The<b><i>Ews/Fli-1</i></b>Fusion Gene Switches the Differentiation Program of Neuroblastomas to Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumors

Rorie, Checo J.; Thomas, Venetia; Chen, Pengchin; Pierce, Heather; O’Bryan, John P.; Weissman, Bernard E.

doi:10.1158/0008-5472.can-03-3274

Cited by 92 publications

(63 citation statements)

References 75 publications

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“…A further hallmark of ESFT is a consistent high-level expression of the cellsurface glycoprotein CD99 (Kovar et al, 1990;Ambros et al, 1991). Introduction of EWS-FLI1 into neuroblastoma or rhabdomyosarcoma cells turns on CD99 expression (Rorie et al, 2004;Hu-Lieskovan et al, 2005) while silencing of EWS-FLI1 in ESFT cells does not affect high-level CD99 expression (our unpublished results). Thus, the functional relation between EWS-FLI1 and CD99 in ESFT remains unclear.…”

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confidence: 59%

Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing's sarcoma cells

et al. 2005

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High CD99 expression levels and rearrangements of the EWS gene with ETS transcription factor genes characterize the Ewing's sarcoma family of tumors (ESFT). CD99 is a cell surface glycoprotein whose engagement has been implicated in cell proliferation as well as upregulation and transport of several transmembrane proteins in hematopoietic cells. In ESFT, antibody ligation of CD99 induces fast homotypic cell aggregation and cell death although its functional role in these processes remains largely unknown. Here, using an RNAi approach, we studied for the first time the consequences of modulated CD99 expression in six different ESFT cell lines, representing the most frequent variant forms of EWS gene rearrangement. CD99 suppression resulted in growth inhibition and reduced migration of ESFT cells. Among genes whose expression changes in response to CD99 modulation, the potassium-channel modulatory factor KCMF1 was consistently upregulated. In a series of 22 primary ESFT, KCMF1 expression levels inversely correlated with CD99 abundancy. Cells forced to express ectopic KCMF1 showed a similar reduction in migratory ability as CD99 silenced ESFT cells. Our results suggest that in ESFT, high CD99 expression levels contribute to the malignant properties of ESFT by promoting growth and migration of tumor cells and identify KCMF1 as a potential metastasis suppressor gene downregulated by high constitutive CD99 expression in ESFT.

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confidence: 59%

Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing's sarcoma cells

et al. 2005

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“…In the last years, several studies have also used microarrays to identify EWS/FLI1 target genes, although none of them reported DAX1 as being up-regulated by EWS/ FLI1. 9,11,[34][35][36] These discrepancies could be a consequence of the technical differences between the platforms employed (cDNA arrays versus oligonucleotide microarrays), different methods of data normalization, different criteria to select the most relevant genes and mainly, differences among the cellular systems used in each study. DAX1 (NR0B1) is an unusual orphan nuclear receptor that was isolated as the gene responsible for the X-linked adrenal hypoplasia congenita (AHC), 37 a genetic disease in which patients display adrenal insufficiency and electrolyte imbalance as a consequence of inactivating mutations in the DAX1 gene.…”

Section: Discussionmentioning

confidence: 99%

The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors

Mendiola¹,

Carrillo²,

García³

et al. 2005

Int. J. Cancer

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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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“…19 and human cancer cell lines. [20][21][22] The most extensively used cell line for the analysis of EWS-ETS function has been NIH3T3 immortalized murine fibroblast cells. 23 NIH3T3 cells have been used to demonstrate the oncogenic potential of EWS-ETS fusions, 8 to study the histologic changes induced by the fusion proteins, 24 to determine EWS-ETS gene targets, 25 and to analyze structure-function relationships within the chimeric oncoproteins.…”

Section: Introductionmentioning

confidence: 99%

Expression of EWS-ETS Fusions in NIH3T3 Cells Reveals Significant Differences to Ewing’s Sarcoma

et al. 2006

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Ewing's sarcomas contain specific chromosomal translocations that fuse EWS to ETS family members, including FLI, ERG, FEV, ETV1 and ETV4. Prior work has suggested that functional differences exist between some of these EWS-ETS fusions. However, as the cell of origin of Ewing's sarcoma is unknown, this prior work was conducted in NIH3T3 cells, which have not been validated as an appropriate model for the study of EWS-ETS fusions. To determine if NIH3T3 cells are a good model for Ewing's sarcoma, we introduced all five EWS-ETS fusions into these cells, and analyzed their phenotypes and gene expression patterns. EWS-FLI, EWS-ERG, and EWS-FEV caused NIH3T3 cells to exhibit anchorage independent growth whereas EWS-ETV1 and EWS-ETV4 did not. In contrast, all the EWS-ETS fusions induced tumor formation in a xenograft model. We defined the core transcriptional profile of the EWS-ETS fusions using cDNA microarrays, and compared these to data derived from patient-derived Ewing's sarcoma cell lines. The NIH3T3 model did not recapitulate the gene expression pattern of bona fide Ewing's sarcoma. Based on these results, we conclude that while there may be functional differences between the various EWS-ETS fusions, the NIH3T3 cell model is inadequate to study the gene expression pattern induced by EWS-ETS proteins in Ewing's sarcoma. Thus, data derived from the NIH3T3 model system needs to be appropriately validated before they can be accepted as relevant to the human disease.

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TheEws/Fli-1Fusion Gene Switches the Differentiation Program of Neuroblastomas to Ewing Sarcoma/Peripheral Primitive Neuroectodermal Tumors

Cited by 92 publications

References 75 publications

Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing's sarcoma cells

Suppression of KCMF1 by constitutive high CD99 expression is involved in the migratory ability of Ewing's sarcoma cells

The orphan nuclear receptor DAX1 is up-regulated by the EWS/FLI1 oncoprotein and is highly expressed in Ewing tumors

Expression of EWS-ETS Fusions in NIH3T3 Cells Reveals Significant Differences to Ewing’s Sarcoma

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