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

Abstract

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...

Cholecystokinin Down-Regulation by RNA Interference Impairs Ewing Tumor Growth

“…We stably transfected HeLa TeT-On cells, which express high levels of the reverse tetracyclinecontrolled transactivator, with a tetracycline-responsive expression vector encoding the EWS/FLI1 type 1 cDNA (HeLa TeT-E/F) or with empty vector (HeLa pTET) as a control. In this manner, EWS/FLI1 expression may be induced in HeLa TeT-E/F cells by tetracycline, or its analogue doxycycline, in a dose-and time-dependent fashion (19). RNA from HeLa pTeT and HeLa…”

“…Cloning of EWS/FLI1 and FLI1 cDNAs in expression vectors and establishment of cell lines are described in detail elsewhere (19). Briefly, we used two different cellular systems to achieve inducible and regulated expression of EWS/FLI1 and native FLI1.…”

NR0B1 Is Required for the Oncogenic Phenotype Mediated by EWS/FLI in Ewing's Sarcoma

“…NR0B1 is an orphan nuclear receptor that is mutated in adrenal hypoplasia congenita and hypogonadotropic hypogonadism (12). Interestingly, NR0B1 has recently been suggested to be an EWS/FLI target gene, however, the functional relevance of this observation was not reported (16). Because NR0B1 expression most correlated to EWS/FLI knock-down in our system, we analyzed the function of NR0B1 in the development of Ewing's sarcoma.…”

Array CGH and gene-expression profiling reveals distinct genomic instability patterns associated with DNA repair and cell-cycle checkpoint pathways in Ewing's sarcoma

“…Previous works have focused their research on array expression profiles of ES cell lines and primary tumours in order to identify tumour classification profiles, new EWS/FLI1 targets or to reveal distinct expression signatures associated to different outcomes (Khan et al, 2001;Ohali et al, 2004;Bandres et al, 2005;Mendiola et al, 2006). A more recent genomic approach is the use of array-based comparative genomic hybridization (aCGH), which has been successfully used for the detection of genomic imbalances in human tumours (Pinkel and Albertson, 2005) in a most precise manner than chromosome-based conventional techniques.…”