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 ؊/؊ ␥C ؊/؊ 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.epigenetic regulation ͉ Ewing tumor ͉ stemness E wing tumors (ET) are highly malignant tumors with an approximate incidence of 3.3/10 6 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 ident...

Osteoarthritis is a degenerative joint disease that ranks among the leading causes of adult disability. Mechanisms underlying osteoarthritis pathogenesis are not yet fully elucidated, putting limits to current disease management and treatment. Based on the phenomenological evidence for dysregulation within the glycome of chondrocytes and the network of a family of adhesion/growth-regulatory lectins, that is, galectins, we tested the hypothesis that Galectin-1 is relevant for causing degeneration. Immunohistochemical analysis substantiated that Galectin-1 upregulation is associated with osteoarthritic cartilage and subchondral bone histopathology and severity of degeneration (p < 0.0001, n = 29 patients). In vitro, the lectin was secreted and it bound to osteoarthritic chondrocytes inhibitable by cognate sugar. Glycan-dependent Galectin-1 binding induced a set of disease markers, including matrix metalloproteinases and activated NF-κB, hereby switching on an inflammatory gene signature (p < 10−16). Inhibition of distinct components of the NF-κB pathway using dedicated inhibitors led to dose-dependent impairment of Galectin-1–mediated transcriptional activation. Enhanced secretion of effectors of degeneration such as three matrix metalloproteinases underscores the data’s pathophysiological relevance. This study thus identifies Galectin-1 as a master regulator of clinically relevant inflammatory-response genes, working via NF-κB. Because inflammation is critical to cartilage degeneration in osteoarthritis, this report reveals an intimate relation of glycobiology to osteoarthritic cartilage degeneration.

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Although p53 is the most frequently mutated gene in cancer, half of human tumors retain wild-type p53, whereby it is unknown whether normal p53 function is compromised by other cancer-associated alterations. One example is Ewing's sarcoma family tumors (ESFT), where 90% express wild-type p53. ESFT are characterized by EWS-FLI1 oncogene fusions. Studying 6 ESFT cell lines, silencing of EWS-FLI1 in a wild-type p53 context resulted in increased p53 and p21 WAF1/CIP1 levels, causing cell cycle arrest. Using a candidate gene approach, HEY1 was linked to p53 induction. HEY1 was rarely expressed in 59 primary tumors, but consistently induced upon EWS-FLI1 knockdown in ESFT cell lines. The NOTCH signaling pathway targets HEY1, and we show NOTCH2 and NOTCH3 to be expressed in ESFT primary tumors and cell lines. Upon EWS-FLI1 silencing, NOTCH3 processing accompanied by nuclear translocation of the activated intracellular domain was observed in all but one p53-mutant cell line. In cell lines with the highest HEY1 induction, NOTCH3 activation was the consequence of JAG1 transcriptional induction. JAG1 modulation by specific siRNA, NOTCH-processing inhibition by either GSI or ectopic NUMB1, and siRNA-mediated HEY1 knockdown all inhibited p53 and p21 WAF1/CIP1 induction. Conversely, forced expression of JAG1, activated NOTCH3, or HEY1 induced p53 and p21 WAF1/CIP1 . These results indicate that suppression of EWS-FLI1 reactivates NOTCH signaling in ESFT cells, resulting in p53-dependent cell cycle arrest. Our data link EWS-FLI1 to the NOTCH and p53 pathways and provide a plausible basis both for NOTCH tumor suppressor effects and oncogenesis of cancers that retain wild-type p53.

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-1α 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-1α-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. Cancer Res; 70(10); 4015-23. ©2010 AACR.