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

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- to 6-week-old severe combined immu- were obtained from our cell bank and cultured in RPMI with 10% fetal nodeficient mice were injected s.c. with 2 Â 106 RD, TC32, or RD-EF leaky bovine serum (Invitrogen). Fusion gene status was tested by RT-PCR. cells expressing comparable levels of EWS-FLI1 as EFT cell lines (n = 5). Among the 16 EFT, 36 rhabdomyosarcoma, 10 neuroblastoma, and 20 Tumor growth was monitored over time, and the mice were sacrificed when osteosarcoma samples used for gene expression analysis, EFT samples the tumors reach 1.5 cm in diameter. EF expression in the tumors was express either EWS-FLI1 or EWS-ERG, whereas the alveolar rhabdomyo- confirmed by quantitative reverse transcription-PCR (QRT-PCR). sarcoma samples express either PAX3-FKHR or PAX7-FKHR. TC32 and Electron microscopy. Electron microscopy (EM) was carried out by the TC71 are EFT lines with type I EWS-FLI1 fusion. RD is an embryonal standard procedure at the CHLA EM laboratory. Briefly, about 1-mm cubes rhabdomyosarcoma line. CHP126 is a neuroblastoma line. from xenograft tumor samples were fixed with 2% glutaraldehyde in Microarray analysis. For tumor samples, tissues that had >90% tumor phosphate buffer (pH 7.4), post-fixed with 1% osmium tetroxide, and cells were chosen. For cultured cell lines, RNA was harvested when cell embedded in epon (Embed-812, Electron Microscopy Sciences, Hatfield, PA). confluence was 50% to 60%. Total RNA were extracted (RNA STAT-60, Tel- One-micrometer-thick sections, cut from the hardened epon blocks and Test), cleaned (RNeasy mini kit, Qiagen), and quantitated. Synthesis of stained with 1% methylene blue and 0.5% basic fuchsin, were examined cDNA, biotin-labeled cRNA, fragmentation, target hybridization, washing, under the light microscope before ultrastructural examination. Ultrathin staining, and scanning probe arrays followed Affymetrix’s manual at CHLA sections from the areas of interest were mounted on one-hole grids, stained microarray core facility. The Affymetrix HU95av2 arrays contain probes for with uranyl acetate/lead citrate, and examined and photographed with a f12,600 human full-length genes and ESTs. Chip performance, background Philip CM-12 transmission electron microscope. levels and presence/absence calls were first assessed using Microarray Suite Immunohistochemistry. Paraffin-embedded tissue blocks were sec- software (Affymetrix, Santa Clara, CA). Cell files were normalized with tioned (4 Am) and deparaffinized. Sections were boiled 15 minutes in H2O ProbeProfiler software (Corimbia, Berkeley, CA) as described before (16). for antigen retrieval, quenched with 3% hydrogen peroxide for 5 minutes. Bioinformatical analyses were done with Genetrix analysis software A M.O.M. Immunodetection Kit (Vector Laboratories, Burlingame, CA) was (Epicenter Software, Pasadena, CA). used for detecting monoclonal mouse anti-human antibodies (MyoD1, Real-time quantitative reverse transcription-PCR. Total cellular RNA DAKO, Carpinteria, CA; Myogenin, DAKO; Muscle Actin, Cell Marque, Hot was isolated using RNA STAT-60 (Tel-Test) when cells reach 50% to 60% Springs, AR; Desmin, DAKO; MIC2, DAKO; and TAU, Neomarkers) confluence. cDNA was synthesized from 2 Ag of DNase I (Invitrogen)– following manufacturer’s protocol. A Vectastain Elite ABC Kit was used treated total RNA in a 42-AL reaction volume using oligo-dT and Superscript for polyclonal rabbit anti-human antibodies (CCK, Neomarkers). Slides II (Invitrogen) for 60 minutes at 42jC following suppliers’ instructions. PCR were incubated in primary antibodies (1:100 dilution) 1 hour followed by primers were designed with MacVector 7.0 (Accelrys, San Diego, CA). The 30 minutes incubation with biotinylated anti-mouse or anti-rabbit IgG sequences are: secondary antibody. Sections were exposed to diaminobenzidine peroxidase

Targets Forward primers Reverse primers

MSX1 5V-TGCTCCAGTTTCACCTCTTTGC-3V 5V-AACCTCTCTGCCCTCAGTTTCC-3V EWS-FLI1 5V-CGACTAGTTATGATCAGAGCAGT-3V 5V-CCGTTGCTCTGTATTCTTACTGA-3V CoREST 5V-ATGGCAACAGCAGCAGCAACTC-3V 5V-GGCAATGGCAATGTATTCATCC-3V NPR 5V-TGCTGGGTCAAGTGTCTCATCATAC-3V 5V-CAAGTGCTGTCACCTCCTTCCTAAG-3V JAK1 5V-CAGGTCTCCCACAAACACATCG-3V 5V-ACCAGGTCTTTATCCTCCAAGTAGC-3V CITED2 5V-TCTGTCTTGGCTTTGGCGTTC-3V 5V-ATTAGGGCGTTGAAGGCGTG-3V MAPT 5V-TGTGGCTCATTAGGCAACATCC-3V 5V-TCTGTCTTGGCTTTGGCGTTC-3V Cyclin D1 5V-CGCACGATTTCATTGAACACTT-3V 5V-CGGATTGGAAATAC TTCACAT-3V Cyclin D3 5V-CCTCTGTGCTACAGATTATACCTTTGC-3V 5V-TTGCAC TGCAGCCCCAAT-3V h-Actin 5V-GCACCCCGTGCT GCTGAC-3V 5V-CAGTGGTACGGCCAGAGG-3V

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Figure 1. A global gene expression change and induction of EFT markers after EWS-FLI1 expression in RD cells. A, Western blot analyses of RD-EF cells after tetracycline or ethanol treatments. The same blots probed with antibodies against FLI1 (top) or Actin (bottom). B, schematic illustration of treatments at different time points for microarray analysis and the number of replicates at each time. E, ethanol treatments; T, tetracycline treatments. C, PCA of induced RD-EF samples and tumor samples. D, expression of known EFT markers and identified EWS-FLI1 targets in RD-EF samples and tumor samples by microarray analysis. Red line, tetracycline-treated samples; green line, ethanol-treated controls.

substrate (Sigma, St. Louis, MO) to give a brown stain and counterstained clone. EWS-FLI1 RNA could be detected as early as 3 hours by with Mayer’s hematoxylin. After washing with PBS and mounted, the QRT-PCR (Supplementary Fig. 1A). Western blotting analysis sections were examined and photographed with a Nikon epifluorescent revealed a rapid induction of EWS-FLI1 proteins as early as microscope. 6 hours and reached the peak level at 36 hours (Fig. 1A). The level RNA interference. Small interfering RNA (siRNA) against EWS-FLI1 of EWS-FLI1 expression induced in the model system is breakpoint (siEFBP2) and a nontargeting control siRNA (C8) were obtained comparable to that in wild-type EFT cell lines (Fig. 3B and D; from Dharmacon Research (Lafayette, CO). Sequence of siEFBP2 was reported before (18). siEFBP2 or C8, complexed with TransIT-TKO (Mirus) Supplementary Fig. 1A). Fluorescent microscopy showed that in Opti-MEM I (Invitrogen) following manufacturer’s directions, were applied induced EWS-FLI1 was localized to nucleus (data not shown). to RD-EF cells or TC32 cells 30% confluent in RPMI with 10% fetal bovine Ethanol (solvent of tetracycline) treatment of RD-EF cells did not serum but without antibiotics in 6-well plates, to give a final concentration of induce EWS-FLI1 expression (Fig. 1A). 100 nmol/L. For RD-EF cells, 12 hours after transfection, the cells were Because EWS-FLI1 functions as an aberrant transcription factor, induced by tetracycline or ethanol for another 24 hours before RNA was to investigate the molecular consequences of EWS-FLI1 expression harvested. For TC32 cells, the same transfection was repeated after 24 hours in RD cells, we undertook a transcriptome-wide gene expression and RNA was harvested after another 24 hours. Gene expression was assessed analysis using Affymetrix HU95av2 gene chips, on samples by QRT-PCR. harvested 0, 6, 12, 18, 24, and 36 hours after tetracycline or ethanol treatments (2-6 biological replicates at each time point, Results Fig. 1B). An additional set of RNA was harvested at 0, 6, 9, 18, 24, Establishment of the RD-EF model system. We expressed the and 36 hours for confirmatory QRT-PCR using h actin as an EWS-FLI1 fusion gene in RD cells by using the TREx system from internal control. We also compared the microarray data of the Invitrogen, which allowed us to identify expression levels that are model system with a pool of data from 83 tumor samples, nonlethal but associated with profound effects on patterns of gene including 16 EFT, 36 rhabdomyosarcoma, 20 osteosarcoma, and expression and cell differentiation. Several stable clones with high 10 neuroblastoma (Fig. 1C). inducibility and low leakage were selected. Among them, B101 EWS-FLI1 expression in RD cells revealed a shift in global (RD-EF) was used for the subsequent experiments because gene expression pattern with induction of EFT markers. To tetracycline treatment led to strong induction of EWS-FLI1 in this provide visual representation of the samples based on gene www.aacrjournals.org 4635 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 expression, we used principal component analysis (PCA; ref. 19) Detailed analysis of the differentially expressed genes revealed to locate the two-dimensional views that capture the greatest several noteworthy genes previously identified as EFT markers amount of variability in the data, using all 12,625 probe sets (CD99, ref. 20; CCK, ref. 3; and STEAP, ref. 21), or EWS-FLI1 interrogating 10,500 genes. Continued expression of EWS-FLI1 downstream genes (c-MYC, ref. 22; ID2, refs. 23, 24; and Cyclin, chimeric gene induced a progressive shift away from both RD cells ref. 25), all highly expressed in EFT, and also highly up-regulated and ethanol-treated controls (Fig. 1C). All of the controls cluster in tetracycline-treated RD-EF cells (Figs. 1D and 2C, m-r). together without an apparent pattern. However, tetracycline- Interestingly, the up-regulation of c-MYC and ID2 (6 hours) treated samples cluster in a distinct pattern that correlates preceded Cyclin D1 (18 hours), which implied that these two directly with the induced time. Longer exposure to tetracycline genes are early response genes and Cyclin D1 is possibly a resulted in a greater accumulation of EWS-FLI1 protein, and secondary target of EWS-FLI1. EWS-FLI1 down-regulates trans- data points separated progressively further from that of the forming growth factor hRII (TGFhRII; ref. 26) and P21 (27). It controls. These data indicated a fundamental difference in gene seemed that ethanol itself could regulate the expression of expression pattern between the EWS-FLI1–expressing cells and TGFhRII. However, tetracycline-treated RD-EF cells showed more the controls. down-regulation than ethanol-treated cells. P21 expression

Figure 2. Cultured RD-EF with EWS-FLI1 expression and xenograft tumors formed by EWS-FLI1 expressing RD showed the phenotype of typical EFT. A, light microscopy revealed a consistent morphological change after EWS-FLI1 induction in RD cells. B, EM study showed that xenograft tumors formed by EWS-FLI1–expressing RD cells lost muscle differentiation features that normally can be found in tumors formed by wild-type RD cells, and acquired certain degree of neural features, such as neurites and dense core granules (dcg). C, H&E staining (a-c) and immunohistochemistry (d-r) of Desmin, muscle-specific actin (HHF35), MAPT, CCK, and CD99 on xenograft tumors formed by RD, EWS-FLI1–expressing RD, or TC32 cells.

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Figure 3. The appearance of an Ewing-like neuroectodermal phenotype at the expense of the myogenic differentiation program in the induced RD-EF cells. A, microarray analyses of MAPT, Keratin 18, and CCK after EWS-FLI1 induction in RD cells. E, ethanol treatments; T, tetracycline treatments. B, Western blot analyses of MAPT protein level after EWS-FLI1 induction in RD cells. C, expression of important muscle differentiation associated genes in RD-EF cells after tetracycline or ethanol treatments by microarray analyses. E, ethanol treatments; T, tetracycline treatments. D, down-regulation of MyoD and Myogenin in tetracycline-treated RD-EF cells by Western blot.

decreased dramatically up to 18 hours. Thus, expression of EWS- marked by the appearance of neuritic processes containing dense FLI1 resulted in a marked shift in global gene expression, with core granules (Fig. 2B). H&E staining of RD-EF xenografts induction and suppression of several known EFT markers and showed a structureless array of biphasic cell population (small EWS-FLI1 target genes. dark and large light types) with scant cytoplasm (Fig. 2C, b), Cultured RD-EF cells and RD-EF xenograft tumors that which is typical of EFT tumors (Fig. 2C, c). On the other hand, express EWS-FLI1 acquired the phenotype of Ewing’s family RD xenografts still showed typical rhabdomyosarcoma tumor tumors. We monitored RD-EF cell morphology preinduction and phenotype (a diffuse infiltrate of small round-to-spindled cells in a post-induction. Figure 2A illustrates the effect of only 24 hours of collagenous stroma, Fig. 2C, a). Clearly, the EWS-FLI1 gene has a expression of EWS-FLI1 in this cellular context. The spindle-shaped potent neural differentiating effect at the expense of the existing RD (typical for cell lines from skeletal muscle lineage) gradually tissue differentiation. This dual effect has not been previously became polygonal with frequent cellular processes. By 24 hours, the recognized. morphology of the tetracycline-treated RD-EF cells resembled EFT Appearance of an Ewing-like neuroectodermal phenotype cells (TC32) far more than untreated RD-EF or wild-type RD cells. was accompanied by diminished myogenic differentiation in Ethanol treatment did not show this effect. This data was in induced RD-EF cells. A genetic basis for these observations was accordance with the observation in C2C12-EF cells (10), which explored by gene expression analysis. Two cytoskeleton structural exhibited a cuboidal appearance after EWS-FLI1 expression, and genes, microtubule associated protein tau (MAPT) and keratin 18, suggested that EWS-FLI1 expression mediated this morphologic were up-regulated >8-fold after EWS-FLI1 expression (Fig. 3A; change of RD. P < 0.001). MAPT is expressed exclusively in the axons of neurons The impression of probable neural induction observed by and promotes microtubule assembly and stability. Both EFT and phase-contrast microscopy, was confirmed by electron microscopy neuroblastoma but not rhabdomyosarcoma express MAPT at high and H&E staining of tumors derived from xenografts of RD cells, levels. Keratin 18 is an epithelial structural protein. The up- TC32 EFT cells, and RD cells expressing EWS-FLI1. RD cells regulation of these two genes implied that EWS-FLI1 induced a normally form bona fide rhabdomyosarcoma tumors with marked neuroectodermal phenotype in mesodermal RD cells, consistent terminal rhabdomyogenesis, whereas EFT cells are largely with the morphologic results reported above. Western blot and undifferentiated with scant neural differentiation marked by immunohistochemical analysis showed that the MAPT protein clusters of cytoplasmic dense core granules. Strikingly, RD cells started to accumulate at 24 hours and reached peak level at that express EWS-FLI1 show near complete suppression of the 48 hours (Figs. 2C, j-l and 3B), which suggested that MAPT takes myogenic phenotype with concomitant neural differentiation, part in the morphologic change induced by EWS-FLI1 expression. www.aacrjournals.org 4637 Cancer Res 2005; 65: (11). June 1, 2005

Table 1. Genes (n = 109) significantly upregulated in RD-EF and also highly expressed in EFT

Affy ID Gene name Symbol P

T6 33113_at Cbp/p300-interacting transactivator CITED2 1.03eÀ12 37724_at V-myc myelocytomatosis viral oncogene homologue (avian) MYC 3.28eÀ10 1973_s_at 9.63eÀ10 41215_s_at Inhibitor of DNA binding 2, ID2 2.80eÀ09 dominant-negative HLH protein 41216_r_at 0.00000001 37393_at Hairy and enhancer of split 1, (Drosophila) HES1 0.00000008 31862_at Wingless-type MMTV integration site family, member 5A WNT5A 0.00000031 1669_at 0.00000074 37651_at REST corepressor RCOR 0.00000099 40144_at Protein tyrosine phosphatase, nonreceptor type substrate 1 PTPNS1 0.000011 1135_at G protein–coupled receptor kinase 5 GPRK5 0.00008 41352_at Sialyltransferase 1 (h-galactoside alpha-2,6-sialyltransferase) SIAT1 0.0001 41126_at Solute carrier family 1, member 4 SLC1A4 0.00036 T12 1916_s_at V-fos FBJ murine osteosarcoma viral oncogene homologue FOS 7.33eÀ12 40049_at Death-associated protein kinase 1 DAPK1 8.83eÀ10 36976_at Cadherin 11, type 2, OB-cadherin (osteoblast) CDH11 0.00000001 2087_s_at 0.00000005 31790_at START domain containing 13 STARD13 0.00000001 36119_at Caveolin 1, caveolae protein, 22 kDa CAV1 0.00000001 38763_at Sorbitol dehydrogenase SORD 0.00000002 38546_at Interleukin 1 receptor accessory protein IL1RAP 0.00000031 1457_at Janus kinase 1 (a protein tyrosine kinase) JAK1 0.00000064 41594_at 0.00000068 36882_at Homeo box D9 HOXD9 0.00000079 36120_at Follicular lymphoma variant translocation 1 FVT1 0.0000013 34877_at Human HepG2 partial cDNA, clone hmd3f07m5 0.0000014 339_at Caveolin 2 CAV2 0.0000015 39382_at Tripartite motif-containing 2 TRIM2 0.0000056 41643_at SMA5 SMA5 0.0000085 41642_at 0.00012 38855_s_at Olfactomedin 1 OLFM1 0.0000099 41717_at Fatty acid desaturase 1 FADS1 0.000011 39372_at 0.000013 41718_g_at 0.000025 41720_r_at 0.000075 41719_i_at 0.0002 39373_at 0.0004 40075_at Synaptotagmin I SYT1 0.000064 35720_at KIAA0893 protein KIAA0893 0.00012 39966_at Chondroitin sulfate proteoglycan 5 (neuroglycan C) CSPG5 0.00015 36502_at PFTAIRE protein kinase 1 PFTK1 0.00017 35785_at GABA(A) receptor–associated protein like 1 GABARAPL1 0.00018 33296_at Likely homologue of mouse glucuronyl C5-epimerase GLCE 0.00023 41691_at KIAA0794 protein KIAA0794 0.00036 39636_at Homo sapiens mRNA full-length insert 0.00039 cDNA clone EUROIMAGE 362780 41242_at UDP-N-acteylglucosamine pyrophosphorylase 1 UAP1 0.00061 35362_at Myosin X MYO10 0.00064 T18

38326_at Putative lymphocyte G0-G1 switch gene G0S2 2.98eÀ13 41435_at Protein tyrosine phosphatase, receptor type, PPFIA3 1.54eÀ10 f polypeptide (PTPRF) 310_s_at Microtubule-associated protein tau MAPT 2.27eÀ10 35334_at Glycogenin 2 GYG2 2.49eÀ10 40199_at Msh homeo box homologue 1 (Drosophila) MSX1 2.55eÀ10 215_g_at 0.0000023

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Table 1. Genes (n = 109) significantly upregulated in RD-EF and also highly expressed in EFT (Cont’d)

Affy ID Gene name Symbol P

37706_at Golgi apparatus protein 1 GLG1 0.00000005 1251_g_at RAP1, GTPase activating protein 1 RAP1GA1 0.00000007 1737_s_at Insulin-like growth factor binding protein 4 IGFBP4 0.00000007 39781_at 3.60eÀ09 39428_at Lymphocyte adaptor protein LNK 0.00000021 40297_at Six transmembrane epithelial antigen of the prostate STEAP 0.0000004 35305_at X-prolyl aminopeptidase (aminopeptidase P) 1, soluble XPNPEP1 0.00000047 37416_at Ras homologue gene family, member H ARHH 0.00000086 39049_at Chromosome 6 open reading frame 9 C6orf9 0.0000012 32225_at ATPase, Na+/K+ transporting, A 1 polypeptide ATP1A1 0.0000018 39751_at Zinc finger, DHHC domain containing 3 ZDHHC3 0.0000025 36330_at Cysteine conjugate-beta lyase; cytoplasmic CCBL1 0.0000029 37861_at CD1E antigen, e polypeptide CD1E 0.000005 38151_at Loss of heterozygosity, 11, chromosomal region 2, gene A LOH11CR2A 0.0000086 32598_at NEL-like 2 (chicken) NELL2 0.0000087 38418_at Cyclin D1 (PRAD1: parathyroid adenomatosis 1) CCND1 0.000011 2020_at 0.000016 2017_s_at 0.000053 32331_at Adenylate kinase 3 AK3 0.00026 642_s_at Presenilin 1 (Alzheimer disease 3) PSEN1 0.00045 39376_at Homeodomain interacting protein kinase 1-like protein Nbak2 0.00055 40810_at SWI/SNF-related, matrix-associated, subfamily c, member 1 SMARCC1 0.00061 T24 38017_at CD79A antigen (immunoglobulin-associated a) CD79A 4.52eÀ13 31432_g_at Fc fragment of IgG, receptor, transporter, A FCGRT 4.14eÀ11 31431_at 4.40eÀ11 37863_at Early growth response 2 (Krox-20 homologue, Drosophila) EGR2 3.92eÀ10 38354_at CCAAT/enhancer-binding protein (C/EBP), beta CEBPB 3.73eÀ09 36567_at 36567_at 0.00000002 36568_at Highly similar to Homo sapiens BNPI mRNA for brain-specific 0.00000004 Na-dependent inorganic phosphate cotransporter. 40468_at Formin-binding protein 1 FNBP1 0.00000023 39175_at Phosphofructokinase, platelet PFKP 0.00000074 40607_at Dihydropyrimidinase-like 2 DPYSL2 0.0000019 41138_at CD99 antigen CD99 0.0000022 106_at Runt-related transcription factor 3 RUNX3 0.0000025 38700_at Cysteine and glycine-rich protein 1 CSRP1 0.0000037 38836_at Neuronal pentraxin receptor NPTXR 0.0000056 38019_at Casein kinase 1, epsilon CSNK1E 0.000007 41585_at KIAA0746 protein KIAA0746 0.0000071 810_at Rho guanine nucleotide exchange factor (GEF) 1 ARHGEF1 0.000021 41222_at Signal transducer and activator of transcription 6 STAT6 0.000042 39158_at Activating transcription factor 5 ATF5 0.000044 675_at Hypothetical protein MGC27165 MGC27165 0.000061 35828_at Cysteine-rich protein 2 CRIP2 0.00015 33244_at Chimerin (chimaerin) 2 CHN2 0.00023 32095_at Importin 13 IPO13 0.00023 837_s_at Malic enzyme 1, NADP(+)-dependent, cytosolic ME1 0.00033 39172_at Hypothetical protein FLJ14547 FLJ14547 0.00074 41766_at Mannosidase, a, class 2A, member 2 MAN2A2 0.0011 T36 33956_at Lymphocyte antigen 96 LY96 0.000002 160029_at Protein kinase C, beta 1 PRKCB1 0.000047 37203_at Carboxylesterase 1 (monocyte/macrophage serine esterase 1) CES1 0.00017 32321_at Major histocompatibility complex, class I, E HLA-E 0.00029 37572_at Cholecystokinin CCK 0.00046 36565_at Zinc finger protein 183 (RING finger, C3HC4 type) ZNF183 0.0012

NOTE: Genes in bold font are common to RD-EF cells, HNFF-EF cells, and EFT cells. Genes that are inducted at different time points in RD-EF cells are shown separately in the table.

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Moreover, the up-regulation of CCK (Figs. 1D and 2C, m-o), induced by EWS-FLI1 included neuronal pentraxin receptor, whose expression differentiates the parasympathetic neural synaptotagmin I, SMA5, presenilin 1, as well as GABARAPL, phenotype of EFT from the sympathetic neural phenotype of XPNPEP1, DPYSL2, CSRP1, and OLFM1. neuroblastoma, clearly indicated that a specific Ewing-like Further inspection revealed that 20 of the 109 up-regulated genes parasympathetic neural phenotype was imposed in RD cell by are well-known WNT signaling components, involving both EWS-FLI1 expression. canonical and noncanonical WNT signaling pathways (Fig. 5A; We also examined the effect of EWS-FLI1 expression on ref. 30). This observation implied that WNT signaling is possibly an myogenesis, cognizant of the profound loss of morphologic important mechanism whereby EWS-FLI1 invokes its biological evidence of myogenesis noted in Fig. 2. Consistent with the effect, especially because WNT signaling is important in neural morphology, we found a striking loss of expression of key genes crest development and aberrant WNT signaling has been implied in necessary for myogenesis, especially the myogenic transcription diverse human cancers. factors MyoD and myogenin, the myogenic intermediate filament Confirmatory QRT-PCR was done on selected neural genes desmin, and muscle-associated genes cholinergic receptor a and (Fig. 4B). The data showed a high correlation (almost identical cholinergic receptor d. Two other myogenic transcription factors, patterns) between microarray and QRT-PCR results. Because the myf5 and myf6, were unaffected by EWS-FLI1 expression (Fig. 3C). RNA for microarray and for QRT-PCR analyses was from separate Interestingly, this expression pattern of myogenic transcription experiments, this consistency confirmed the reliability of the factors is reminiscent of what was seen in biphenotypic sarcomas microarray results as well as the reproducibility of the RD-EF expressing EWS-FLI1 (13). Protein level confirmation of the down- system. In addition, we also used siRNAs against the EWS-FLI1 regulation of MyoD and Myogenin by Western blot is illustrated in breakpoint (siEFBP2, sequence from ref. 18) to inhibit EWS-FLI1 Fig. 3D, where a nearly inverse relationship between levels of EWS- before induction. Twelve hours after transfection of siEFBP2 or a FLI1 protein and these two transcription factors was noted. scrambled control siC8, RD-EF cells were treated by tetracycline Immunohistochemical staining is consistent with the microarray or ethanol for 24 hours before RNA was harvested. EWS-FLI1 and Western results (Fig. 2C, d-i). Moreover, Cyclin D3, the major expression level in siEFBP2-transfected cells induced by tetracy- D-type cyclin expressed in rhabdomyosarcoma (17) and associated cline was only 20% of those in siC8-transfected cells induced by with muscle differentiation (28, 29), was down-regulated to an tetracycline. In accordance with the EWS-FLI1 level, the almost undetectable level after EWS-FLI1 induction, in parallel expression of the neural genes was also lower in tetracycline- with the increase of cyclin D1 (the major D-type cyclin in EFT; Figs. induced siEFBP2 cells than the siC8 controls (Fig. 4C). 1D and 3C; Supplementary Fig. 1B, ref. 17). These data indicate that Correlation of EWS-FLI1 expression and the induced targets EWS-FLI1 expression induces a profound down-regulation of the was also validated by introducing siEFBP2 into an EFT cell line muscle differentiation program. TC32. EWS-FLI1 RNA level was reduced >60% in siEFBP2- Comparison of up-regulated genes in RD-EF system and transfected TC32 cells compared with siC8-transfected cells highly expressed genes in Ewing’s family tumors revealed (Fig. 4D). Thus, the expression of the neural genes was also genes crucial in neural crest development and WNT signaling reduced. These results showed that the induction of these genes, pathway. In an attempt to specify genes that were significantly assessed by QRT-PCR, was EWS-FLI1 dependent. The level of up-regulated by EWS-FLI1, we first identified a list of 865 ‘‘EFT modulation of the neural genes by EWS-FLI1 in TC32 cells was signature genes’’ of the 12,625 probe sets on the HU95av2 arrays, less dramatic. This is likely due to the observed lesser inhibition that are highly associated with primary EFT from patients (P V of EWS-FLI1 expression in these cells. Greater inhibition of EWS- 0.001) and are expressed at least 2-fold higher than in the rest of FLI1 expression is probably needed to fully abrogate its the tumors. PCA analysis showed that at 24 and 36 hours, modulation of downstream genes. tetracycline-treated RD-EF cells were completely separated from Up-regulation of neural crest genes was confirmed in the controls and the EWS-FLI1 protein level was stable and another cell lineage expressing EWS-FLI1. Our result suggests comparable with the original EFT cell lines. Thus, we compared that EWS-FLI1 is a potent differentiation factor that blocks a the gene expression pattern of the T24 and T36 samples with the preexisting commitment to myogenesis in rhabdomyosarcoma controls, including untreated and all of the ethanol-treated whereas imposing neural differentiation. We sought to determine samples, and selected 370 genes that are highly associated with whether neural differentiation is unique to the skeletal muscle T24 and T36 (P V 0.001). Of these EWS-FLI1–induced genes, 109 cellular milieu found in RD cells, or is a generalizable effect of (30%) are also signature genes of the EFT (Table 1; Fig. 4D; EWS-FLI1. The fact that these genes are also highly expressed in Supplementary Fig. 2). Hierarchical clustering of the RD-EF primary EFT precludes the possibility that this is merely a samples separated these 109 genes in a temporal manner (Fig. 4A). nonspecific effect after the muscle differentiation program was Two main gene expression patterns within the time course were inhibited. However, because cell context is very important in identified: genes that were up-regulated at early time points (T6 extrapolating EWS-FLI1’s function, we have pooled our RD-EF and T12) and late time points (T18, T24, and T36). We screened gene expression data with comparable expression profiles the gene lists by searching the literature as well as by their Gene derived from primary, untreated EFT, and a similar data set Ontology Annotation. Strikingly, a marked number (30%) of EWS- derived from EWS-FLI1 transfectants in a human normal FLI1–up-regulated genes are important for neural crest develop- foreskin fibroblast model (HNFF-EF) kindly provided by the ment, such as EGR2 (Krox20), MSX1, CITED2, c-MYC, ID2, authors (8). Using a cutoff of P < 0.001 (and expression >2-fold Cadherin 11, RUNX3, and Rho family members [ARHH (RhoH) increased in the case of primary EFT), we have done a multiple t test and ARHGEF1]. This is grossly disproportionate to the relative analysis of each data set to identify the genes that meet these abundance of such genes by gene ontology code (P < 0.001) and selection criteria and compared results in a Venn diagram (Fig. 5B). strongly suggests that a primary function of EWS-FLI1 is to A final 46 genes common to all three datasets was identified invoke a form of neurogenesis. Other neural associated genes (highlighted in Table 1). These genes are of especial interest,

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Figure 4. Comparison of up-regulated genes in RD-EF and highly expressed genes in EFT revealed important EWS-FLI1 target genes. A, hierarchical clustering of microarray samples based on 109 genes common between EWS-FLI1 induced genes in RD-EF cells and EFT signature genes. C, all untreated and ethanol-treated controls shown in Fig. 1C. B, selected genes that are highly expressed in EFT and induced RD-EF cells are validated by QRT-PCR analysis. Left, microarray data; right, QRT-PCR data. C, QRT-PCR target validation by RNA interference technology in RD-EF cells. siRNA against EWS-FLI1 breakpoint (siEFBP2) or a control siRNA (C8) was introduced into RD-EF cells 12 hours before tetracycline or ethanol treatments for another 24 hours. Expression level of genes in siC8 transfected, tetracycline-treated cells was taken as 1. T24 C8-12, RD-EF cells were transfected with siC8 for 12 hours before tetracycline treatment for another 24 hours; T24 EFBP2-12, RD-EF cells were transfected with siEFBP2 for 12 hours before tetracycline treatment for another 24 hours; E24 EFBP2-12, RD-EF cells were transfected with siEFBP2 for 12 hours before ethanol treatment for another 24 hours. D, QRT-PCR target validation by RNA interference technology in TC32 EFT cells. Correlation of EWS-FLI1 expression and the induced targets was validated by transfecting siEFBP2 to TC32 EFT cells. Gene expression in siEFBP2-transfected cells was divided by the expression value in siC8-transfected cells.

because they are clearly up-regulated by expression of EWS-FLI1, Discussion independently of cellular background, normal or malignant. The traditional view of oncogenes in tumor pathogenesis is to Strikingly, the majority of the neural crest differentiation–related promote proliferation, increase survival, and block the differenti- genes identified in RD-EF model were also up-regulated by EWS- ation program of the target cells. However, chimeric fusion genes FLI1 expression in this human fibroblast background. Fourteen of that result from various chromosomal translocations are different the 46 genes are associated with neurogenesis, and even more from traditional oncogenes in that they are often associated with strikingly, of the seven genes most strongly associated with the specific tumor types (i.e., EWS-FLI1 in EFT, PAX3/7-FKHR in EWS-FLI1 expression in the model systems (P < 0.000000001), the rhabdomyosarcoma and BCR-ABL in acute lymphoblastic leuke- majority (5) are involved in neural differentiation, particularly mia, ALL). This specificity implies that the presence of the fusion neural crest development (Table 1; ref. 31). Thus, there is a strong gene may determine the differentiation program that the particular association between neurogenesis and EWS-FLI1 expression. These tumor type manifests. Transgenic mouse model studies of data clearly indicate that neural differentiation induced by Philadelphia chromosome-positive (BCR-ABLp190) ALL (Ph+- expression of EWS-FLI1 is independent of cellular background in ALL) indicated that the target of leukemic transformation in which the gene is expressed. It excludes the possibility of a Ph+-ALL is normal pluripotent hematopoietic stem cells rather nonspecific neural up-regulation as a result of the down-regulation than committed progenitor cells and the presence of BCR-ABLp190 of myogenesis and indicates a general effect of EWS-FLI1 on imposes the B-cell differentiation program of the precursor stem differentiation. cells but prevents further development of the committed B-cell www.aacrjournals.org 4641 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 precursors (32). It results in the accumulation of abnormal cells epidermal lineage (38). CITED2 is a coactivator of another neural organized as a hierarchy, which failed to differentiate into crest marker TFAP2. The expression of CITED2 is essential for the functional B lymphocytes. Similarly, when PAX3-FKHR was survival of neuroepithelial cells and disruption of CITED2 gene is introduced into NIH3T3 cells, cDNA microarray analysis indicated embryonic lethal because of defects in the development of heart and profound activation of the myogenic transcription program, neural tube (39–41). Cadherin 11 and Rho family GTPases are both including up-regulation of MyoD and myogenin (33). PAX3 related to delamination of neural crest cells from the neural tube transduced cells did not show this activation. (42–44). RUNX3 is an important regulator of the axonal projections Our study showed that EWS-FLI1 has a profound effect on cell of a subpopulation of dorsal root ganglion neurons (45). differentiation as well as proliferation. It can induce an Ewing-like Expression of neural structural genes, such as neuronal pentraxin neural crest phenotype in multiple cellular contexts whereas receptor, synaptotagmin I, and MAPT, is also highly induced. CCK is a blocking the existing cell differentiation program, which strongly neuropeptide and is highly expressed in EFT (46). This is almost supports the hypothesis that this tumor-specific fusion protein acts unique among tumor cells and served as evidence that EFT as a cell lineage determinator, rather than a pure ‘‘oncogene.’’ originated from parasympathetic progenitors. Another tumor type Otherwise conspicuous terminal skeletal muscle differentiation is of neural crest sympathetic origin, neuroblastoma, does not express nearly completely lost in RD cells expressing EWS-FLI1, supplanted this gene. This was recently further corroborated by published by marked neural differentiation as documented by the appearance observations that expression of EWS-FLI1 in a neuroblastoma of neurites, dense core granules, and a number of neural genes, as cellular background (11) suppresses sympathetic neural differenti- noted above. Krox20, MSX1 (34), and c-MYC are all neural crest ation, the hallmark feature of neuroblastoma cells, whereas MIC2 markers during development. Krox20 knockout mice have severely (CD99), c-MYC, and keratin 18 were all markedly up-regulated, defective myelination of peripheral nerves (35). MSX1À/À mice have similar to what we observed in this study. These data exclude the deficiencies in neural crest derivatives (36). C-MYC was shown an possibility of a nonspecific neural up-regulation and indicate a essential early regulator of neural crest development and knock- general effect of EWS-FLI1 on differentiation. The high expression of down of c-MYC by antisense ODN resulted in a loss of neural crest all these critical neural crest associated genes provided further precursors in Xenopus embryos (37). Overexpression of ID2 can drive evidence for a parasympathetic neural crest differentiation program ectodermal cells into a neural crest phenotype instead of the in EFT.

Figure 5. Possible signaling pathway and gene regulation network initiated by EWS-FLI1. A, identified EWS-FLI1 target genes that are also called WNT signaling pathway components, including both canonical and noncanonical pathways. B, Venn diagram comparison of genes up-regulated in both RD-EF cells and HNFF-EF cells after EWS-FLI1 expression, as well as genes highly expressed in EFT cells revealed 46 genes common in all three groups. C, gene regulation network initiated by EWS-FLI1. Genes in red were up-regulated by EWS-FLI1. Genes in blue were down-regulated by EWS-FLI1. Genes in square boxes are called Wnt signaling targets.

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Because James Ewing described Ewing’s sarcoma >80 years ago, neural crest marker during development, was found to little has been known about the cell of origin of this group of dedifferentiate terminally differentiated murine myotubes by tumors. Our results support the speculation that EFT probably repressing MyoD, Myogenin, cyclin D3, p21 and up-regulating originates from primitive multipotent progenitor cells that are cyclin D1 with minimal effect on cell proliferation (49, 50). capable of differentiating into neural crest derivatives. EWS-FLI1 Similar effects seen after EWS-FLI1 expression in RD cells lead fusion subsequently imposes a neural crest parasympathetic us to speculate that EWS-FLI1 function is at least partially lineage direction to the cells but inhibits terminal differentiation. mediated by MSX1. However, MSX1 is not likely a direct target Eventually secondary genetic alterations lead to the malignancy of because its up-regulation starts at 18 hours after EWS-FLI1 the cells. Interestingly, EFT members represent a continuum of induction, when MYOD, CYCLIN D3, and P21 were already different degrees of neural differentiation. Bone marrow stromal down-regulated. A possible mediator of MSX1 up-regulation by stem cells (MSC), which are classic mesodermal derivatives, have EWS-FLI1 was CITED2, induced as early as 6 hours. It has been been shown multidifferentiated in addition to being multipotent shown that MSX1 is a potent inhibitor of its own promoter and could be induced to differentiate into neurons (47). region and this autosuppression could be counteracted by CBP/ Considering that most EFT occurs in bone and soft tissue, MSC p300, which is the necessary partner of CITED2 for TFAP2 serves as one possible source of the cells susceptible of EWS-FLI1 coactivation (51, 52). transformation. A similar argument may be true in soft tissue, The striking incidence of WNT signaling–associated genes where pluripotent stem cells have also been described. identified in this analysis cannot be due to chance alone. WNT Despite several years of effort by multiple investigators, the signaling has been found to be very important in neural crest basic mechanism whereby EWS-FLI1 expression results in the development (53). WNTs could function as endogenous neural tumor entity that we recognize as EFT remains unknown. Little crest inducers in avian embryos, and inhibition of this pathway was revealed about how expression of a single chimeric could block neural crest precursor formation in Xenopus (54). ‘‘oncogene’’ can lead to such a profound genome-wide gene However, the classic WNT/h-catenin mediated signaling through expression shift. This study does not permit us to distinguish TCF/LEF target genes is not likely active in EFT tumors direct from indirect targets of EWS-FLI1. However, by time (Supplementary Fig. 2). Can EWS-FLI1 hijack the classic WNT course study and microarray analysis, we were able to infer a signaling downstream of h-catenin? Or does it activate a multitude of sequential downstream events and have identified noncanonical WNT pathway, whose mechanism is still largely an important but heretofore unidentified signaling pathway unknown? Or has a completely different, currently undescribed, operative in Ewing’s tumors, both in vivo and in vitro (Fig. 5B). pathway been used? Increased knowledge of the noncanonical C-MYC and ID2 were among the earliest up-regulated genes and WNT pathways will help to answer these questions. ID2 has been reported to be a direct target of both c-MYC and EWS-FLI1 (24, 48). It is possible that EWS-FLI1 can up-regulate Acknowledgments both genes directly and overexpression of c-MYC can promote expression of ID2 to a higher degree. ID2 inactivates RB and Received 11/4/2004; revised 3/1/2005; accepted 3/22/2005. Grant support: Molecular Pathology from Las Madrinas at CHLA (S. Hu- subsequently promotes G1 phase transition. In addition, as a Lieskovan) and the Director’s Challenge UO1 (T.J. Triche, Ewing’s tumor gene dominant-negative bHLH transcription factor, ID2 antagonizes expression data for comparison). the expression of myogenic transcription factors MyoD and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Myogenin, as well as the cell cycle inhibitor P21. A previous with 18 U.S.C. Section 1734 solely to indicate this fact. study showed that P21 is a direct target of EWS-FLI1 (27) and We thank Darkin Chan and Minerva Mongeotti (EM Laboratory) for EM processing; Morgan Wu (clinical pathology laboratory) for immunohistochemical staining; Betty its expression was also down-regulated dramatically by EWS- Schaub, Xuan Chen, and Sitara Waidyaratne (Microarray Core) for microarray FLI1 in our system. Another up-regulated gene, MSX1, an early processing; and Dr. George Mcnamara (Imaging Core) for his expertise at microscopy.

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Cancer Res 2005; 65: (11). June 1, 2005 4644 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. 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, et al.

Cancer Res 2005;65:4633-4644.

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