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Proc Natl Acad Sci U S A. 2009 Mar 31; 106(13): 5324–5329.
Published online 2009 Mar 16. doi:  10.1073/pnas.0810759106
PMCID: PMC2656557
Medical Sciences

EZH2 is a mediator of EWS/FLI1 driven tumor growth and metastasis blocking endothelial and neuro-ectodermal differentiation

Abstract

Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2−/−γC−/− mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor.

Keywords: epigenetic regulation, Ewing tumor, stemness

Ewing tumors (ET) are highly malignant tumors with an approximate incidence of 3.3/106 in children under the age of 15. ET are characterized by early metastases, and metastatic spread is commonly hematogeneous. ET were originally described by Ewing in 1921 as endothelioma of the bone (1), and we confirmed this endothelial signature by microarray analysis (2). ET are molecularly defined by ews/ets translocations. Translocation-derived chimeric transcription factors yield transactivation, transformation, and the highly malignant phenotype. In mice, EWS/FLI1 transforms bone marrow derived or mesenchymal progenitor cells, and generates tumors (3, 4), which have features of ET. Also, inhibition of EWS/FLI1 expression may allow ET cells to recover the phenotype of their presumed mesenchymal stem cell (MSC) progenitor (5). Multipotent MSCs represent a leading candidate for primary transformation in ET. We revealed a relationship of ET to both endothelial and fetal neural crest-derived cells (2), after having demonstrated neuroectodermal histogenesis of ET in 1985 (6). Based on our recent study, we postulated in 2004 that the ET stem cell is arrested at early mesenchyme development from the neuroectodermal germ layer, and, thus, the ET stem cell is a neuronal crest-derived stem cell at transition to mesenchymal endothelial development, residing in the bone marrow. However, ectopic EWS/FLI1 expression resulted in a neural phenotype, raising the possibility that transdifferentiation or lineage promiscuity may be an alternative to the MSC histogenetic origin hypothesis of ET (7).

We used high density DNA microarrays for the identification of ET specific gene expression profiles compared with 133 normal tissues of diverse origin (normal body atlas, NBA), and identified 37 genes that were highly up-regulated or specifically expressed in ET (2). The histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2), one of these up-regulated genes, is part of the polycomb repressor complex 2 (PRC2), together with embryonic ectoderm development (EED) protein and suppressor of Zeste (SUZ12). Within this complex, EZH2 exhibits methyltransferase activity, and silences target genes by methylating lysine 27 on histone 3 (H3K27). EZH2 is already active at gastrulation, and maintains a stemness expression signature (8). In tumors, it may recruit DNA-methyltransferase to promoter regions, resulting in specific gene switch-off (9). Varambally et al. (10) demonstrated that EZH2 mediated gene silencing could be reverted by histone deacetylase inhibitors (HDACis), because PRC2 complexes interact through EED with HDAC2 to mediate their suppressive activity (11).

Here, we analyzed the pathogenetic role of EZH2 in ET in vitro and in vivo. We identified EZH2 to be induced by EWS/FLI1 presumably due to EWS/FLI1 binding to the EZH2 promoter in vivo. Inhibition of EZH2 expression by RNA interference suppressed contact independent growth in vitro, and significantly delayed tumor development and metastasis in mice. Expression profiling and differentiation assays of ET after EZH2 RNA interference and HDAC-inhibitor treatment with Trichostatin A revealed that EZH2 maintains a stemness signature in ET by inhibiting endothelial and neuroectodermal differentiation.

Results

Histone Methyltransferase EZH2 Is Strongly Up-Regulated in ETs.

In a previous microarray analysis, we identified the histone methyltransferase EZH2 as a strongly up-regulated gene in ET (2). We observed high levels of EZH2 expression in ET, as well as in normal thymus, testis, and fetal tissue (Fig. S1). Also, real-time RT-PCR demonstrated that other embryonal tumors including acute lymphoblastic leukemia and neuroblastoma showed a significantly lower expression of EZH2 (Fig. S2). Down-regulation of EWS/FLI1 by specific siRNA (12) in ET cell lines SK-N-MC and SBSR-AKS (SI Materials and Methods) led to suppression of EZH2 expression in both cell lines (Fig. 1A). Expression of EED and SUZ12 components of the PRC2 complex was not affected. Subsequently, we overexpressed EWS/FLI1 cDNA under the control of a stem cell virus LTR (pMSCVneo, Clontech) in human MSC V54.2 and L87 (13). Western blot analysis identified several lines with good EWS/FLI1 expression (Fig. 1B). When these infectants were analyzed for EZH2 expression, we observed an increase of EZH2 that appeared EWS/FLI1 dose-dependent (Fig. 1C). The up-regulation of EZH2 expression in ET suggested that the EZH2 gene might be a direct target of EWS/FLI1. ETS transcription factors bind to the core consensus binding motif GGAA/T. Analysis of the promoter region of the EZH2 gene identified among others an evolutionary conserved ETS recognition site, starting −1081 bp of the TSS (accession no. NT_007914). ChIPs for this and several other regions were performed with anti-FLI1 antibody, and normalized to IgG-IPs and nonspecific binding to an unrelated genomic region. The anti-FLI1 antibody detects only EWS/FLI1 in ET cell lines. Real-time quantitative PCR revealed that endogenous EWS/FLI1 bound specifically to the conserved ETS recognition sequence at −1081 bp of the EZH2 promoter in vivo (Fig. 1D).

Fig. 1.
EZH2 expression and regulation by EWS/FLI1. (A) Transient transfection of EWS/FLI1 specific siRNA down-regulates EZH2 expression. EWS/FLI1 #I and EWS/FLI1 #II represent different siRNAs (see Materials and Methods; control siRNA: non silencing siRNA). ...

Suppression of EZH2 Expression in ET Inhibits Contact Independent Growth.

Bracken et al. (14) identified EZH2 to be amplified in human tumors, and suggested EZH2 to be a downstream mediator of E2F driven proliferation. To further elucidate the role of EZH2 overexpression in the pathology of ET, we down-regulated EZH2 with several specific siRNAs (SI Materials and Methods and Fig. S3) in ET cell lines A673 and SBSR-AKS. Transient siRNA transfection enabled a down-regulation of EZH2 expression to levels 20–25% of normal values (Fig. S3). Stable shRNA infectants [generated by cloning of one of the siRNAs that tested positive into pSIREN (EZH2-7, see Materials and Methods) and subsequent retroviral gene transfer of pSIRENEZH2 (Clontech, see Materials and Methods) constructs into A673 cells (Fig. 2A)] lost their ability of contact independent growth in colony forming assay (Fig. 2B). Also, cell proliferation of ET lines tested was blocked by 200 nM of HDACi Trichostatin A (TSA), whereas growth of neuroblastoma and ALL lines was not inhibited (Fig. S4).

Fig. 2.
Effects of EZH2 down-regulation on in vitro and in vivo tumor growth and metastasis. (A) Stable EZH2 shRNA infectants, generated by retroviral gene transfer of pSIREN-shEZH2 constructs (see Materials and Methods; pSIRENn.siRNA: non silencing siRNA) into ...

Down-Regulation of EZH2 Inhibits Tumor Growth in Vivo.

Based on these results, we asked whether down-regulation of EZH2 in ET might affect tumorigenic growth in vivo. We injected stable pSIRENEZH2 infected A673 cells and the respective controls (as identified in Fig. 2A) s.c. into immunodeficient Rag2−/−γC−/− mice, and analyzed tumor growth. As shown in Fig. 2C, suppression of EZH2 expression resulted in a significant delay of tumor growth that appeared dose-dependent, because the clone with the stronger EZH2 down-regulation revealed the most significant delay in tumor growth (see also Fig. 2A). Similarly, the metastatic behavior of these cells was strongly influenced by the level of EZH2 expression. Although A673 control infectants injected into the tail vein strongly colonized the lungs and liver, pSIRENEZH2 infectants almost completely lost their ability to colonize the lung (Fig. 2D), and only A673pSIRENEZH2-2 cells with a lower level of EZH2 mRNA reduction still metastasized into lung and liver tissue. Metastases were also detected in kidney (2/4 mice) and subclavicular connective tissue (1/4 mice) only with control infectants.

Blockade of HDAC and EZH2 Increases the Expression of Genes Involved in Endothelial and Neuroectodermal Differentiation.

To further elucidate downstream targets regulated by EZH2 in ET, we transiently down-regulated EZH2 in A673 cells, and compared their expression pattern with HDAC-inhibitor TSA treated cells in a microarray analysis. To limit off target artifacts, 2 independent assays and different siRNAs were used. Significance analysis of microarrays (SAM) identified 270 probe sets comprising 259 genes that were up-regulated after EZH2 siRNA or TSA treatment of A673 cells (Fig. 3A; the 100 most significant genes are shown in Table S1), respectively. An independent analysis considering present/absent calls with a fold change cutoff >1.7, and conventional t test values (P < 0.05; see Materials and Methods) identified 37 probe sets comprising 36 genes, of which 23 were up-regulated (Fig. S5 and Table S2). Interestingly, both analyses identified a number of genes important for neural and endothelial function, as well as development (arrows in Fig. 3A; Fig. S5). For example, down-regulation of EZH2 resulted in the induction of epithelial membrane protein 1 (EMP1), Ephrin receptor B2 (EPHB2), glial fibrillary acidic protein (GFAP), growth associated protein 43 (GAP43), or protocadherin 7 (PCDH7). Other modulated genes included activated leukocyte cell adhesion molecule (ALCAM) and nerve growth factor receptor (NGFR), which were induced or down-regulated by EZH2siRNA/TSA treatment, respectively. NGFR early on was described as an essential marker of neuroectodermal stem cells (15). Also, this expression signature identified by SAM clustered neuronal tissues, MSCs, and endothelial cells separate from other normal tissue (Fig. S6). TSA or EZH2 siRNA-mediated up-regulation of ALCAM, EPHB2, EMP1, GFAP, and GAP43, as well as NGFR suppression, was confirmed by real-time RT-PCR in independent assays (Table S3). A similar modulation of these genes was observed after siRNA-mediated suppression of EED or SUZ12 (Fig. 3B), suggesting their regulation by PRC2 complex. Several mechanisms, including direct recruitment of the PRC2 complex to the promoters of genes, could be relevant for the regulation of these genes. Recently, it has been suggested that noncoding RNAs may direct transcriptional coregulators to their site of action (16). To test this approach, we silenced Argonaute-1 (AGO1), and subsequently observed down-regulation of NGFR expression, but no regulation of EPHB2, EMP1, or GAP43 (Fig. 3B). These findings may suggest that noncoding RNA might be involved in mediating the regulatory effects of EZH2 for some of the identified genes.

Fig. 3.
EZH2 blockade in ET induces a number of genes important for epithelial and neuroectodermal differentiation. (A) Microarray data of selected genes after SAM analysis with their normalized fluorescent signal intensities (see Materials and Methods). Combined ...

Down-Regulation of Genes Constituting PRC2 Complex Enable Neuronal As Well As Endothelial Differentiation.

Considering that EZH2 would not only maintain a stemness signature in stem cells but also in ET, EZH2 suppression should increase the ability of this tumor to differentiate. First, we induced neurogenic differentiation with 0.1 mM butylated hydroxyanisole (BHA) in stable A673 shRNA infectants (see Materials and Methods). We observed that A673 cells were able to fully differentiate and express GFAP, a major intermediate filament protein of mature astrocytes (17), only after EZH2 suppression (Fig. 4A). Similarly, when we analyzed ET for their endothelial differentiation potential in tube formation assay, we observed that A673 and MHHES1 cells, which are unable to form tubes under normal conditions, efficiently formed tubular networks under blockade of EZH2 expression (Fig. 4B), indicating an enhanced differentiation potential under EZH2 blockade. Interestingly, similar results were obtained after EED, and, to a lesser extent, after SUZ12 suppression.

Fig. 4.
Blockade of genes of PRC2 complex in ET induces a number of genes important for and enables epithelial and neuroectodermal differentiation. (A) Neurogenic differentiation of stable A673-infectants pSIRENEZH2-1 and pSIRENn.siRNA treated for 5 days with ...

Discussion

Stemness is a salient feature of malignancy. Embryonic tumors provide a unique opportunity to identify molecular mechanisms of stemness in tumors. Here, we demonstrate that EWS/FLI1 maintains an immature signature in ET via the histone methyltransferase EZH2. EZH2 is overexpressed in several types of cancer, and the level of expression correlates with cancer aggressiveness (8, 10, 18). EZH2 is already active at gastrulation, maintains a stemness expression signature (8, 19), and is believed to be a key regulator of stem cell renewal (20) and differentiation (21). Overexpression of EZH2 was shown to bypass the cellular senescence program in mouse embryonic fibroblasts, and to prevent mouse hematopoietic stem cell exhaustion (22).

It has been shown that EWS/ETS fusions underlie transactivation, transformation, and the malignant phenotype of this tumor. Surprisingly, although the chimeric proteins constitute aberrant transcription factors, in cells ectopically expressing EWS/FLI1, an approximately equal number of genes was observed to be either suppressed or activated (23, 24). Our data suggest that the strong expression of EZH2 in ET might be directly mediated by EWS/FLI1. Because EZH2-containing PRC2 can suppress transcription, the repressive activity of EWS/FLI1 may at least in part be mediated by EZH2. Interestingly, the neuroectodermal and endothelial differentiation genes silenced by EZH2 were reexpressed after EZH2 knock down. This flexibility is in contrast to tumor suppressor genes in colon cancer that have been shown to be irreversibly methylated in their promoter region even after depletion of EZH2 (25).

Genes typically expressed in neuronal or endothelial cells were up-regulated by EZH2 suppression. Also, EZH2 suppression inhibited not only tumor growth but also metastatic spread, presumably by induction of differentiation. These observations may indicate a potentially more comprehensive role for EZH2 in tumorigenicity by regulating a reversible state in ET as shown for prostate cancer (26). These findings might suggest that not all changes mediated by EZH2-containing PRC2 are necessarily maintained due to promoter DNA methylation (2729).

Gene regulation by EZH2 might be direct or indirect. To further elucidate functional involvement of PRC2 complex, suppression of other member components including EED and SUZ12 (20) correspondingly induced genes of neuroectodermal and endothelial signature in ET, and increased their differentiation potential. Also, HDAC inhibition by TSA up-regulated a similar set of genes, possibly delineating EZH2 ability to interact with HDAC2 via the EED protein (11). Emerging data suggest that noncoding RNA might be guiding chromatin modifying complexes to epigenetically silenced target genes (16, 30). To test the relevance of this approach, we suppressed AGO1 that is necessary for this process (31). We observed suppression of NGFR expression by ≈50%, but no change in 3 other analyzed genes. These findings suggest that for the majority of PRC2 suppressed genes in our dataset noncoding RNA direction is unlikely to be the main mechanism.

During the preparation of our manuscript, we noticed 2 recent publications that investigated the overexpression of EWS/FLI1 in human MSCs (32, 33). Both observed that this overexpression did not transform MSC and lead to tumorigenicity. The publication by Riggi et al. (33) is in line with our finding that EWS/FLI1 overexpression induces EZH2 expression, and that EZH2 may have a role in tumorigenicity of ET. Our findings indeed go far beyond by demonstrating the role of EZH2 in EWS/FLI1-induced metastasis, and the analysis of the EZH2 regulated gene set in ET.

The neuronal expression pattern in the EZH2 siRNA and the HDAC-inhibitor-treated ET cells is in agreement with the pathogenetic hypothesis that EZH2 blocks the (probably EWS/FLI1-induced) neuronal differentiation program of ET. The MSC/endothelial expression pattern might be indicative of EZH2-mediated suppression of markers typically expressed in the ET stem cell. Whether this cell is derived from primordial streak derived early mesoblasts (e.g., with endothelial differentiation capacity, as suggested by James Ewings initial observation; see ref. 1), or whether EWS/FLI1 can induce transdifferentiation and lineage promiscuity in more differentiated cells requires further investigation. EWS/FLI1 driven overexpression of EZH2 in ET reveals upstream mechanisms of EZH2 deregulation, which to our knowledge have not been previously described in malignancies of adulthood.

Overall, our observations indicate that EZH2 expression is required in ETs to maintain stemness and metastatic spread. These findings suggest that EZH2 and EZH2-mediated events are promising novel targets for ET therapy.

Materials and Methods

Mice.

Immunodeficient Rag2−/−γC−/− mice on a BALB/c background were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan), and maintained in our animal facility under pathogen-free conditions in accordance with the institutional guidelines and approval by local authorities. Experiments were performed in 6–16 week-old mice.

Cell Lines.

ET lines (MHHES1, SK-ES1, SK-N-MC, and TC71), neuroblastoma lines (CHP126, MHHNB11, SH-SY5Y, and SIMA), and pediatric human B cell precursor leukemic lines (NALM6, 697, and cALL2) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). A673 was from ATCC (LGC Standards). SBSR-AKS is a cell line with a type 1 EWS/FLI1 translocation that was established in our lab from an extraosseous inguinal metastasis of a 17-year old female Ewing patient enrolled in our clinical protocol. MSCs L87 and V54.2 were immortalized with SV40 large T-antigen (13). Retrovirus packaging cell line PT67 was from Takara Bio Europe/Clontech.

RNA Interference.

For transient RNA interference, cells were transfected with siRNA. Briefly, 3 × 106 cells were resuspended in a final volume of 12 mL medium/80 mm Petri dish containing 5 nM siRNA and 36 μL transfection medium (HiPerFect, Qiagen), and incubated at 37 °C. To test transfection efficiency and gene knock-down, RNA was extracted, and gene expression assessed by real-time RT-PCR. Unspecific induction of an IFN response was excluded by monitoring induction of IFN responsive genes G1P2 and IFITM1. Small interfering RNAs for EWS/FLI1 were synthesized at MWG Biotech, corresponding to ref. 12, all other siRNAs were purchased from Qiagen. Small interfering RNA sequences are provided in SI Materials and Methods.

Constructs and Retroviral Gene Transfer.

The cDNA containing the EWS/FLI1-coding region was described previously (2). A BglII fragment was subcloned into the retrovial vector pMSCVneo (Takara Bio Europe/Clontech). For stable silencing of EWS/FLI1 or EZH2 expression, oligonucleotides of the short hairpin corresponding to the published EZH2 siRNAs (10), and EWS/FLI1 corresponding to the published EWS/FLI1 siRNA, site II (12), were cloned into retroviral vector pSIREN-RetroQ vector (Takara Bio Europe/Clontech) (for detailed sequences, see SI Materials and Methods). Retroviral constructs were transfected by electroporation into PT67 packaging cells, and viral supernatant isolated 48 h after transfection. Infection of target cells was carried out in the presence of 4 μg/mL polybrene. Stable infectants were isolated after selection in 600 μg/mL G418 (pMSCVneo) or 2.5 μg/mL puromycin (pSIREN-RetroQ), respectively.

RNA Microarray Analysis.

Biotinylated target cRNA was prepared as previously described (2, 34). A detailed protocol is available at www.affymetrix.com. Samples were hybridized to Affymetrix HG U133A microarrays and analyzed by using Affymetrix software Microarray Suite 5.0 (Affymetrix), and scaled to the same target intensity of 500. Subsequent analysis was carried out with signal intensities that were log2 transformed for equal representation of over and under expressed genes, and then median centered to remove biases based on single expression values. Hierarchical clustering (35) was accomplished by use of the Genesis software package (36). For the identification of differentially expressed genes, we used SAM (37).

Real-Time RT-PCR.

Differential gene expression of cDNA or siRNA transfectants was verified by real-time RT-PCR. Quantitative real-time PCR was performed by use of TaqMan Universal PCR Master Mix (Applied Biosystems), and fluorescence detection with an AB 7300 Real-Time PCR System (Applied Biosystems). Gene specific primers and probes were obtained as TaqMan Gene Expression Assay sets from Applied Biosystems, which consisted of a FAM dye-labeled TaqMan MGB probe and 2 unlabeled PCR primers; 20× stock solutions of these reagents were added to the TaqMan Universal PCR Master Mix with cDNA at a final volume of 25 μL. The final concentration of primers and probe was 900 and 250 nM, respectively. A list of used assays is provided in SI Materials and Methods.

Western Blot Analysis.

Protein samples were resolved by SDS/PAGE electrophoresis on 10 or 12% gels, and transferred by semidry blotting onto nitrocellulose membranes (GE Healthcare). The membranes were blocked to prevent nonspecific Ab binding by incubation with specific antibody in Blotto (Pierce) overnight at 4 °C. Membranes were subsequently washed twice in TBST (50 mM Tris·HCl, pH 7.6/0.15 M NaCl/0.05% Tween 20) and then resuspended in a 1/1,000 dilution of HRP-bovine anti-rabbit IgG in Blotto under gentle agitation for 1 h at room temperature. Membranes were washed twice in TBST and once in TBS, and were finally soaked in ECL+ reagent (GE Healthcare) for HRP (60 s), and exposure to autoradiography film for visualization of the bands.

ChIP and Quantitative Real-Time PCR.

ChIP was carried out as described previously with slight modifications (38). In brief, 3 × 107 A673 cells were formaldehyde (1% final) fixed for 10 min. After neutralization with glycine, cell pellets were lyzed in RIPA-buffer with protease inhibitors. Samples were sonicated to an average DNA length of 500 bp. ChIP was carried out with 3 μg of antibody either anti-FLI1 (C-19), or anti-IgG (Santa Cruz) added to 0.5 mg of precleared chromatin. Quantitative real-time PCR by using Sybr Green (Applied Biosystems) with quantitation performed by the ΔΔCt-method (38). FLI1 data at individual genomic loci were normalized to the IgG control to compensate for differences in PCR efficiency, and standardized to 2 nonregulated genomic loci outside of the EZH2 locus. Primer sequences are provided in SI Materials and Methods.

Colony Forming Assay.

Cells were seeded in duplicate at a density of 5 × 103 cells per 1.1 mL methylcellulose-based media (R&D Systems) into a 35-mm plate according to the manufacturer's instruction, and cultured for 14 days at 37 °C and 5% CO2 in a humidified atmosphere.

Differentiation Assays.

For neuron-like or astrocyte-like cell differentiation, cells were cultured in the presence of 2% dimethyl sulfoxide (DMSO, Sigma-Aldrich) and 0.1 mM BHA (Sigma-Aldrich) for 5 days. The differentiated cells were identified after fixation in 4% paraformaldehyde by staining with an antibody directed against glial fibrillary acid protein (GFAP, BD PharMingen), and visualized with a FITC-labeled goat anti-mouse F(ab')2 fragment (Jackson ImmunoResearch Laboratories).

Cellular tube formation was tested by use of a commercial Matrigel Matrix assay (Biocoat, BD Biosciences) according to the manufacturer's instruction. Briefly, cells were seeded at 50,000 cells per well in a 96-well plate, and grown at 37 °C (5% CO2) in a humidified atmosphere. After 16–18 h, cells were stained with Calcein AM Fluorescent Dye (BD Biosciences) 1 μg/mL for 30 min in the dark. Cells were imaged by fluorescence microscopy by using a Nikon Eclipse TS 100 with an attached Nikon Coolpix 5400 camera.

In Vivo Experiments.

For the analysis of in vivo tumor growth, 2–4 × 106 ET cells and derivatives were harvested by trypsinization, washed with Dulbecco's PBS, and injected in a volume of 0.2 mL into immunodeficient Rag2−/−γC−/− mice. To monitor local tumor growth, cells were injected s.c. intrainguinal, and tumor size was determined as described (39). Mice bearing a tumor >10 mm in diameter were considered as positive. To analyze metastatic potential tumor, cells were injected i.v. into the tail vein. Four weeks later, mice were killed, and metastatic spread was monitored in individual organs. In all experiments, tumor and affected tissue after in vivo growth was excised for immunohistology, and local tumors in addition were analyzed for EZH2 activity.

Histology.

Organs were fixed in 4% formaldehyde and paraffin embedded; 3 to 5 μm thick sections from all tissues were cut and stained with hematoxylin and eosin (H&E). All sections were reviewed by 2 pathologists (I.M. and L.Q-M.).

Supplementary Material

Supporting Information:

Acknowledgments.

We thank Ines Volkmer and Colette Zobywalski for expert technical assistance. L87 and V54.2 MSC lines were kindly provided by Peter Nelson (Medical Policlinic, Ludwig-Maximilians University, Munich). This work was supported by unrestricted Special Grants P31/08//A123/07 from the Else-Kröner-Fresenius Stiftung, and KKF8739175 and 1528/TUM11.16-9c/32269 from the Bayerisches Staatsministerium für Wissenschaft und Kunst. This work was also supported by the Bundesministerium für Bildung und Forschung (BMBF/DLR) Kompetenznetz Pädiatrische Onkologie/TP Immun-und Gentherapie Grant GI9965, Wilhelm-Sander Stiftung Grant 2006.109.1, and Austrian Science Fund (FWF) Grant 18046-B12; and is part of the Translational Sarcoma Research Network supported by the BMBF.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. J.J.R. is a guest editor invited by the Editorial Board.

Data deposition: The sequence reported in this paper has been deposited in the Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE12692).

This article contains supporting information online at www.pnas.org/cgi/content/full/0810759106/DCSupplemental.

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