| 1. |
- Andersson, Mattias K, 1979, et al.
(författare)
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Nuclear expression of FLT1 and its ligand PGF in FUS-DDIT3 carrying myxoid liposarcomas suggests the existence of an intracrine signaling loop.
- 2010
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Ingår i: BMC cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Engström, Katarina, 1956, et al.
(författare)
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The myxoid/round cell liposarcoma fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype in transfected human fibrosarcoma cells.
- 2006
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Ingår i: The American journal of pathology. - : Elsevier BV. - 0002-9440 .- 1525-2191. ; 168:5, s. 1642-53
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Tidskriftsartikel (refereegranskat)abstract
- Myxoid/round cell liposarcoma (MLS/RCLS) is the most common subtype of liposarcoma. Most MLS/RCLS carry a t(12;16) translocation, resulting in a FUS-DDIT3 fusion gene. We investigated the role of the FUS-DDIT3 fusion in the development of MLS/RCLS in FUS-DDIT3- and DDIT3-transfected human HT1080 sarcoma cells. Cells expressing FUS-DDIT3 and DDIT3 grew as liposarcomas in severe combined immunodeficient mice and exhibited a capillary network morphology that was similar to networks of MLS/RCLS. Microarray-based comparison of HT1080, the transfected cells, and an MLS/RCLS-derived cell line showed that the FUS-DDIT3- and DDIT3-transfected variants shifted toward an MLS/RCLS-like expression pattern. DDIT3-transfected cells responded in vitro to adipogenic factors by accumulation of fat and transformation to a lipoblast-like morphology. In conclusion, because the fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype when expressed in a primitive sarcoma cell line, MLS/RCLS may develop from cell types other than preadipocytes. This may explain the preferential occurrence of MLS/RCLS in nonadipose tissues. In addition, development of lipoblasts and the typical MLS/RCLS capillary network could be an effect of the DDIT3 transcription factor partner of the fusion oncogene.
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| 3. |
- Göransson, Melker, 1974
(författare)
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Functional characterization of the liopsarcoma-associated fusion oncigene FUS-DDIT3
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fusion genes represent a growing class of translocation-derived potent oncogenes that frequently show tumor type-specific expression. We have studied the myxoid/round cell liposarcoma (MLS/RCLS)-specific FUS-DDIT3 fusion, with the aim of functionally characterizing this fusion oncogene in sarcoma development. FUS-DDIT3 is the result of a chromosomal translocation t(12;16)(q13;p11) which fuses the 5' end of FUS (TLS) with the entire DDIT3 (CHOP). As a result, the fusion gene is transcriptionally controlled by the constitutively active FUS promoter. The causative role of the FUS-DDIT3 fusion in initiation of MLS/RCLS has been demonstrated in transgenic mice. Ectopic expression of DDIT3 and the FUS-DDIT3 proteins have been shown to counteract differentiation and abrogate adipocyte development. This results in partially committed pre-adipocytes, a cell population with the potential to progress towards liposarcoma development. We have developed an experimental system, consisting of genetically modified human fibrosarcoma HT1080 cells stably expressing FUS-DDIT3, C-terminally truncated FUS or DDIT3, coupled with the Green Fluorescent Protein (GFP). By using this system we have been able to study the localization of these proteins, their interaction partners and the different gene expression profiles induced by their expression. We have found that the FUS-DDIT3-GFP fusion protein localizes to well-defined nuclear structures. This enabled us to study the interaction between FUS-DDIT3 and other nuclear proteins. We have found that FUS-DDIT3 associates with the splicing machinery and proposed a model in which this fusion gene disturbs the normal splicing process, an idea later confirmed by others in splicing assays. By microarray analysis, we identified the IL6 and IL8 genes as FUS-DDIT3 targets and demonstrated for the first time that DDIT3 and FUS-DDIT3 initiate opposing transcriptional regulation of the IL8 gene. IL6 is a multi-functional cytokine that has been shown to act as an autocrine growth factor in human prostate cancer cells and the existence of an IL6 autocrine loop has been implicated in the oncogenesis of multiple myeloma. In addition, it has been shown that IL6 plays a pivotal role for proliferation and invasion of malignant fibrous histiocytoma. It is possible that aberrant IL6 expression has similar functions in MLS/RCLS. Immuno-histochemical analysis involving 5 primary non-irradiated and 12 secondary and/or irradiated human MLS/RCLS showed that high expression of the G1 cyclins, cyclin D and E, and their associated kinases CDK4 and 2, is a recurrent pattern in these tumors. Furthermore, we have recently demonstrated, via luciferase assay experiments, the importance of NFkappaB for IL8 expression in FUS-DDIT3-carrying cells, and we found a direct physical connection between the FUS-DDIT3 protein and nuclear IkappaB zeta. The NFkappaB system controls genes involved in proliferation, migration and apoptosis. Modification of this system by FUS-DDIT3 would result in disturbance of vital functions in normal cells. In summary, we have shown that the FUS-DDIT3 fusion oncogene is capable of affecting multiple cellular processes and pathways. The potency of this fusion oncogene in liposarcoma development may be explained by this ability to affect several different cellular systems and thus induce multiple hits in the neoplastic pathway.
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| 4. |
- Göransson, Melker, 1974, et al.
(författare)
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Myxoid liposarcoma FUS-DDIT3 fusion oncogene induces C/EBP β-mediated interleukin 6 expression
- 2005
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 115:4, s. 556-560
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Tidskriftsartikel (refereegranskat)abstract
- The myxoid/round cell liposarcoma oncogene FUS-DDIT3 is the result of a translocation derived gene fusion between the splicing factor FUS and DDIT3. In order to investigate the downstream targets of DDIT3, and the transforming effects of the FUS-DDIT3 fusion protein, we have introduced DDIT3-GFP and FUS-DDIT3-GFP constructs into a human flbrosarcoma cell line. The gene expression profiles of stable transfectants were compared to the original fibrosarcoma cell line by microarray analysis. We here report that the NFκB and C/EBP β controlled gene IL6 is upregulated in DDIT3- and FUS-DDIT3-expressing fibrosarcoma cell lines and in myxoid liposarcoma cell lines. Strong expression of the tumor associated multifunctional cytokine interleukin 6 was confirmed both at mRNA and protein level. Knockdown experiments using siRNA against CEBPB transcripts showed that the effect of FUS-DDIT3 on IL6 expression is C/EBP β dependent. Chromatin immunoprecipitation revealed direct interaction between the IL6 promoter and the C/EBP β protein. In addition, the effect of DDIT3 and FUS-DDIT3 on the expression of other acute phase genes was examined using real-time PCR. We demonstrate for the first time that DDIT3 and FUS-DDIT3 show opposite transcriptional regulation of IL8 and suggest that FUS-DDIT3 may affect the synergistic activation of promoters regulated by C/EBP β and NFκB.
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| 5. |
- Göransson, Melker, 1974, et al.
(författare)
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The myxoid liposarcoma FUS-DDIT3 fusion oncoprotein deregulates NF-kappaB target genes by interaction with NFKBIZ.
- 2009
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Ingår i: Oncogene. - : Springer Science and Business Media LLC. - 1476-5594 .- 0950-9232. ; 28:2, s. 270-8
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Tidskriftsartikel (refereegranskat)abstract
- FUS (also called TLS), EWSR1 and TAF15 (also called TAF2N) are related genes involved in tumor type-specific fusion oncogenes in human malignancies. The FUS-DDIT3 fusion oncogene results from a t(12;16)(q13;p11) chromosome translocation and has a causative role in the initiation of myxoid/round cell liposarcomas (MLS/RCLS). The FUS-DDIT3 protein induces increased expression of the CAAT/enhancer-binding protein (C/EBP) and nuclear factor-kappaB (NF-kappaB)-controlled gene IL8, and the N-terminal FUS part is required for this activation. Chromatin immunoprecipitation analysis showed that FUS-DDIT3 binds the IL8 promoter. Expression studies of the IL8 promoter harboring a C/EBP-NF-kappaB composite site pinpointed the importance of NF-kappaB for IL8 expression in FUS-DDIT3-expressing cells. We therefore probed for possible interaction of FUS-DDIT3 with members of the NF-kappaB family. The nuclear factor NFKBIZ colocalizes with FUS-DDIT3 in nuclear structures, and immunoprecipitation experiments showed that FUS-DDIT3 binds the C-terminal of NFKBIZ. We also report that additional NF-kappaB-controlled genes are upregulated at the mRNA level in FUS-DDIT3-expressing cell lines and they can be induced by NFKBIZ. Taken together, the results indicate that FUS-DDIT3 deregulates some NF-kappaB-controlled genes through interactions with NFKBIZ. Similar mechanisms may be a part of the transformation process in other tumor types carrying FUS, EWSR1 and TAF15 containing fusion oncogenes.
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| 6. |
- Olofsson, Anita, 1943, et al.
(författare)
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Abnormal expression of cell cycle regulators in FUS-CHOP carrying liposarcomas.
- 2004
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Ingår i: International journal of oncology. - 1019-6439. ; 25:5, s. 1349-55
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Tidskriftsartikel (refereegranskat)abstract
- Myxoid/round cell liposarcomas (MLS/RCLS) are characterized by chromosome translocations that result in formation of FUS-CHOP or EWSR1-CHOP fusion oncogenes. More than 95% of the tumors carry one of these fusion genes. FUS-CHOP transforms 3T3 cells and causes MLS/RCLS-like tumors in transgenic mice. The fusion oncoproteins act as abnormal transcription factors and are believed to induce abnormal expression of growth controlling genes as part of their transforming activities. The aim of this study was to search for recurrent abnormal expression patterns of cell cycle regulating proteins and growth factor receptors. A series of 14 MLS/RCLS, 2 MLS/RCLS derived cell lines and a FUS-CHOP transfected human sarcoma cell line were analyzed using immunohistochemistry, Western blotting, and cDNA microarray based screening. The results revealed a highly abnormal expression pattern of several growth controlling proteins. The G1 cyclins D1 and E and their associated kinases CDK4 and CDK2 were strongly overexpressed in all of the tumors. High expression levels were also found for Cdk4/6 inhibitor P16 and CDK2 inhibitors P27 and P57. The growth factor tyrosine kinase receptors PDGFRB and EGFR were present in most cells of all investigated tumors. We conclude that deregulation of G1 controlling proteins is common in MLS/RCLS and that aberrant expression of these proteins is of importance in the pathogenesis of this tumor type.
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