PDF file - 5409KB, DKK2 knock-down increases apoptosis in vitro

PDF file - 3443KB, DKK2 enhances chondrogenic and osteogenic differentiation potential

PDF file - 2784KB, DKK2 expression in different small-round-blue cell tumors

<p>PDF file - 5289KB, DKK2 knock-down has no effect on angiogenesis</p>

PDF file - 150KB, Supplementary materials and methods, supplementary references and supplementary figure legends

PDF file - 7894KB, DKK2 expression in clinical samples

PDF file - 4007KB, DKK2 seems to be a Wnt agonist in ES

<p>PDF file - 5289KB, DKK2 knock-down has no effect on angiogenesis</p>

Abstract Ewing sarcoma (ES), a malignancy of bone and soft tissue that affects children and young adults, is characterized by early metastasis to lung and bone and by oncogenic ews/ets translocations. We previously observed the histone methyltransferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be regulated by EWS-FLI1 and highly increased in ES. We hypothesized non-coding RNAs to contribute to epigenetic processes and malignancy of ES. Here we show by ChIP on chip analysis that EZH2 occupies promoter regions of putative tumor suppressor miRNAs, oncogenic miRNAs and differentiation-associated miRNAs in ES and investigated their functional relevance by miRNA mimic, RNA interference, and functional assays. We show that hsa-miR-203a-3p (miR-203), a promoter of epidermal differentiation and trigger to exit cell cycle, was found epigenetic silenced in ES. We not only observed miR-203 up-regulation in different ES lines after EZH2 knock down or treatment with the EZH2 inhibitors GSK126 but also after histone de-acetylase inhibition (HDACi) via Trichostatin A (TSA) as well as MS-275. Similarly, treatment of ES lines with the DNA-methylation inhibitor 5′-azacytidine resulted in a strong up-regulation of miR-203 in ES. On the other hand, de-regulated expression of miR-203 after transient transfection with a miR-203-mimic identified a feed back loop resulting in a down-regulation of EWS-FLI1 and EZH2 in ES cell lines as compared to controls. In functional assays we observed that an increased expression of miR-203 suppressed contact dependent as well as independent growth in ES. In addition, in vitro invasiveness was strongly impaired after over-expression of miR-203. Taken together, we demonstrate the essential need for epigenetic suppression of miR-203 expression in ES that otherwise would suppress critical features of ES malignancy such as contact independent growth and invasiveness of ES. These results further provide a rationale for epigenetic combination therapy directed to reverse miR-203 suppression. Citation Format: Tim Hensel, Esther Heid, Stephanie Plehm, Stefan Burdach, Günther H. Richter. Epigenetic suppression of miRNA-203 promotes Ewing sarcoma malignancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 492. doi:10.1158/1538-7445.AM2015-492

Ewing sarcoma, an osteolytic malignancy that mainly affects children and young adults, is characterized by early metastasis to lung and bone. In this study, we identified the pro-metastatic gene DKK2 as a highly overexpressed gene in Ewing sarcoma compared with corresponding normal tissues. Using RNA interference, we showed that DKK2 was critical for malignant cell outgrowth in vitro and in an orthotopic xenograft mouse model in vivo. Analysis of invasion potential in both settings revealed a strong correlation of DKK2 expression to Ewing sarcoma invasiveness that may be mediated by the DKK effector matrix metalloproteinase 1 (MMP1). Furthermore, gene expression analyses established the ability of DKK2 to differentially regulate genes such as CXCR4, PTHrP, RUNX2, and TGFb1 that are associated with homing, invasion, and growth of cancer cells in bone tissue as well as genes important for osteolysis, including HIF1a, JAG1, IL6, and VEGF. DKK2 promoted bone infiltration and osteolysis in vivo and further analyses defined DKK2 as a key factor in osteotropic malignancy. Interestingly, in Ewing sarcoma cells, DKK2 suppression simultaneously increased the potential for neuronal differentiation while decreasing chondrogenic and osteogenic differentiation. Our results provide strong evidence that DKK2 is a key player in Ewing sarcoma invasion and osteolysis and also in the differential phenotype of Ewing sarcoma cells. Cancer Res; 73(2); 967–77. 2012 AACR.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. To identify novel candidates for targeted therapy, we performed a comprehensive transcriptome analysis identifying MondoA (MLXIP) - a transcription factor regulating glycolysis - to be overexpressed in ALL compared to normal tissues. Using microarray-profiling, gene-set enrichment analysis, RNA interference and functional assays we show that MondoA overexpression increases glucose catabolism and maintains a more immature phenotype, which is associated with enhanced survival and clonogenicity of leukemia cells. These data point to an important contribution of MondoA to leukemia aggressiveness and make MondoA a potential candidate for targeted treatment of ALL.

Abstract Ewing tumors comprise the second most common type of bone-associated cancer in children and are characterized by oncogenic EWS/FLI1 fusion proteins and early metastasis. Compelling evidence suggests that elevated levels of intracellular oxidative stress contribute to enhanced aggressiveness of numerous cancers, possibly including Ewing tumors. Using comprehensive microarray analyses and RNA interference, we identified the six-transmembrane epithelial antigen of the prostate 1 (STEAP1)—a membrane-bound mesenchymal stem cell marker of unknown function—as a highly expressed protein in Ewing tumors compared with benign tissues and show its regulation by EWS/FLI1. In addition, we show that STEAP1 knockdown reduces Ewing tumor proliferation, anchorage-independent colony formation as well as invasion in vitro and decreases growth and metastasis of Ewing tumor xenografts in vivo. Moreover, transcriptome and proteome analyses as well as functional studies revealed that STEAP1 expression correlates with oxidative stress responses and elevated levels of reactive oxygen species that in turn are able to regulate redox-sensitive and proinvasive genes. In synopsis, our data suggest that STEAP1 is associated with the invasive behavior and oxidative stress phenotype of Ewing tumors and point to a hitherto unanticipated oncogenic function of STEAP1. Mol Cancer Res; 10(1); 52–65. ©2011 AACR.

Abstract Although microRNAs (miRNA) were shown to be involved in cancer progression of many tumor entities, little is known about the role of non-coding RNAs in Ewing Tumor (ET) pathogenesis. The highly malignant ET comprise the second most common type of bone associated cancers in children and are characterized by oncogenic ews/ets translocations. We previously observed the histone methyltransferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be regulated by EWS-FLI1 and highly increased in ET. Here we demonstrate that EZH2 occupies promotor regions of putative tumor suppressor miRNAs and differentiation associated miRNAs in ET. We show that EZH2 mediates silencincig of miRNA expression via histone modification as well as DNA methylation and that these miRNAs are reactivated upon EZH2 knock down or treatment with inhibitors histone deacetylation and DNA methylation. In addition, we show that the well-known oncogenic miRNA221 is highly expressed in ET and processed in the presence of Argonaute (AGO) protein 2. Using RNA interference we observed that AGO1 and AGO2 mediated processes affect cellular invasiveness and anchorage-independent proliferation in vitro and influence ET growth and metastasis in immunodeficient Rag2−/−γC−/− mice, confirming an involvement of non-coding RNAs in ET pathogenesis. Taken together, these data illuminate the hitherto unknown epigenetic regulation of miRNA expression by EZH2 in ET and a general involvement of noncoding RNA in ET-pathogenesis, opening the avenue for new therapeutic modalities i.e. the implementation of miRNA therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C23.

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 methyl-transferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. EZH2’s suppressive activity 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, GAP43). These data suggest that EZH2 might play a central role in Ewing Tumor pathology by shaping the oncogenicity and stem cell phenotype of this tumor.

Chromatin modifications are increasingly recognized as a key mechanism in cancer. The histone methyl-transferase Enhancer of Zeste, Drosophila, Homolog 2 (EZH2), the enzymatic subunit of the polycomb PRC2 complex methylates histone H3K27, thereby, mediating gene silencing. EZH2 is over-expressed in a variety of tumor tissue including breast and prostate. Ewing Tumors (ET), alias Peripheral NeuroEctodermal Tumors (PNET), are highly malignant tumors molecularly defined by ews/ets translocations. We found EWS-FLI1 bound to the EZH2 promoter in vivo. Other components of the PRC2 complex, like EED or SUZ12 were not deregulated in ET. Down-regulation of EZH2 by RNA interference suppressed tumor development and metastasis in vivo and microarray analysis of EZH2 knock down revealed an EZH2-maintained, undifferentiated, reversible phenotype in ET. EZH2 suppression resulted in a generalized loss of H3K27me3 as well as increase in H3 acetylation. ChIP-Chip assays for H3K27me3 verified such genes that had specifically lost H3K27me3 upon EZH2 silencing, suggesting that stemness features are preserved via epigenetic mechanisms. Taken together, the genetic EWS-Fli1 translocation is intimately linked to global and gene specific epigenetic alterations in ET biology. EZH2 mediates neuroectodermal and endothelial embryonal tumor stem cell growth and metastatic spread induced by a translocation derived chimeric transcription factor.

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(-/-)gamma(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.