Functional characterization of the liopsarcoma-associated fusion oncigene FUS-DDIT3

Sammanfattning: 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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