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

Abstract

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.Ewing's family tumor (EFT) is a rare childhood cancer arising mainly in bone and soft tissue. Since EFT has a poor prognosis, it is important to elucidate the underlying pathogenic mechanisms for establishing a more effective therapeutic strategy. EFT is characterized by the presence of chimeric genes composed of EWS and ets transcription factor genes (ETS) formed by specific chromosomal translocations, i.e., EWS/FLI1, t(11;22)(q24;q12); EWS/ERG, t(21;22)(q12;q12); EWS/ETV1, t(7;22)(p22;q12); EWS/E1AF, t(17;22)(q12;q12); and EWS/FEV, t(2;22)(q33;q12) (26). The products of these chimeric genes behave as aberrant transcriptional regulators and are believed to play a crucial role in the onset and progression of EFT (3,36). Indeed, recent studies have revealed that the induction of EWS/FLI1 proteins can trigger transformation in certain cell types, including NIH 3T3 cells (36), C2C12 myoblasts (12), and murine primary bone marrow-derived mesenchymal progenitor cells (MPCs) (6, 45, 52). However, studies have also indicated that overexpression of EWS/ FLI1 provokes apoptosis and growth arrest in mouse normal embryonic fibroblasts and primary human fibroblasts (10, 31), hence hampering understanding of the precise role of EWS/ ETS proteins in the development of EFT. The function of EWS/ETS proteins would be greatly influenced by cell type, and thus the cells that can originate EFTs might be more susceptible to the tumorigenic effects of EWS/ETS.Although the cell origin of EFT is still unknown, the expression of neuronal markers in spite of the occurrence in bone and soft tissues has kept open the debate as to a potential mesenchymal or neuroectodermal origin. As described ...

EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation

“…During the preparation of our manuscript, we noticed 2 recent publications that investigated the overexpression of EWS/FLI1 in human MSCs (32,33). Both observed that this overexpression did not transform MSC and lead to tumorigenicity.…”

Characterization of Ewing sarcoma associated cancer/testis antigens

“…Expression data were obtained from the Gene Expression Omnibus (GEO) database. 42 The used data sets 19,[43][44][45][46][47][48][49][50][51][52][53][54][55] are summarized in Table 1. Primary data analysis was performed with Expression Console (Affymetrix) applying the Microarray Suite 5 algorithm.…”

“…In order to investigate the impact of EFT-specific fusion proteins on the expression of CTA, GEO data sets from EWS-ETS transgenic MSC were analyzed. 19,42 Expression data sets from EWSR1-FLI1 transgenic MSC and expression data sets from EWSR1-ERG transgenic MSC were analyzed. Probe sets with signal intensities below 100 in the experimental situations were excluded from analysis.…”

Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles

“…To analyze the data, we used agglomerative hierarchical clustering and principal component analysis (PCA) using NIA Array Analysis Tools provided by the National Institute on Aging (Bethesda, MD). 11 Reverse Transcriptase-PCR RNA was converted to cDNA and amplified using the Titan One Tube RT-PCR System (Roche Diagnostics, Indianapolis, IN). The products were resolved by electrophoresis on a 1.5% agarose gel and visualized with ethidium bromide.…”