The molecular hallmark of the Ewing's family of tumors is the presence of balanced chromosomal translocations, leading to the formation of chimerical transcription factors (that is, EWS/FLI1) that play a pivotal role in the pathogenesis of Ewing's tumors by deregulating gene expression. We have recently demonstrated that DAX1 (NR0B1), an orphan nuclear receptor that was not previously implicated in cancer, is induced by the EWS/ FLI1 oncoprotein and is highly expressed in Ewing's tumors, suggesting that DAX1 is a biologically relevant target of EWS/FLI1-mediated oncogenesis. In this study we demonstrate that DAX1 is a direct transcriptional target of the EWS/FLI1 oncoprotein through its binding to a GGAA-rich region in the DAX1 promoter and show that DAX1 is a key player of EWS/FLI1-mediated oncogenesis. DAX1 silencing using an inducible model of RNA interference induces growth arrest in the A673 Ewing's cell line and severely impairs its capability to grow in semisolid medium and form tumors in immunodeficient mice. Gene expression profile analysis demonstrated that about 10% of the genes regulated by EWS/ FLI1 in Ewing's cells are DAX1 targets, confirming the importance of DAX1 in Ewing's oncogenesis. Functional genomic analysis, validated by quantitative RT-PCR, showed that genes implicated in cell-cycle progression, such as CDK2, CDC6, MCM10 or SKP2 were similarly regulated by EWS/FLI1 and DAX1. These findings indicate that DAX1 is important in the pathogenesis of the Ewing's family of tumors, identify new functions for DAX1 as a cell-cycle progression regulator and open the possibility to new therapeutic approaches based on DAX1 function interference.
Purpose:Tumors of the Ewing family are characterized by chromosomal translocations that yield chimeric transcription factors, such as EWS/FLI1, which regulate the expression of specific genes that contribute to the malignant phenotype. In the present study, we show that cholecystokinin (CCK) is a new target of the EWS/FLI1oncoprotein and assess its functional role in Ewing tumor pathogenesis. Experimental Design: Relevant EWS/FLI1 targets were identified using a combination of cell systems with inducible EWS/FLI1expression, Ewing tumors and cell lines, microarrays, and RNA interference with doxycycline-inducible small hairpin RNA (shRNA) vectors. A doxycyclineinducible CCK-shRNA vector was stably transfected in A673 and SK-PN-DW Ewing cell lines to assess the role of CCK in cell proliferation and tumor growth. Results: Microarray analysis revealed that CCK was up-regulated by EWS/FLI1 in HeLa cells. CCK was overexpressed in Ewing tumors as compared with other pediatric malignancies such as rhabdomyosarcoma and neuroblastoma, with levels close to those detected in normal tissues expressing the highest levels of CCK. Furthermore, EWS/FLI1 knockdown in A673 and SK-PN-DW Ewing cells using two different doxycycline-inducible EWS/FLI1-specific shRNA vectors down-regulated CCK mRNA expression and diminished the levels of secreted CCK, showing that CCK is a EWS/FLI1 specific target gene in Ewing cells. A doxycycline-inducible CCK-specific shRNA vector successfully down-regulated CCK expression, reduced the levels of secreted CCK in Ewing cell lines, and inhibited cell growth and proliferation in vitro and in vivo. Finally, we show that Ewing cell lines and tumors express CCK receptors and that the growth inhibition produced by CCK silencing can be rescued by culturing the cells with medium containing CCK. Conclusions: Our data support the hypothesis that CCK acts as an autocrine growth factor stimulating the proliferation of Ewing cells and suggest that therapies targeting CCK could be promising in the treatment of Ewing tumors.Ewing family of tumors is a group of highly malignant tumors arising mainly in bone that most often affects children and young adults in the first two decades of life. Despite the use of multimodal therapy (chemotherapy, radiation therapy, and surgery), the long-term disease-free survival rate of Ewing tumor patients is still disappointingly low, particularly in the high-risk groups with metastasis at diagnosis or unfavorable localization (reviewed in ref. 1). The lack of new effective drugs in the treatment of Ewing tumors, together with the side effects that high-dose regimens have on young patients who have a long life expectancy, supports the need for innovative therapeutic strategies that should mainly include targeted therapies against molecules critical for the pathogenesis and progression of these tumors.The molecular hallmark of Ewing family of tumors is the presence of balanced chromosomal translocations leading to the formation of chimeric transcription factors. These aberrant...
The Ewing family of tumors harbors chromosomal translocations that join the N-terminal region of the EWS gene with the C-terminal region of several transcription factors of the ETS family, mainly FLI1, resulting in chimeric transcription factors that play a pivotal role in the pathogenesis of Ewing tumors. To identify downstream targets of the EWS/FLI1 fusion protein, we established 293 cells expressing constitutively either the chimeric EWS/ FLI1 or wild type FLI1 proteins and used cDNA arrays to identify genes differentially regulated by EWS/FLI1. DAX1 (NR0B1), an unusual orphan nuclear receptor involved in gonadal development, sex determination and steroidogenesis, showed a consistent up-regulation by EWS/FLI1 oncoprotein, but not by wild type FLI1. Specific induction of DAX1 by EWS/FLI1 was confirmed in two independent cell systems with inducible expression of EWS/ FLI1. We also analyzed the expression of DAX1 in Ewing tumors and derived cell lines, as well as in other nonrelated small round cell tumors. DAX1 was expressed in all Ewing tumor specimens analyzed, and in seven out of eight Ewing tumor cell lines, but not in any neuroblastoma or embryonal rhabdomyosarcoma. Furthermore, silencing of EWS/FLI1 by RNA interference in a Ewing tumor cell line markedly reduced the levels of DAX1 mRNA and protein, confirming that DAX1 up-regulation is dependent upon EWS/FLI1 expression. The high levels of DAX1 found in Ewing tumors and its potent transcriptional repressor activity suggest that the oncogenic effect of EWS/FLI1 may be mediated, at least in part, by the up-regulation of DAX1 expression. ' 2005 Wiley-Liss, Inc.Key words: Ewing tumors; EWS/FLI1 oncoprotein; orphan nuclear receptor DAX1; cDNA arrays Ewing sarcoma and peripheral primitive neuroectodermal tumors (PNETs) are referred to as the Ewing family of tumors, a group of highly malignant tumors arising in children, adolescents and young adults. 1 Ewing tumors are characterized by specific balanced chromosomal translocations, leading to the formation of chimeric proteins that join the N-terminal region from the EWS gene product in frame with the C-terminal portion of out of five different members of the ETS family of transcription factors: FLI1, ERG or FEV (ERG subfamily) and E1AF or ETV1 (PEA3 subfamily). The EWS/FLI1 combination is most frequent and is present in nearly 85% of all cases. 2 A large body of evidence has shown the oncogenic potential of EWS/FLI1 protein. Thus, the ectopic expression of EWS/FLI1 in murine fibroblasts promotes anchorage-independent growth in vitro 3 and accelerates tumorigenesis in vivo. 4 Moreover, EWS/FLI1 knock-down using a variety of techniques inhibits the growth of Ewing tumor-derived cell lines both in vitro and in vivo. 5-9 These data, together with the observation that EWS/ETS chimeric proteins are present in virtually all Ewing tumors, have provided strong evidence that these fusion genes are necessary for the development of these neoplasms. However, EWS/FLI1 appears to be toxic for mouse embryo fibroblasts 10 and p...
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