Article

Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells

Department of Pediatrics, University of Michigan, Ann Arbor, Michigan, United States of America.
PLoS ONE (Impact Factor: 3.23). 04/2011; 6(4):e19305. DOI: 10.1371/journal.pone.0019305
Source: PubMed

ABSTRACT

Ewing sarcoma family tumors (ESFT) are aggressive bone and soft tissue tumors that express EWS-ETS fusion genes as driver mutations. Although the histogenesis of ESFT is controversial, mesenchymal (MSC) and/or neural crest (NCSC) stem cells have been implicated as cells of origin. For the current study we evaluated the consequences of EWS-FLI1 expression in human embryonic stem cell-derived NCSC (hNCSC). Ectopic expression of EWS-FLI1 in undifferentiated hNCSC and their neuro-mesenchymal stem cell (hNC-MSC) progeny was readily tolerated and led to altered expression of both well established as well as novel EWS-FLI1 target genes. Importantly, whole genome expression profiling studies revealed that the molecular signature of established ESFT is more similar to hNCSC than any other normal tissue, including MSC, indicating that maintenance or reactivation of the NCSC program is a feature of ESFT pathogenesis. Consistent with this hypothesis, EWS-FLI1 induced hNCSC genes as well as the polycomb proteins BMI-1 and EZH2 in hNC-MSC. In addition, up-regulation of BMI-1 was associated with avoidance of cellular senescence and reversible silencing of p16. Together these studies confirm that, unlike terminally differentiated cells but consistent with bone marrow-derived MSC, NCSC tolerate expression of EWS-FLI1 and ectopic expression of the oncogene initiates transition to an ESFT-like state. In addition, to our knowledge this is the first demonstration that EWS-FLI1-mediated induction of BMI-1 and epigenetic silencing of p16 might be critical early initiating events in ESFT tumorigenesis.

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    • "Among them are EZH2 and BMI-1 and, as Inmaculada Hernandez and Jaume Mora reported, RING1B (RNF2), which affects genes of heme biosynthesis, endothelial and neural development, and which they found to protect Ewing sarcoma cells from NFκB induced apoptosis through regulation of the sodium channel NaV1.6. Summarizing available evidence, Elizabeth Lawlor speculated that BMI- 1 positive cells may provide the permissive environment for fusion gene activity, as has previously been reported for E2A-PBX1 in hematopoietic stem cells[16], and more recently demonstrated by her own group for EWS-FLI1 in neural crest derived stem cells (NCSC)[17]. The chimeric oncogene would then perpetuate the progenitor-like state by hijacking the developmental transcription program. "
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    ABSTRACT: Despite multimodal treatment, long term outcome for patients with Ewing sarcoma is still poor. The second "European interdisciplinary Ewing sarcoma research summit" assembled a large group of scientific experts in the field to discuss their latest unpublished findings on the way to the identification of novel therapeutic targets and strategies. Ewing sarcoma is characterized by a quiet genome with presence of an EWSR1-ETS gene rearrangement as the only and defining genetic aberration. RNA-sequencing of recently described Ewing-like sarcomas with variant translocations identified them as biologically distinct diseases. Various presentations adressed mechanisms of EWS-ETS fusion protein activities with a focus on EWS-FLI1. Data were presented shedding light on the molecular underpinnings of genetic permissiveness to this disease uncovering interaction of EWS-FLI1 with recently discovered susceptibility loci. Epigenetic context as a consequence of the interaction between the oncoprotein, cell type, developmental stage, and tissue microenvironment emerged as dominant theme in the discussion of the molecular pathogenesis and inter- and intra-tumor heterogeneity of Ewing sarcoma, and the difficulty to generate animal models faithfully recapitulating the human disease. The problem of preclinical development of biologically targeted therapeutics was discussed and promising perspectives were offered from the study of novel in vitro models. Finally, it was concluded that in order to facilitate rapid pre-clinical and clinical development of novel therapies in Ewing sarcoma, the community needs a platform to maintain knowledge of unpublished results, systems and models used in drug testing and to continue the open dialogue initiated at the first two Ewing sarcoma summits.
    Full-text · Article · Jan 2016 · Oncotarget
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    • "The EWS-FLI-GFP expression vector was generously provided by Dr. Elizabeth R. Lawlor (University of Michigan, Ann Arbor, MI, USA). Lentiviral transductions were performed following a previously described protocol [14]. EWS-GFP cells were cultured in DMEM supplemented with 10% (v/v) Hyclone FBS and 1% penicillin/streptomycin. "
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    ABSTRACT: Monolayer cultures of tumor cells and animal studies have tremendously advanced our understanding of cancer biology. However, we often lack animal models for human tumors, and cultured lines of human cells quickly lose their cancer signatures. In recent years, simple 3D models for cancer research have emerged, including cell culture in spheroids and on biomaterial scaffolds. Here we describe a bioengineered model of human Ewing's sarcoma that mimics the native bone tumor niche with high biological fidelity. In this model, cancer cells that have lost their transcriptional profiles after monolayer culture re-express genes related to focal adhesion and cancer pathways. The bioengineered model recovers the original hypoxic and glycolytic tumor phenotype, and enables re-expression of angiogenic and vasculogenic mimicry features that favor tumor adaptation. We propose that differentially expressed genes between the monolayer cell culture and native tumor environment are potential therapeutic targets that can be explored using the bioengineered tumor model.
    Full-text · Article · Apr 2014 · Biomaterials
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    • "To determine if the expression of putative miR-31 target genes was altered in ES we used Affymetrix GeneChip Human 1.0 ST arrays to profile a subset of 20 primary ES that had been used for miRNA profiling as above. Transcript summarized data from ES were compared to adult bone marrow MSCs as previously described [13]. All raw cel files were normalized together using gcRMA and transcript summarized in Partek Genomics Suite (Partek, St. Louis, MO, USA). "
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    ABSTRACT: Ewing sarcoma, the second most common bone tumor in children and young adults, is an aggressive malignancy with a strong potential to metastasize. Ewing sarcoma is characterised by translocations encoding fusion transcription factors with an EWSR1 transactivation domain fused to an ETS family DNA binding domain. microRNAs are post-transcriptional regulators of gene expression and aberrantly expressed microRNAs have been identified as tumor suppressors or oncogenes in most cancer types. To identify potential oncogenic and tumor suppressor microRNAs in Ewing sarcoma, we determined and compared the expression of 377 microRNAs in 40 Ewing sarcoma biopsies, 6 Ewing sarcoma cell lines and mesenchymal stem cells, the putative cellular origin of Ewing sarcoma, from 6 healthy donors. Of the 35 differentially expressed microRNAs identified (fold change >4 and q<0.05), 19 were higher and 16 lower expressed in Ewing sarcoma. In comparisons between Ewing sarcoma samples with EWS-FLI or EWS-ERG translocations, with differing dissemination characteristics and of primary samples and metastases no significantly differential expressed microRNAs were detected using various stringency criteria. For miR-31, the microRNA with lowest expression in comparison to mesenchymal stem cells, functional analyses were performed to determine its potential as a tumor suppressor in Ewing sarcoma. Two of four miR-31 transfected Ewing sarcoma cell lines showed a significantly reduced proliferation (19% and 33% reduction) due to increased apoptosis in one and increased length of G1-phase in the other cell line. All three tested miR-31 transfected Ewing sarcoma cell lines showed significantly reduced invasiveness (56% to 71% reduction). In summary, we identified 35 microRNAs differentially expressed in Ewing sarcoma and demonstrate that miR-31 affects proliferation and invasion of Ewing sarcoma cell lines in ex vivo assays.
    Full-text · Article · Mar 2014 · PLoS ONE
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