Research Article EWS-FLI1 Fusion Protein Up-regulates Critical Genes in Neural Crest Development and Is Responsible for the Observed Phenotype of Ewing’s Family of Tumors Siwen Hu-Lieskovan, Jingsong Zhang, Lingtao Wu, Hiroyuki Shimada, Deborah E. Schofield, and Timothy J. Triche Department of Pathology and Laboratory Medicine, Children’s Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, California Abstract Ewing’s family tumors (EFT), a group of poorly differentiated Tumor-specific translocations are common in tumors of pediatric and young adult cancers of bone and soft tissue, which mesenchymal origin. Whether the translocation determines harbors characteristic translocations in virtually all cases. These the phenotype, or vice versa, is debatable. Ewing’s family translocations fuse a heretofore unknown gene, termed EWS,on tumors (EFT) are consistently associated with an EWS-FLI1 chromosome 22, with a member of the ETS family of developmen- translocation and a primitive neural phenotype. Histogenesis tally regulated genes, most commonly FLI1 on chromosome 11 (2). and classification are therefore uncertain. To test whether These fusion proteins function as aberrant transcription factors. EWS-FLI1 fusion gene expression is responsible for the Numerous investigations have now documented the near universal primitive neuroectodermal phenotype of EFT, we established association of one of these translocations with the tumor. a tetracycline-inducible EWS-FLI1 expression system in a The occurrence of EWS-FLI1 (EWS-ETS) translocation(s) has rhabdomyosarcoma cell line RD. Cell morphology changed enabled grouping of a spectrum of seemingly unrelated tumors with after EWS-FLI1 expression, resembling cultured EFT cells. various degrees of neuroectodermal differentiation into one family: Xenografts showed typical EFT features, distinct from tumors from typical undifferentiated Ewing’s sarcoma to poorly differenti- formed by parental RD. Neuron-specific microtubule gene ated atypical Ewing’s sarcoma to differentiated peripheral primitive MAPT, parasympathetic marker cholecystokinin, and epithelial neuroectodermal tumor (pPNET). The cell lineage that EFT marker keratin 18 were up-regulated. Conversely, myogenesis originates from is still somewhat enigmatic. However, a parasym- was diminished. Comparison of the up-regulated genes in RD- pathetic neural crest origin has been suggested because some of the EF with the Ewing’s signature genes identified important EWS- tumors express limited degree of neural markers (e.g., cholecysto- FLI1 downstream genes, many involved in neural crest kinin [CCK]; ref. 3) and they can be induced to undergo neural differentiation. These results were validated by real-time differentiation by various differentiating agents (4, 5). reverse transcription-PCR analysis and RNA interference EWS-FLI1 has been considered a traditional ‘‘oncogene’’ (i.e., pro- technology using small interfering RNA against EWS-FLI1 moting the proliferation and blocking the differentiation of a com- breakpoint. The present study shows that the neural phenotype mitted neural crest precursor cell). Indeed, early experiments that of Ewing’s tumors is attributable to the EWS-FLI1 expression down-regulated expression of the chimeric gene resulted in dimin- and the resultant phenotype resembles developing neural crest. ished proliferation (6). However, it was later discovered that simple Such tumors have a limited neural phenotype regardless of transfection of the EWS-FLI1 gene was generally lethal and certainly tissue of origin. These findings challenge traditional views of did not accelerate cell proliferation (7, 8). This in fact has been true of histogenesis and tumor origin. (Cancer Res 2005; 65(11): 4633-44) most such chimeric oncogenes when simply transfected into normal or tumor cell backgrounds and indicates that secondary genetic Introduction alterations are required for EWS-FLI1–mediated transformation. Mesenchymal tumors often harbor characteristic chromosome In contrast to its role in oncogenesis, EWS-FLI1 seems to inhibit translocations (1). The consistency and tumor specificity of these tissue-specific differentiation. Forced EWS-FLI1 expression translocations imply a close relation between the fusion proteins as a inhibited osteogenic and adipogenic differentiation in marrow result of the translocations and certain tumor phenotypes. One stromal cells (9), myogenic differentiation in C2C12 cells (10), and possible explanation is that a given translocation can only occur in a sympathetic neural differentiation in neuroblastoma cells (11). certain determined cell lineage where the right cellular background Interestingly, tumors formed by EWS-FLI1–transformed NIH3T3 exists to tolerate and cooperate with the fusion protein. Alterna- cells, an immortalized murine fibroblast line, acquired a certain tively, distinct fusions may occur in common multipotent undiffer- degree of neural features and a small round cell morphology, which entiated precursor cells and influence cell development, is typical of EFT but distinct from fibrosarcomas (12). This suggests subsequently driving the cells towards different phenotypes or a possible role of EWS-FLI1 in inhibiting tissue-associated lineages. A good model to investigate these possibilities is the differentiation but promoting an Ewing-specific neurectodermal differentiation program in these tumors. Further evidence is a group of biphenotypic soft tissue sarcomas. They contain the same Note: Supplementary data for this article are available at Cancer Research Online EWS-FLI1 or EWS-ERG fusions and manifest a lesser degree of (http://cancerres.aacrjournals.org/). Requests for reprints: Timothy J. Triche, Department of Pathology and Laboratory myogenic differentiation than rhabdomyosarcoma with no trans- Medicine, Children’s Hospital Los Angeles, Keck School of Medicine, University of locations whereas displaying some neural features (13–15). Southern California, Box 43, 4650 Sunset Boulevard, Los Angeles, CA 90027. Phone: 323-669-4516; Fax: 323-667-1123; E-mail: [email protected]. In this study, we established a tetracycline-regulated EWS-FLI1 I2005 American Association for Cancer Research. expression model in RD, an embryonal rhabdomyosarcoma cell line www.aacrjournals.org 4633 Cancer Res 2005; 65: (11). June 1, 2005 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research with marked myogenic differentiation, to test the hypothesis that PCR was done as described before (17). PCR conditions were 95jC for EWS-FLI1 fusion protein is responsible for the observed primitive 900 seconds, 40 cycles of 95jC for 15 seconds, 55jC (or 60jC) for j j neuroectodermal phenotype of EFT, by the regulation of genes 30 seconds, 72 C for 30 seconds, and a final denaturing stage from 60 C j involved in cell proliferation and differentiation. to 95 C. All PCR products were analyzed on 1% agarose gel and single band was observed except negative controls. The reproducibility was evaluated by at least three PCR measurements. The expression level of target gene was Materials and Methods normalized to internal h-actin and the mean and SD of the target/h-actin Transfection and selection of clones. EWS-FLI1 type I fusion cDNA was ratios were calculated for sample-to-sample comparison. cloned into pcDNA4/TO (Invitrogen, San Diego, CA), a tetracycline-inducible Antibodies and Western blot analysis. Cells were lysed in immuno- expression vector, and named pcDNA4/TO-EF. The construct was verified by precipitation buffer and centrifuged for 15 minutes at 14,000 Â g. DNA sequencing. Effectene (Qiagen, Chatsworth, CA) was used for all trans- Solubilized proteins in the supernatant were quantified using bicincho- fections following manufacturer’s suggestions. To establish the tetracycline- ninic acid protein assay (Bio-Rad, Richmond, CA). Sixty micrograms of regulated system (TREx, Invitrogen), RD cells were first stably transfected total cellular protein were loaded per lane, separated by precast SDS-PAGE with pcDNA6/TR and monoclones were selected and maintained in (Invitrogen), and transferred to a polyvinylidene difluoride membrane tetracycline-free medium containing 5 Ag/mL Blasticidin (Invitrogen). The (Millipore, Bedford, MA). Blots were blocked with 0.5% I-Block in PBS inducibility of each clone was tested by transient transfection with pcDNA4/ (pH 8.0), with 0.1% Tween 20 before addition of primary antibodies, and TO/LacZ and stained for h-gal (Invitrogen). Two of 40 pcDNA6/TR clones horseradish peroxidase–conjugated secondary antibodies (Santa Cruz were chosen for second stable transfection with pcDNA4/TO-EF. Tetracy- Biotechnology,SantaCruz,CA).Boundsecondaryantibodieswere cline-free medium containing 5 Ag/mL Blasticidin and 400 Ag/mL Zeocin detected using an enhanced chemiluminescence system (Amersham (Invitrogen) was used for selecting and maintaining monoclones. Induction Pharmacia Biotech, Piscataway, NJ). Monoclonal antibodies against FLI1, of EWS-FLI1 was accomplished by adding 1 Ag/mL tetracycline to media and MyoD, and Myogenin were from BD PharMingen (San Diego, CA) and tested by reverse transcription-PCR (RT-PCR) and Western blot. MAPT from Neomarkers (Fremont, CA). Polyclonal h-actin antibody was Tumors and cell lines. Frozen primary tumor tissues were obtained from Santa Cruz Biotechnology. before therapy from Children’s Hospital Los Angeles (CHLA). All cell lines Xenograft experiments. Four-