EWS/ETS Regulates the Expression of the Dickkopf Family in Ewing Family Tumor Cells

BACKGROUND: The Dickkopf (DKK) family comprises a set of proteins that function as regulators of Wnt/beta-catenin signaling and has a crucial role in development. Recent studies have revealed the involvement of this family in tumorigenesis, however their role in tumorigenesis is still remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: We found increased expression of DKK2 but decreased expression of DKK1 in Ewing family tumor (EFT) cells. We showed that EFT-specific EWS/ETS fusion proteins enhance the DKK2 promoter activity, but not DKK1 promoter activity, via ets binding sites (EBSs) in the 5' upstream region. EWS/ETS-mediated transactivation of the promoter was suppressed by the deletion and mutation of EBSs located upstream of the DKK2 gene. Interestingly, the inducible expression of EWS/ETS resulted in the strong induction of DKK2 expression and inhibition of DKK1 expression in human primary mesenchymal progenitor cells that are thought to be a candidate of cell origin of EFT. In addition, using an EFT cell line SK-ES1 cells, we also demonstrated that the expression of DKK1 and DKK2 is mutually exclusive, and the ectopic expression of DKK1, but not DKK2, resulted in the suppression of tumor growth in immuno-deficient mice. CONCLUSIONS/SIGNIFICANCE: Our results suggested that DKK2 could not functionally substitute for DKK1 tumor-suppressive effect in EFT. Given the mutually exclusive expression of DKK1 and DKK2, EWS/ETS regulates the transcription of the DKK family, and the EWS/ETS-mediated DKK2 up-regulation could affect the tumorigenicity of EFT in an indirect manner

Inducible Expression of Chimeric EWS/ETS Proteins Confers Ewing's Family Tumor-Like Phenotypes to Human Mesenchymal Progenitor Cells▿ †

Ewing's family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlled EWS/ETS-inducible system in human bone marrow-derived mesenchymal progenitor cells (MPCs). Ectopic expression of both EWS/FLI1 and EWS/ERG proteins resulted in a dramatic change of morphology, i.e., from a mesenchymal spindle shape to a small round-to-polygonal cell, one of the characteristics of EFT. EWS/ETS also induced immunophenotypic changes in MPCs, including the disappearance of the mesenchyme-positive markers CD10 and CD13 and the up-regulation of the EFT-positive markers CD54, CD99, CD117, and CD271. Furthermore, a prominent shift from the gene expression profile of MPCs to that of EFT was observed in the presence of EWS/ETS. Together with the observation that EWS/ETS enhances the ability of cells to invade Matrigel, these results suggest that EWS/ETS proteins contribute to alterations of cellular features and confer an EFT-like phenotype to human MPCs

The expression pattern of the DKK family in Ewing's family tumor (EFT).

<p>A, B, Real-time RT-PCR analysis using pediatric tumor cell lines for DKK1 (A) and DKK2 (B) expression. EFT: Ewing's family tumor, NB: neuroblastoma, BL: Burkitt lymphoma, RMS: Rhabdomyosarcoma, EC: embryonal carcinoma. Data are normalized to the mRNA level in SCCH196 (for DKK1) and Ramos (for DKK2) which is arbitrarily set to 1. Signal intensity was normalized using that of a control housekeeping gene (human GAPDH gene). Data are relative values with the SD for triplicate wells.</p

The effect of ectopic DKK1 expression on tumor cell growth <i>in vivo</i>.

<p>A, Examples of Immuno-deficient mice that have been injected with SK-ES1-DEST (DEST#1) and SK-ES1-DKK1 (DKK1#1) (left panels). The image was taken at 28 days after injection. Right panels indicate the xenografts of mice injected with DEST#1 and DKK1#1. Red circles indicate the positions of tumors. B, Tumor growth rates from mice injected with DEST#1 and DKK1#1. Diamond symbols indicate the tumor volume of mice injected with DEST#1; Box symbols indicate the tumor volume of mice injected with DKK1#1.</p

The change in expression pattern of the DKK family with EWS/ETS's induction in UET-13 cells.

<p>A, The expression pattern of DKKs in hMPCs and EFT cells. DKK1 and DKK2 mRNAs in the H4-1, UEET-12, UET-13 and EFT cell lines were detected by RT-PCR. As an internal control, a human GAPDH gene was used. B, The expression of DKK2 protein in hMPCs and EFT cells. For the detection of secreted DKK2, cells were cultured for 48 hours and then the culture medium was analyzed by Western blotting. C, D, E, F, Relative level of DKK1 (C, D) and DKK2 (E, F) in UET-13 transfectants in the absence or presence of tetracycline. UET-13 transfectants were treated with or without 3 µg/ml of tetracycline for the indicated periods. Real-time RT-PCR was performed to investigate the expression pattern of the DKK family. Signal intensity was normalized using that of a control housekeeping gene (human GAPDH gene). Data are relative values with the SD for triplicate wells and normalized to the mRNA level at 0 hour which is arbitrarily set to 1.</p