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...

Summary We examined the leukemic stem cell potential of blasts at different stages of maturation in childhood acute lymphoblastic leukemia. Human leukemic bone marrow was transplanted intrafemorally into NOD/scid mice. Cells sorted using the B precursor differentiation markers CD19, CD20 and CD34 were isolated from patient samples and engrafted mice before serial transplantation into primary or subsequent (up to quaternary) recipients. Surprisingly, blasts representative of all the different maturational stages were able to reconstitute and re-establish the complete leukemic phenotype in vivo. Sorted blast populations mirrored normal B precursor cells with transcription of a number of stage-appropriate genes. These observations have informed a model for leukemia-propagating stem cells in childhood ALL.

Background:Novel treatment strategies are needed to cure disseminated Ewing sarcoma. Primitive neuroectodermal features and a mesenchymal stem cell origin are both compatible with aberrant expression of the ganglioside antigen GD2 and led us to explore GD2 immune targeting in this cancer.Methods:We investigated GD2 expression in Ewing sarcoma by immunofluorescence staining. We then assessed the antitumour activity of T cells expressing a chimeric antigen receptor specific for GD2 against Ewing sarcoma in vitro and in vivo.Results:Surface GD2 was detected in 10 out of 10 Ewing sarcoma cell lines and 3 out of 3 primary cell cultures. Moreover, diagnostic biopsies from 12 of 14 patients had uniform GD2 expression. T cells specifically modified to express the GD2-specific chimeric receptor 14. G2a-28ζ efficiently interacted with Ewing sarcoma cells, resulting in antigen-specific secretion of cytokines. Moreover, chimeric receptor gene-modified T cells from healthy donors and from a patient exerted potent, GD2-specific cytolytic responses to allogeneic and autologous Ewing sarcoma, including tumour cells grown as multicellular, anchorage-independent spheres. GD2-specific T cells further had activity against Ewing sarcoma xenografts.Conclusion:GD2 surface expression is a characteristic of Ewing sarcomas and provides a suitable target antigen for immunotherapeutic strategies to eradicate micrometastatic cells and prevent relapse in high-risk disease.

Purpose: S100 proteins are implicated in metastasis development in several cancers. In this study, we analyzed the prognostic role of mRNA levels of all S100 proteins in early stage non^small cell lung cancer (NSCLC) patients as well as the pathogenetic of S100A2 in the development of metastasis in NSCLC. Experimental Design: Microarray data from a large NSCLC patient cohort was analyzed for the prognostic role of S100 proteins for survival in surgically resected NSCLC. Metastatic potential of the S100A2 gene was analyzed in vitro and in a lung cancer mouse model in vivo. Overexpression and RNAi approaches were used for analysis of the biological functions of S100A2. Results: High mRNA expression levels of several S100 proteins and especially S100A2 were associated with poor survival in surgically resected NSCLC patients. Upon stable transfection into NSCLC cell lines, S100A2 did not alter proliferation. However, S100A2 enhanced transwell migration as well as transendothelial migration in vitro. NOD/SCID mice injected s.c. with NSCLC cells overexpressing S100A2 developed significantly more distant metastasis (64%) than mice with control vector transfected tumor cells (17%; P < 0.05). When mice with S100A2 expressing tumors were treated i.v. with shRNA against S100A2, these mice developed significantly fewer lung metastasis than mice treated with control shRNA (P = 0.021). Conclusions: These findings identify S100A2 as a strong metastasis inducer in vivo. S100A2 might be a potential biomarker as well as a novel therapeutic target in NSCLC metastasis.

Open questions in the pathogenesis of childhood acute lymphoblastic leukemia (ALL) are which hematopoietic cell is target of the malignant transformation and whether primitive stem cells contribute to the leukemic clone. Although good-prognosis ALL is thought to originate in a lymphoid progenitor, it is unclear if this applies to high-risk ALL. Therefore, immature CD34 + CD19 À bone marrow cells from 8 children with ALL/t(9;22) and 12 with ALL/t(4;11) were purified and analyzed by fluorescence in situ hybridization, reverse transcription-PCR (RT-PCR), and colony assays. Fiftysix percent (n = 8, SD 31%) and 68% (n = 12, SD 26%) of CD34 + CD19 À cells in ALL/t(9;22) and ALL/t(4;11), respectively, carried the translocation. In addition, 5 of 168 (3%) and 22 of 228 (10%) myeloerythroid colonies expressed BCR/ABL and MLL/AF4. RT-PCR results were confirmed by sequence analysis. Interestingly, in some patients with ALL/t(4;11), alternative splicing was seen in myeloid progenitors compared with the bulk leukemic population, suggesting that these myeloid colonies might be part of the leukemic cell clone. Fluorescence in situ hybridization analysis, however, shows that none of these myeloid colonies (0 of 41 RT-PCRpositive colonies) originated from a progenitor cell that carries the leukemia-specific translocation. Thus, leukemic, translocation-positive CD34 + CD19 À progenitor/stem cells that were copurified by cell sorting were able to survive in these colony assays for up to 28 days allowing amplification of the respective fusion transcripts by sensitive RT-PCR. In conclusion, we show that childhood high-risk ALL/t(9;22) and t(4;11) originate in a primitive CD34 + CD19 À progenitor/stem cell without a myeloerythroid developmental potential. (Cancer Res 2005; 65(4): 1442-9)