ABSTRACT
Hypoxia is an important condition in the tumor cell microenvironment and approximately 1% to 1.5% of the genome is transcriptionally responsive to hypoxia with hypoxia-inducible factor-1 (HIF-1) as a major mediator of transcriptional activation. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. Because, by immunohistochemistry, HIF-1alpha expression was readily detectable in 18 of 28 primary Ewing's sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, we studied the effect of hypoxia on ESFT cell lines in vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is upregulated by hypoxia in a HIF-1alpha-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis-related genes, hypoxia stimulated the invasiveness and soft agar colony formation of ESFT cells in vitro. Our data represent the first transcriptome analysis of hypoxic ESFT cells and identify hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity with far-reaching consequences for the malignant properties of ESFT.
Subject(s)
Biomarkers, Tumor/genetics , Bone Neoplasms/genetics , Gene Expression Regulation, Neoplastic/physiology , Hypoxia/metabolism , Oncogene Proteins, Fusion/genetics , Proto-Oncogene Protein c-fli-1/genetics , Sarcoma, Ewing/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Bone Neoplasms/pathology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Profiling , Gene Regulatory Networks , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunoenzyme Techniques , Oligonucleotide Array Sequence Analysis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Protein EWS , Reverse Transcriptase Polymerase Chain Reaction , Sarcoma, Ewing/pathology , Transcriptional ActivationABSTRACT
Ewing's sarcoma family of tumors (ESFT) are a clinically and scientifically very demanding group of tumors in children and young adults with still unknown histogenesis. The rate-limiting oncogenic mutation in this disease has been identified as a chromosomal translocation, t(11;22)(q24;q12), that leads to the expression of a chimeric transcription factor, EWS-FLI1. We have studied the downstream pathway of EWS-FLI1 by a dual strategy including the isolation of direct target genes from ESFT chromatin and the monitoring of transcriptomic changes after silencing of EWS-FLI1 by RNA interference. This study has lead to the identification of several directly EWS-FLI1-regulated genes and the characterization of their genomic distribution. By comparing several ESFT cell lines, not only variation in overall gene expression patterns downstream of EWS-FLIl was observed, but also differential regulation of directly EWS-FLI1-bound genes. Interestingly, there was variation between members of the same functional gene families. Studies on CD99, another diagnostic hallmark of ESFT, in relation to EWS-FLI1 provided additional evidence for context dependence of fusion protein function. Together, our study represents a first approach to the separation of essential molecular consequences from noise generated by the EWS-FLI1 gene rearrangement in ESFT.
Subject(s)
Bone Neoplasms/physiopathology , Oncogene Proteins, Fusion/physiology , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/physiology , RNA-Binding Protein EWS/genetics , Sarcoma, Ewing/physiopathology , Bone Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Humans , Oncogene Proteins, Fusion/genetics , Sarcoma, Ewing/genetics , Translocation, GeneticABSTRACT
The chimeric protein EWS-FLI1, arising from chromosomal translocation in Ewing's sarcoma family tumors (ESFT), acts as an aberrant tumorigenic transcription factor. The transforming activity of EWS-FLI1 minimally requires an ETS DNA binding domain and the EWS NH(2) terminus. Proteins interacting with the EWS portion differ between germ-line and chimeric EWS despite their sharing identical sequences in this domain. We explored the use of the phage display technology to isolate anti-EWS-FLI1 specific single-chain antibody fragments (scFvs). Using recombinant EWS-FLI1 as bait, 16 independent specific antibody clones were isolated from combinatorial phage display libraries, of which six were characterized in detail. Despite differing in their complementarity-determining region sequences, all six scFvs bound to the same epitope spanning residues 51 to 75 within the shared minimal transforming EWS domain. Whereas all six scFvs bound efficiently to cellular EWS, reactivity with ESFT-expressed EWS-FLI1 was weak and restricted to denatured protein. One scFv, scFv-I85, when expressed as an intrabody, efficiently suppressed EWS-dependent coactivation of hepatocyte nuclear factor 4- and OCT4-mediated transcription in vivo but no effect on known EWS-FLI1 target genes was observed. These data suggest that a prominent EWS epitope exposed on recombinant EWS-FLI1 structurally differs between germ-line and chimeric EWS in mammalian cells and that this region is functionally involved in the transcriptional activity of EWS. Thus, we have generated a tool that will prove useful to specifically differentiate between normal and rearranged EWS in functional studies.
Subject(s)
Immunoglobulin Fragments/immunology , Oncogene Proteins, Fusion/immunology , Proto-Oncogene Protein c-fli-1/immunology , Sarcoma, Ewing/genetics , Sarcoma, Ewing/immunology , Amino Acid Sequence , Antibody Specificity , Carcinoma, Hepatocellular , Cell Line, Tumor , Epitopes/immunology , Humans , Immunoglobulin Fragments/chemistry , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/metabolism , Liver Neoplasms , Molecular Sequence Data , Neuroblastoma , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , Protein Binding , Protein Folding , Proto-Oncogene Protein c-fli-1/chemistry , Proto-Oncogene Protein c-fli-1/genetics , Proto-Oncogene Protein c-fli-1/metabolism , RNA-Binding Protein EWS , Transcriptional Activation , TransfectionABSTRACT
In all, 85% of Ewing's sarcoma family tumors (ESFT), a neoplasm of unknown histogenesis, express EWS-FLI1 transcription factor gene fusions. To characterize direct target genes avoiding artificial model systems, we cloned genomic DNA from ESFT chromatin precipitating with EWS-FLI1. We now present a comprehensive list of 99 putative transcription factor targets identified, for the first time, by a hypothesis-free approach based on physical interaction. Gene-derived chromatin fragments co-precipitating with EWS-FLI1 were nonrandomly distributed over the human genome and localized predominantly to the upstream region and the first two introns of the genes. At least 20% of putative direct EWS-FLI1 targets were neural genes. One-third of genes recovered showed a significant ESFT-specific expression pattern and were found to be altered upon RNAi-mediated knockdown of EWS-FLI1. Among them, MK-STYX, encoding a MAP kinase phosphatase-like protein, was consistently expressed in ESFT. EWS-FLI1 was found to drive MK-STYX expression by binding to a single ETS binding motif within the first gene intron. MK-STYX serves as precedence for successful recovery of direct EWS-FLI1 targets from the authentic ESFT cellular context, the most relevant system to study oncogenic mechanisms for the discovery of new therapeutic targets in this disease.
