Tumor-associated chromosomal translocations lead to the formation of chimeric fusions between the EWS gene and one of ®ve dierent ETS transcription factors in Ewing's family tumors (EFTs). The resultant EWS/ETS proteins promote oncogenesis in a dominant fashion in model systems and are necessary for continued growth of EFT cell lines. EWS belongs to a family of genes that encode proteins that may serve as adapters between the RNA polymerase II complex and RNA splicing factors. EWS/ETS fusions have biochemical characteristics of aberrant transcription factors and appear to promote abnormal cellular growth by transcriptionally modulating a network of target genes. Early evidence suggests that EWS/ETS proteins may also impact gene expression through alteration in RNA processing. Elucidation of EWS/ETS target gene networks in the context of other signaling pathways will hopefully lead to biology based therapeutic strategies for EFT. Oncogene (2001) 20, 5747 ± 5754.

EWS/FLI1, a fusion gene found in Ewing's sarcoma, encodes a transcriptional regulator and promotes cellular transformation by modulating the transcription of specific target genes. We have found that EWS/FLI1 and structurally related fusion proteins upregulate manic fringe (MFNG), a recently described member of the Fringe gene family instrumental in somatic development. MFNG is also expressed in human tumour-derived cell lines expressing EWS/FLI1. Overexpression of MFNG in NIH 3T3 cells renders them tumorigenic in mice with severe combined immunodeficiency disease (SCID). These data demonstrate that part of the oncogenic effect of EWS/FLI1 is to transcriptionally deregulate a member of a family of morphogenic genes.

Ewing's sarcomas express chimeric transcription factors resulting from a fusion of the amino terminus of the EWS gene to the carboxyl terminus of one of ®ve ETS proteins. While the majority of tumors express EWS/ FLI1 fusions, some Ewing's tumors contain variant chimeras such as EWS/ETV1 that have divergent ETS DNA-binding domains. In spite of their structural dierences, both EWS/ETS fusions up regulate EAT-2, a previously described EWS/FLI1 target gene. In contrast to EWS/FLI1, NIH3T3 cells expressing EWS/ ETV1 cannot form colonies in soft agar though coexpression of a dominant negative truncated ETV1 construct attenuates EWS/FLI1 mediated anchorage independent growth. When EWS/ETV1 or EWS/FLI1 expressing NIH3T3 cells are injected into SCID mice, tumors form more often and faster than with NIH-3T3 cells with empty vector controls. The tumorigenic potency of each EWS/ETS fusion is linked to its Cterminal structure, with the FLI1 C-terminus confering a greater tumorigenic potential than the corresponding ETV1 domain. The resulting EWS/ETV1 and EWS/ FLI1 tumors closely resemble each other at both a macroscopic and a microscopic level. These tumors dier greatly from tumors formed by NIH3T3 cells expressing activated RAS. These data indicate that in spite of their structural dierences, EWS/ETV1 and EWS/FLI1 promote oncogenesis via similar biologic pathways.

The EWS/FLI1 fusion gene found in Ewing's sarcoma and primitive neuroectodermal tumor, is able to transform certain cell lines by acting as an aberrant transcription factor. The ability of EWS/FLI1 to modulate gene expression in cells transformed and resistant to transformation by EWS/FLI1, was assessed by Representational Di erence Analysis (RDA). We found that the cyclin selective ubiquitin conjugase murine E2-C, was up regulated in NIH3T3 cells transformed by EWS/FLI1 but not in a nontransformed NIH3T3 clone expressing EWS/FLI1. We also found that mE2-C is upregulated in NIH3T3 cells transformed by other genes including activated cdc42, v-ABL and c-myc. We demonstrated that expression of mE2-C in both the EWS/FLI1 transformed and parent NIH3T3 lines varies with the cell cycle. Finally, dominant-negative mE2-C, created by changing a catalytic cysteine to serine, inhibits the in vitro ubiquitination and degradation of cyclin B in human HeLa cell extracts. These data suggest that part of the biologic e ect of EWS/FLI1 could be to transcriptionally modulate genes involved in cell cycle regulation.

Specific chromosomal translocations involving the ews gene and one of five members of the ets family of transcription factors create ews/ets fusion genes that are found in ϳ85% of Ewing's family of tumors. ews/ets fusion genes consistently maintain an intact and functional ets DNA binding domain (DBD) in all of these cases. We demonstrate here, however, that EWS/FLI1, the most prevalent EWS/ETS fusion, activates oncogenic pathways independent of its DBD. In in vivo tumor assays, EWS/FLI1 molecules with either point mutations or a large deletion in the ets DBD retain the ability to accelerate tumors in NIH 3T3 cells, whereas they lose the ability to bind DNA in vitro. Additionally, whereas inhibition of DBD functions of EWS/FLI1 with a dominant negative form of FLI1 is sufficient to inhibit anchorage-independent growth in NIH 3T3 cells, it is ineffective in inhibiting tumor growth in SCID mice. Usage of this dominant negative construct in a Ewing's tumor cell line, however, does reduce the rate of tumor formation, supporting the need for a functional DBD in this context. Together, these results suggest that EWS/FLI1 induces both DBD-dependent and DBD-independent oncogenic pathways.The production of chimeric fusion genes through chromosomal translocation is a common theme in the development of neoplasia. The resultant gene products often utilize domainassociated functions derived from the native participants of the fusions but in novel, atypical contexts. Such is the case for the Ewing's family of tumors, in which the ews gene on chromosome 22 is fused to one of five members of the ets family of transcription factors (FLI1, ERG, ETV1, E1AF, or FEV) (1). The consequent ews/ets fusion genes are thought to utilize amino-terminal EWS transactivation domains and intact carboxyl-terminal ets DNA binding domains (DBDs) 1 to form aberrant transcription factors that induce neoplastic phenotypes through altered gene regulation (2).EWS is a member of the TET family of RNA-binding proteins, which also includes TLS and TAF II 68 (3). These genes are commonly involved in chromosomal translocations that lead to the development of cancer, particularly sarcomas. In addition to Ewing's sarcoma, TET translocations are associated with at least five other sarcomas including desmoplastic small round cell tumors, clear cell sarcoma, and myxoid liposarcoma (1). A pervasive feature of all of the TET translocations is that a TET family member is fused to an intact DBD of a transcription factor. This observation supports the hypothesis that TET translocation products, including EWS/ETS fusions, utilize their DBDs to bind DNA and directly alter transcriptional activity.Although the physiological functions of wild type EWS are not well defined, EWS may have a role in coupling transcription and RNA processing. The amino-terminal transcriptional activation domain of EWS includes protein-protein interaction domains that direct binding to RNA polymerase II subunits (4, 5). In addition, an interaction with splicing factor U1C has been mapped to ...