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 ؊/؊ ␥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...

EZH2 promotes a bi-lineage identity in basal-like breast cancer cells

“…EZH2-silenced cells give rise to slow growing tumors, as has been previously shown (18)(19)(20)(21).…”

“…In breast cancers its expression is associated with high grade, ERnegative status, the basal-like subtype, and poor prognosis (13,15,16). EZH2 promotes cancer cell proliferation, anchorage-independent growth and invasiveness (13,(17)(18)(19). In vivo, EZH2 inhibition reduces tumor growth rates to various extents (18)(19)(20)(21).…”

“…EZH2 promotes cancer cell proliferation, anchorage-independent growth and invasiveness (13,(17)(18)(19). In vivo, EZH2 inhibition reduces tumor growth rates to various extents (18)(19)(20)(21). However, the functions of EZH2 in controlling cancer cell differentiation remain poorly understood.…”

The posterior HOXD locus: Its contribution to phenotype and malignancy of Ewing sarcoma

“…Consistent with these results, suppression of DKK2 by specific shRNA [5] in different ES lines resulted in a significant down-regulation of HOXD10, HOXD11 and HOXD13 ( Figure 1F). Further, down-regulation of TCF4, a transcription factor in the canonical WNT/β-catenin pathway, resulted in a partial inhibition of posterior www.impactjournals.com/oncotarget Posterior HOXD genes contribute to chondrogenic as well as bone associated gene expression in ES ES are bone or soft tissue neoplasms with a prominent immature stemness phenotype maintained by epigenetic repressors BMI1 and EZH2 [27,28]. Further, posterior HOXD genes regulated by EZH2 during development are known to influence the ossification pattern of bones [22], so it seemed relevant to investigate whether the expression of posterior HOXD genes in ES may influence their differentiation capacity.…”

Total body MRI-governed involved compartment irradiation combined with high-dose chemotherapy and stem cell rescue improves long-term survival in Ewing tumor patients with multiple primary bone metastases

“…[1][2][3][4][5] They are defined by a reciprocal chromosomal t(11;22)(q24;q12) translocation and the expression of ews/ets fusion genes, which permit a specific molecular genetic diagnosis of ET. 6,7 ET requires risk-adapted treatment and stage is the main risk factor.…”