DAX1, a direct target of EWS/FLI1 oncoprotein, is a principal regulator of cell-cycle progression in Ewing's tumor cells

Abstract

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.

Tumorigenic Role of Orphan Nuclear Receptor NR0B1 in Lung Adenocarcinoma

“…Kinsey et al 30 and Garcia-Aragoncillo et al 40 reported that NR0B1 expression is related to cell proliferative activity in Ewing's sarcoma cell lines. In contrast, the knocked-down expression of NR0B1 did not affect the cell proliferation of A549 cells.…”

“…The proportion of CpG methylation was inversely correlated with the NR0B1 expression level in clinical samples. Very recently, Garcia-Aragoncillo et al 40 reported that EWS/FLI1 directly binds to the GGAArich region in NR0B1 promoter and transactivates its activity. It is well known that transcription factors may bind to their cognate recognition sequences when the promoter region is relaxed in the unmethylated state.…”

Axolotl Nanog activity in mouse embryonic stem cells demonstrates that ground state pluripotency is conserved from urodele amphibians to mammals

“…Among the factors identified (Fig. 2E) that are exclusively associated with Nanog dimers, Nac1, Sall4 and Dax1 are known to enhance proliferation in other cell types (Garcia- Aragoncillo et al, 2008;Ma et al, 2009;Yuri et al, 2009), and we presume that their association with Nanog in ESCs plays a similar role. Interestingly, the WR domain is exclusive to eutherian mammals (see Fig.…”

Role of Orphan Nuclear Receptor DAX-1/NR0B1 in Development, Physiology, and Disease

“…In Ewing tumors, the EWS/FLI1 oncoprotein directly regulates DAX-1 expression by binding to a polymorphic GGAA-rich region in its promoter [160,161]. Moreover, DAX-1 expression levels in Ewing tumor cell lines correlated positively with the number of GGAA repeats present in their DAX-1 promoter.…”

“…DAX-1 was also reported to physically interact with and modulate the transcriptional activity of the EWS/FLI1 oncoprotein [59]. DAX-1 has an important role in regulating Ewing tumor cells growth since its knockdown blocks cell proliferation in vitro, causing accumulation in the G1 phase of the cell cycle, and significantly impairs the growth of Ewing tumor cells grafted in immunodeficient mice [160]. Importantly, the effect of DAX-1 knockdown on Ewing tumor cells proliferation was observed in the presence of continued expression of the EWS/FLI-1 oncoprotein, indicating the main role of DAX-1 in mediating EWS/FLI1 effects.…”