Variability in Gene Expression Patterns of Ewing Tumor Cell Lines Differing in EWS-FLI1 Fusion Type

Abstract

Type 1 and type 2 EWS-FLI1 fusion products result from variation in breakpoint locations arising from the t(11;22)(q24;q12) recurrent chromosomal translocation in Ewing's sarcoma family tumors (EFT). Previously, studies from our institution (updated in the present communication at a median follow-up of more than 6 years) and others suggested a prognostic difference for EFT patients with localized disease depending on the type of EWS-FLI1 fusion present in the tumor. It has been suggested that the observed clinical discrepancies result from different transactivation potentials of the various EWS-FLI1 fusion proteins. In an attempt to identify genes whose expression levels are differentially modulated by structurally different EWS-FLI1 transcription factors, we have used two related PCR-based subtractive approaches, cDNA representational difference analysis (cDNA-RDA) and linker-capture subtraction (LCS) to compare transcript representations in cDNA pools of type 1 versus type 2 EFT cell lines. About 800 clones obtained by the two approaches were analyzed by dot blot hybridization to cDNA pools. Eighty-six clones showing the highest variability in signal intensities on the dot blots were further hybridized to individual EFT cell line RNAs on Northern blots, and four of them were additionally studied by real-time quantitative PCR (RTQ-PCR). Although interindividual variations in gene expression patterns in the range of one- to several-fold were observed, no correlation to specific EWS-FLI1 fusion types could be identified. Among the genes differentially expressed in individual EFT cell lines are several previously implicated in tumor growth, invasion, and metastasis. Although our data may have revealed candidate genes whose composite expression pattern may be relevant for the biology of individual EFT, they do not support a role of distinct EWS-FLI1 fusion types for EFT prognosis based on different transactivation potentials.