of the different subtypes, with MLS cells reacting particularly sensitive in MTT assays. Flow cytometric analyses revealed that this effect was due to a significant decrease in mitotic activity and an induction of apoptosis. SRC inhibition by Dasatinib resulted in dephosphorylation of SRC itself, its interacting partners FAK and IGF-IR as well as its downstream target AKT. Consistent with a particular role of SRC in cell motility, Dasatinib reduced the migratory and invasive potential of MLS cells in Boyden chamber and Matrigel chamber assays. In summary, we provide evidence that SRC activation plays an important role in LS biology and therefore represents a potential therapeutic target, particularly in MLS and PLS.
Assuntos
Lipossarcoma Mixoide/tratamento farmacológico , Lipossarcoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Quinases da Família src/antagonistas & inibidores , Apoptose/efeitos dos fármacos , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Humanos , Mitose/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/metabolismoRESUMO
The three FET (FUS, EWSR1, and TAF15) family RNA binding proteins are expressed in all tissues and almost all cell types. The disordered N-terminal parts are always present in FET fusion oncoproteins of sarcomas and leukemia. Mutations in FUS and TAF15 cause aggregation of FET proteins in neurological disorders. Here we used recombinant proteins in pulldown experiments and mass spectrometry to identify major interaction partners of the FET N-terminal parts. We report that FUS, EWSR1, and TAF15 form homo- and heterocomplexes as major binding partners and identify an evolutionarily conserved N-terminal motif (FETBM1) that is required for this interaction. The binding is RNA and DNA independent and robust up to 1 M of NaCl. The localization of FETBM1 and its target sequences supports a simple model for FET protein aggregation as reported in neurological disorders such as amyotrophic lateral sclerosis, frontotemporal dementia, and essential tremor. The FETBM1 localization also explains the binding of normal full-length FET proteins to their oncogenic fusion proteins.
Assuntos
Proteínas de Ligação a Calmodulina/química , Proteínas de Fusão Oncogênica/química , Proteína FUS de Ligação a RNA/química , Proteínas de Ligação a RNA/química , Fatores Associados à Proteína de Ligação a TATA/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a Calmodulina/metabolismo , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteína EWS de Ligação a RNA , Proteína FUS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismoRESUMO
Fused in sarcoma (FUS) is a multifunctional protein involved in transcriptional control, pre-mRNA processing, RNA transport and translation. The domain structure of FUS reflects its functions in gene regulation and its ability to interact with other proteins, RNA and DNA. By use of a recombinant fragment of FUS in pull-down experiments followed by mass spectrometry analysis we have identified a novel interaction between the FUS N-terminal and the cytolinker plectin. An in situ proximity ligation assay confirmed that FUS-plectin interactions take place in the cytoplasm of cells. Furthermore, plectin deficient cells showed an altered subcellular localization of FUS and a deregulated expression of mRNAs bound to FUS. Our results show that plectin is important for normal FUS localization and function. Mutations involving FUS are causative factors in sarcomas and leukemias and also hereditary forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Plectin deficiency causes epidermolysis bullosa, a disease involving the skin and neuromuscular system. The novel FUS-plectin interaction offers new perspectives for understanding the role of FUS and plectin mutations in the pathogenesis of these diseases.
Assuntos
Plectina/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Camundongos , Plectina/química , Plectina/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Transporte Proteico , RNA Mensageiro/metabolismo , Proteína FUS de Ligação a RNA/químicaRESUMO
BACKGROUND: The single cell represents the basic unit of all organisms. Most investigations have been performed on large cell populations, but understanding cell dynamics and heterogeneity requires single-cell analysis. Current methods for single-cell analysis generally can detect only one class of analytes. METHODS: Reverse transcription and the proximity ligation assay were coupled with quantitative PCR and used to quantify any combination of DNA, mRNAs, microRNAs (miRNAs), noncoding RNAs (ncRNAs), and proteins from the same single cell. The method was used on transiently transfected human cells to determine the intracellular concentrations of plasmids, their transcribed mRNAs, translated proteins, and downstream RNA targets. RESULTS: We developed a whole-cell lysis buffer to release unfractionated DNA, RNA, and proteins that would not degrade any detectable analyte or inhibit the assay. The dynamic range, analytical sensitivity, and specificity for quantifying DNA, mRNAs, miRNAs, ncRNAs, and proteins were shown to be accurate down to the single-cell level. Correlation studies revealed that the intracellular concentrations of plasmids and their transcribed mRNAs were correlated only moderately with translated protein concentrations (Spearman correlation coefficient, 0.37 and 0.31, respectively; P < 0.01). In addition, an ectopically expressed gene affected the correlations between analytes and this gene, which is related to gene regulation. CONCLUSIONS: This method is compatible with most cell-sampling approaches, and generates output for the same parameter for all measured analytes, a feature facilitating comparative data analysis. This approach should open up new avenues in molecular diagnostics for detailed correlation studies of multiple and different classes of analytes at the single-cell level.
Assuntos
DNA/análise , Reação em Cadeia da Polimerase/métodos , Proteínas/análise , RNA/análise , Soluções Tampão , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Humanos , MicroRNAs/análise , RNA Mensageiro/análise , RNA não Traduzido/análise , Proteína FUS de Ligação a RNA/análise , Proteína FUS de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Análise de Célula ÚnicaRESUMO
The therapeutic options for patients with relapsed or metastatic myxoid liposarcoma (MLS) remain scarce and there is currently no targeted therapy available. Inhibition of the HSP90 family of chaperones has been suggested as a possible therapeutic option for patients with MLS. However, the clinical effect of different HSP90 inhibitors vary considerably and no comparative study in MLS has been performed. Here, we evaluated the effects of the HSP90 inhibitors 17-DMAG, AUY922 and STA-9090 on MLS cell lines and in an MLS patient-derived xenograft (PDX) model. Albeit all drugs inhibited in vitro growth of MLS cell lines, the in vivo responses were discrepant. Whereas 17-DMAG inhibited tumor growth, AUY922 surprisingly led to increased tumor growth and a more aggressive morphological phenotype. In vitro, 17-DMAG and STA-9090 reduced the activity of the MAPK and PI3K/AKT signaling pathways, whereas AUY922 led to a compensatory upregulation of downstream ERK. Furthermore, all three tested HSP90 inhibitors displayed a synergistic combination effect with trabectidin, but not with doxorubicin. In conclusion, our results indicate that different HSP90 inhibitors, albeit having the same target, can vary significantly in downstream effects and treatment outcomes. These results should be considered before proceeding into clinical trials against MLS or other malignancies.
RESUMO
Myxoid liposarcoma is one of the most common sarcoma entities characterized by FET fusion oncogenes. Despite a generally favorable prognosis of myxoid liposarcoma, chemotherapy resistance remains a clinical problem. This cancer stem cell property is associated with JAK-STAT signaling, but the link to the myxoid-liposarcoma-specific FET fusion oncogene FUS-DDIT3 is not known. Here, we show that ectopic expression of FUS-DDIT3 resulted in elevated levels of STAT3 and phosphorylated STAT3. RNA sequencing identified 126 genes that were regulated by both FUS-DDIT3 expression and JAK1/2 inhibition using ruxolitinib. Sixty-six of these genes were connected in a protein interaction network. Fifty-three and 29 of these genes were confirmed as FUS-DDIT3 and STAT3 targets, respectively, using public chromatin immunoprecipitation sequencing data sets. Enriched gene sets among the 126 regulated genes included processes related to cytokine signaling, adipocytokine signaling, and chromatin remodeling. We validated CD44 as a target gene of JAK1/2 inhibition and as a potential cancer stem cell marker in myxoid liposarcoma. Finally, we showed that FUS-DDIT3 interacted with phosphorylated STAT3 in association with subunits of the SWI/SNF chromatin remodeling complex and PRC2 repressive complex. Our data show that the function of FUS-DDIT3 is closely connected to JAK-STAT signaling. Detailed deciphering of molecular mechanisms behind tumor progression opens up new avenues for targeted therapies in sarcomas and leukemia characterized by FET fusion oncogenes.
RESUMO
FET fusion oncoproteins containing one of the FET (FUS, EWSR1, TAF15) family proteins juxtaposed to alternative transcription-factor partners are characteristic of more than 20 types of sarcoma and leukaemia. FET oncoproteins bind to the SWI/SNF chromatin remodelling complex, which exists in three subtypes: cBAF, PBAF and GBAF/ncBAF. We used comprehensive biochemical analysis to characterize the interactions between FET oncoproteins, SWI/SNF complexes and the transcriptional coactivator BRD4. Here, we report that FET oncoproteins bind all three main SWI/SNF subtypes cBAF, PBAF and GBAF, and that FET oncoproteins interact indirectly with BRD4 via their shared interaction partner SWI/SNF. Furthermore, chromatin immunoprecipitation sequencing and proteomic analysis showed that FET oncoproteins, SWI/SNF components and BRD4 co-localize on chromatin and interact with mediator and RNA Polymerase II. Our results provide a possible molecular mechanism for the FET-fusion-induced oncogenic transcriptional profiles and may lead to novel therapies targeting aberrant SWI/SNF complexes and/or BRD4 in FET-fusion-caused malignancies.
Assuntos
Montagem e Desmontagem da Cromatina , Sarcoma , Proteínas de Ciclo Celular/metabolismo , Cromatina , Proteínas Cromossômicas não Histona/genética , Humanos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteômica , Fatores de Transcrição/metabolismoRESUMO
Myxoid liposarcoma (MLS) represents a common subtype of liposarcoma molecularly characterized by a recurrent chromosomal translocation that generates a chimeric FUS-DDIT3 fusion gene. The FUS-DDIT3 oncoprotein has been shown to be crucial in MLS pathogenesis. Acting as a transcriptional dysregulator, FUS-DDIT3 stimulates proliferation and interferes with adipogenic differentiation. As the fusion protein represents a therapeutically challenging target, a profound understanding of MLS biology is elementary to uncover FUS-DDIT3-dependent molecular vulnerabilities. Recently, a specific reliance on the Hippo pathway effector and transcriptional co-regulator YAP1 was detected in MLS; however, details on the molecular mechanism of FUS-DDIT3-dependent YAP1 activation, and YAP1´s precise mode of action remain unclear. In elaborate in vitro studies, employing RNA interference-based approaches, small-molecule inhibitors, and stimulation experiments with IGF-II, we show that FUS-DDIT3-driven IGF-IR/PI3K/AKT signaling promotes stability and nuclear accumulation of YAP1 via deregulation of the Hippo pathway. Co-immunoprecipitation and proximity ligation assays revealed nuclear co-localization of FUS-DDIT3 and YAP1/TEAD in FUS-DDIT3-expressing mesenchymal stem cells and MLS cell lines. Transcriptome sequencing of MLS cells demonstrated that FUS-DDIT3 and YAP1 co-regulate oncogenic gene signatures related to proliferation, cell cycle progression, apoptosis, and adipogenesis. In adipogenic differentiation assays, we show that YAP1 critically contributes to FUS-DDIT3-mediated adipogenic differentiation arrest. Taken together, our study provides mechanistic insights into a complex FUS-DDIT3-driven network involving IGF-IR/PI3K/AKT signals acting on Hippo/YAP1, and uncovers substantial cooperative effects of YAP1 and FUS-DDIT3 in the pathogenesis of MLS.
RESUMO
BACKGROUND: The FUS-DDIT3 fusion oncogene encodes an abnormal transcription factor that has a causative role in the development of myxoid/round-cell liposarcomas (MLS/RCLS). We have previously identified FLT1 (VEGFR1) as a candidate downstream target gene of FUS-DDIT3. The aim of this study was to investigate expression of FLT1 and its ligands in MLS cells. METHODS: HT1080 human fibrosarcoma cells were transiently transfected with FUS-DDIT3-GFP variant constructs and FLT1 expression was measured by quantitative real-time PCR. In addition, FLT1, PGF, VEGFA and VEGFB expression was measured in MLS/RCLS cell lines, MLS/RCLS tumors and in normal adiopocytes. We analyzed nine cases of MLS/RCLS and one cell line xenografted in mice for FLT1 protein expression using immunohistochemistry. MLS/RCLS cell lines were also analyzed for FLT1 by immunofluorescence and western blot. MLS/RCLS cell lines were additionally treated with FLT1 tyrosine kinase inhibitors and assayed for alterations in proliferation rate. RESULTS: FLT1 expression was dramatically increased in transfected cells stably expressing FUS-DDIT3 and present at high levels in cell lines derived from MLS. The FLT1 protein showed a strong nuclear expression in cells of MLS tissue as well as in cultured MLS cells, which was confirmed by cellular fractionation. Tissue array analysis showed a nuclear expression of the FLT1 protein also in several other tumor and normal cell types including normal adipocytes. The FLT1 ligand coding gene PGF was highly expressed in cultured MLS cells compared to normal adipocytes while the other ligand genes VEGFA and VEGFB were expressed to lower levels. A more heterogeneous expression pattern of these genes were observed in tumor samples. No changes in proliferation rate of MLS cells were detected at concentrations for which the kinase inhibitors have shown specific inhibition of FLT1. CONCLUSIONS: Our results imply that FLT1 is induced as an indirect downstream effect of FUS-DDIT3 expression in MLS. This could be a consequence of the ability of FUS-DDIT3 to hijack parts of normal adipose tissue development and reprogram primary cells to a liposarcoma-like phenotype. The findings of nuclear FLT1 protein and expression of corresponding ligands in MLS and normal tissues may have implications for tissue homeostasis and tumor development through auto- or intracrine signaling.
Assuntos
Núcleo Celular/metabolismo , Fibrossarcoma/metabolismo , Lipossarcoma Mixoide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Proteínas da Gravidez/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/patologia , Proliferação de Células , Relação Dose-Resposta a Droga , Fibrossarcoma/genética , Fibrossarcoma/patologia , Imunofluorescência , Humanos , Imuno-Histoquímica , Ligantes , Lipossarcoma Mixoide/genética , Lipossarcoma Mixoide/patologia , Camundongos , Camundongos SCID , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/genética , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , Proteína FUS de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição CHOP/genética , Transfecção , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Differentiation is a complex set of events that can be blocked by rearrangements of regulatory genes producing fusion proteins with altered properties. In the case of myxoid liposarcoma (MLS) tumors, the causative abnormality is a fusion between the CHOP transcription factor and the FUS or EWS genes. CHOP belongs to and is a negative regulator of the large CAAT/enhancer binding protein family whose alpha, beta, and delta members are master genes of adipogenesis. Recent clinical data indicate a peculiar sensitivity of these tumors to the natural marine compound trabectedin. One hypothesis is that the activity of trabectedin is related to the inactivation of the FUS-CHOP oncogene. We find that trabectedin causes detachment of the FUS-CHOP chimera from targeted promoters. Reverse transcription-PCR and chromatin immunoprecipitation analysis in a MLS line and surgical specimens of MLS patients in vivo show activation of the CAAT/enhancer binding protein-mediated transcriptional program that leads to morphologic changes of terminal adipogenesis. The activity is observed in cells with type 1 but not type 8 fusions. Hence, the drug induces maturation of MLS lipoblasts in vivo by targeting the FUS-CHOP-mediated transcriptional block. These data provide a rationale for the specific activity of trabectedin and open the perspective of combinatorial treatments with drugs acting on lipogenic pathways.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Dioxóis/farmacologia , Lipossarcoma Mixoide/patologia , Tetra-Hidroisoquinolinas/farmacologia , Adipogenia/efeitos dos fármacos , Adipogenia/genética , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lipossarcoma Mixoide/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Ligação Proteica/efeitos dos fármacos , Proteína FUS de Ligação a RNA/genética , Trabectedina , Fator de Transcrição CHOP/genéticaRESUMO
Single-cell analysis enables detailed molecular characterization of cells in relation to cell type, genotype, cell state, temporal variations, and microenvironment. These studies often include the analysis of individual genes and networks of genes. The total amount of RNA also varies between cells due to important factors, such as cell type, cell size, and cell cycle state. However, there is a lack of simple and sensitive methods to quantify the total amount of RNA, especially mRNA. Here, we developed a method to quantify total mRNA levels in single cells based on global reverse transcription followed by quantitative PCR. Standard curve analyses of diluted RNA and sorted cells showed a wide dynamic range, high reproducibility, and excellent sensitivity. Single-cell analysis of three sarcoma cell lines and human fibroblasts revealed cell type variations, a lognormal distribution of total mRNA levels, and up to an eight-fold difference in total mRNA levels among the cells. The approach can easily be combined with targeted or global gene expression profiling, providing new means to study cell heterogeneity at an individual gene level and at a global level. This method can be used to investigate the biological importance of variations in the total amount of mRNA in healthy as well as pathological conditions.
Assuntos
Sarcoma/genética , Sarcoma/patologia , Análise de Célula Única , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Poliadenilação/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transcriptoma/genéticaRESUMO
BACKGROUND: The DDIT3 gene encodes a transcription factor belonging to the CCAAT/enhancer binding protein (C/EBP) family. It is normally expressed at very low levels but is activated by cellular stress conditions and induces G1 arrest and, in some cell types, apoptosis. DDIT3 is found as a part of the fusion oncogene FUS-DDIT3 that is causal for the development of myxoid/round-cell liposarcomas (MLS/RCLS). RESULTS: In the present study, we searched for putative interaction partners of DDIT3 and the oncogenic FUS-DDIT3 among G1 cyclins and cyclin-dependent kinases. We found that FUS-DDIT3 and the normal DDIT3 bind CDK2. In addition, CDK2 showed an increased affinity for cytoskeletal proteins in cells expressing FUS-DDIT3 and DDIT3. CONCLUSIONS: We conclude that DDIT3 binds CDK2 and that many of the observed biological effects of DDIT3 may involve interaction with CDK2.
Assuntos
Quinase 2 Dependente de Ciclina/metabolismo , Lipossarcoma Mixoide/metabolismo , Proteínas de Fusão Oncogênica/metabolismo , Fator de Transcrição CHOP/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Quinase 2 Dependente de Ciclina/genética , Humanos , Lipossarcoma Mixoide/genética , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Ligação Proteica , Alinhamento de Sequência , Fator de Transcrição CHOP/química , Fator de Transcrição CHOP/genéticaRESUMO
Chromosome rearrangements resulting in pathogenetically important gene fusions are a common feature of many cancers. They are often potent oncogenic drivers and have key functions in central cellular processes and pathways and encode transcription factors, transcriptional co-regulators, growth factor receptors, tyrosine kinases, and chromatin modifiers. In addition to being useful diagnostic biomarkers, they are also targets for development of new molecularly targeted therapies. Studies in recent decades have shown that several oncogenic gene fusions interact with the insulin-like growth factor (IGF) signaling pathway. For example, the MYB-NFIB fusion in adenoid cystic carcinoma is regulated by IGF1R through an autocrine loop, and IGF1R is a downstream target of the EWSR1-WT1 and PAX3-FKHR fusions in desmoplastic small round cell tumors and alveolar rhabdomyosarcoma, respectively. Here, we will discuss the mechanisms behind the interactions between oncogenic gene fusions and the IGF signaling pathway. We will also discuss the role of therapeutic inhibition of IGF1R in fusion gene driven malignancies.