Modeling Initiation of Ewing Sarcoma in Human Neural
Cornelia von Levetzow2, Xiaohua Jiang1, Ynnez Gwye1, Gregor von Levetzow2, Long Hung1, Aaron
Cooper1, Jessie Hao-Ru Hsu2, Elizabeth R. Lawlor2*
1Department of Pediatrics, Children’s Hospital Los Angeles, Los Angeles, California, United States of America, 2Departments of Pediatrics and Pathology, University of
Michigan, Ann Arbor, Michigan, United States of America
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.
Citation: von Levetzow C, Jiang X, Gwye Y, von Levetzow G, Hung L, et al. (2011) Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells. PLoS
ONE 6(4): e19305. doi:10.1371/journal.pone.0019305
Editor: Laszlo Tora, Institute of Genetics and Molecular and Cellular Biology, France
Received January 14, 2011; Accepted March 29, 2011; Published April 29, 2011
Copyright: ? 2011 von Levetzow et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Grant support for this work was provided by CIRM SEED Grant RS1-00249, R01 CA134604, U01 CA114757, SU2C-AACR-IRG1309, and by CIRM Training
Grants T2-00005 (to CvL and XJ) and T1-00004 (to JH-RH). Philanthropic support from the My Brother Joey, T. J. Martell and Bogart Foundations is gratefully
acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Ewing’s sarcoma family tumors (ESFT) are aggressive bone and
soft tissue tumors that primarily affect children and young adults
. They are largely undifferentiated tumors that are genetically
characterized by expression of fusion oncogenes resulting from
chromosomal translocations involving EWSR1 (EWS), (or rarely
TLS), and FLI1 or another member of the ETS family of
transcription factors . Despite its action as an oncogene in
ESFT, EWS-FLI1 is toxic to most cells, inducing arrest and death
[3,4]. Although experimental inactivation of p53 and p16 in
primary cells can induce tolerance to EWS-FLI1, genetic
mutations in these tumor suppressors are present in only a
minority of ESFT . Thus, alternate mechanisms of tumor
suppressor gene inactivation may exist in the ESFT cell of origin.
The cellular origin of ESFT remains both elusive and controver-
sial. Recent studies have shown that bone marrow-derived human
mesenchymal stem cells (BM-MSC) are permissive for EWS-FLI1
and that ectopic expression of EWS-FLI1 in these cells initiates
transition to an ESFT-like cellular phenotype [6,7]. However, the
immature neural phenotype of many tumors, along with their gene
expression signatures and their disposition to neural differentiation
also implicate a neural crest origin [8,9,10,11]. Like ESFT cells,
neural crest stem cells (NCSC) are highly migratory and invasive and
. Significantly, studies in model organisms have additionally
shown that some MSC are derived from the neural crest [13,14].
Thus, this partially shared ontogeny leads to the unifying hypothesis
that ESFT might arise from malignant transformation of MSC of
either mesodermal or neural crest origin .
Studies in chick embyros have demonstrated that expression of
EWS-FLI1 disrupts normal NCSC development . However,
studies with human neural crest cells have not yet been reported
and have been challenged by the very transient nature of the
neural crest in early embryogenesis and the rarity of NCSC in
post-natal tissues. We recently established an efficient method to
generate NCSC from in vitro differentiating human embryonic
stem cells (hESC) . These cells display the genetic, phenotypic
and functional characteristics of NCSC and differentiate in vitro
and in vivo into neural crest derivatives . For the current study
we used this model to study the consequences of EWS-FLI1
expression in human NCSC and their progeny.
Materials and Methods
All human tumor specimens were obtained in compliance with
HIPAA regulations and following protocol review by the
PLoS ONE | www.plosone.org1 April 2011 | Volume 6 | Issue 4 | e19305
Committee for Clinical Investigation at Children’s Hospital Los
Angeles. Samples were provided to investigators as anonymized
RNA with no links to patient identifiers. The study (05-545) was
reviewed in an expedited manner on 2/12/2007 and was
approved and deemed to meet the criteria for non-human subjects
research and for a waiver of authorization/informed consent. All
animal studies were performed following full protocol review and
approval by the Institutional Animal Care and Usage Committee
(IACUC) of Children’s Hospital Los Angeles (protocol 216-07).
WA-09 hESC were purchased from Wicell (Madison, WI) and
hESC-derived neural crest stem cells (hNCSC) generated as
described . FACS-sorted p75+ hNCSC cells were maintained
in self-renewal medium (DMEM-F12 (1:1) N2 and B27, 20 ng/ml
bFGF, 20 ng/ml EGF, 20 nM IGF-1 (all from Gibco), 0.1 mM b-
mercaptoethanol) on 6-well ultra-low attachment plates (Corning,
Lowell, MA) at a density of 56103cells/ml. To promote generation
of MSC-like cells, hNCSC were plated at a cell density of 10–
206103cells/cm2in self-renewal media on 6-well plates that were
pre-coated with 15 mg/ml Polyornithine (Sigma), 1 mg/ml laminin
(Millipore) and 10 mg/ml fibronectin (Invitrogen). Three unique
human bone marrow-derived MSC (BM-MSC) lines were obtained
from Dr. D. Prockop (Tulane University) and maintained at low
density in aMEM with 10% FBS (Invitrogen), L-Glutamine, NEM
Nonessential Amino Acids and Sodium Pyruvate (all from Cellgro).
All studies were performed with the approval of Institutional
Human Pluripotent Stem Cell Research Oversight Committees.
For non-specific neural crest differentiation of hNCSC, self-
renewal media was changed to DMEM/F12 (1:1) supplemented with
N2, B27 and 5% HycloneH fetal bovine serum (FBS) (Thermo
scientific). For adipogenic differentiation, media of confluent cellswas
changed to aMEM (Cellgro) containing 10% FBS, 10 mg/ml insulin
(Sigma), 10 mM Dexamethasone (Sigma) and 0.5 mM IsoButyl-
MethylXanthine (Sigma). For osteogenic differentiation, media was
changed to Minimum Essential Medium, Alpha 16 (Cellgro)
containing 10% FBS, 10 mM b-glycerolphosphate (Sigma), 0.1 mM
Dexamethasone (Sigma) and 200 mM L-ascorbic acid (Sigma).
Defined media were changed every 3 days and cells fixed in 4%
paraformaldehyde after 21 days. Terminal adipocytic and osteoblas-
tic differentiation was assessed by staining fixed cells with 0.5% Oil
Red-O (Sigma) and 1% Alizarin Red S (Sigma), respectively.
Immunostaining and Western blot
Cells were grown in pre-coated chamber slides (LAB-TEK, Nun
International), fixed in 4% paraformaldehyde and permeabilized
with 0.1% Triton X-100. After rinsing in PBS the slides were
incubated for one hour in blocking solution (1% BSA and 5%
donkey serum in PBS). Blocked slides were incubated with primary
antibodies for 1 hr at 37uC, washed in PBS, incubated with
fluorescent-labeled secondary antibodies for 1 hr and then
visualized with a Lexica DM RXA Upright Fluorescence
Microscope (Applied Spectral Imaging, Inc., Carlsbad, CA).
Primary antibodies used were: V5 (1:500, Invitrogen), GFP
(1:1000, Abeam, Cambridge, MA). Secondary antibodies were:
Alexi fluorophore 488- conjugated Donkey-Anti Rabbit (1:1000,
Molecular Probes- Invitrogen) and Cy3-conjugated Goat Anti-
Mouse (1:500; Jackson Immune Research Laboratories). In some
samples, nuclei were counterstained with 49, 6-diamidino-2-
phenylindole (DAPI). Western blots were performed using
standard procedures and the following antibodies at 1:1000
dilutions: V5 (Invitrogen), BMI-1 (Millipore); EZH2, GAPDH,
Cyclin D1 (Cell Signaling Technology); p16, Actin, (Santa Cruz
Biotechnology); p21 (Abcam); p53 (1:2000, Cell signaling).
Flow cytometry and cell growth assays
Cells were dissociated with Accutase (Millipore) and washed
with L15 medium with 10 mM HEPES and 1 mg/ml BSA.
Resuspended cells were blocked with anti-human Fc-receptor
(Miltenyi Biotec, Germany) and incubated with antibodies for
10 min at 4uC in the dark (phycoerythrin (PE)-conjugated anti-
p75, PE-CD29, PE-CD73, Allophycocyanin (APC)-conjugated
CD44 (Miltenyi Biotec, Germany), PE-CD105 (Santa Cruz
Biotechnology), APC-CD90 (BD Biosciences Pharmingen) and
PE Mouse IgG2ak-, PE Mouse IgG2b-, PE Mouse IgG1k-, APC
Mouse IgG1k-isotope controls (all BD Biosciences Pharmingen).
For the p75 and CD73 double staining cells were incubated with
PE-p75 and CD73 (BD Pharmingen) antibodies followed by a
FITC-conjugated goat anti mouse antibody (Jackson ImmunoR-
esearch, West grove, PA). Cells were washed with staining media
and then analyzed using a FACS Caliber instrument (BD
Biosciences, San Jose, CA). Gates were defined relative to
untransduced and isotope-control cells and FCS Express software
package (De Novo Software) used for data analysis. To isolate GFP
positive populations, dissociated cells were sorted on a FACS
Vantage or FACS Aria (BD Biosciences). Cell cycle analysis was
performed on a FACScan instrument (Becton Dickinson) and data
analyzed using Flow-Jo (Tree Star, Ashland, OR). Beta-galacto-
sidase staining for cellular senescence was performed as previously
described . To assess p53 function cells were exposed to 1500
Rad gamma irradiation and whole cell lysates collected after
EWS-FLI1 expression construct and BMI-1 knockdown
The coding sequence of EWS-FLI1, minus its stop codon, was
PCR amplified from pcDNA/TO-EF (kindly provided by Dr. T.
Triche) and inserted into pENTR/D-TOPO (Invitrogen, Carls-
bad, CA). EWS-FLI1 was then cloned in-frame into pLenti4/TO/
V5-DEST (Invitrogen) by LR recombination according to the
manufacturer’s instructions. EWS-FLI1-V5 was PCR-amplified
from the pLenti4/TO/V5-DEST vector, following site-directed
mutagenesis of the internal EcoRI site (conserving amino acid
sequence), and cloned into the NheI and EcoRI restriction sites of
the pCLS backbone to generate pCLS-EF. The pCLS lentiviral
vector is a derivative of pCL1 , modified to include a foot-and-
mouth-disease virus (FMDV)-derived 2A self-cleaving peptide
sequence in place of the internal ribosomal entry site upstream of
the EGFP reporter gene . Furin cleavage sites (R-A-K-R) were
inserted between EWS-FLI1 and the 2A sequence in order to
achieve full cleavage of the translated EWS-FLI1 protein from
residual 2A peptide [21,22]. Empty vector with 2A-EGFP alone
served as a control (pCLS-CV).
For the BMI-1 knock down experiments, the pCLS lentiviral
backbone was modified as follows: First, the rtTA IRES Puro
expression cassette was amplified from pTRIPZ (Open Biosys-
tems, Huntsville, AL) using the following primers: 59 TC-
and 59 TTTACTTGTACATCAGGCACCGGGCTTGCGG 39.
The resulting PCR product was then cut with AscI and BsrGI
(both New England Biolabs, Ipswich, MA) and ligated into pCLS.
Two additional restriction enzyme sites (AgeI and NheI) were
inserted into the vector upstream of the SFFV U3 promoter by site
directed mutagenesis using 59-phosphorylated oligonucleotides
(fw: 5-GGATCCACCGGTCGCCACCATGAGCGA-39 and rev:
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Finally, the expression cassette containing the TRE/CMV
minimal promoter, tRFP and the shRNA cassette was amplified
from pTRIPZ using the following primers: 59-CCCGGGT-
The resulting PCR product was cut with BspEI and NheI and
ligated into AgeI and NheI sites in the modified pCLS vector to
generate an shRNA-cassette-ready TRE/tRFP/rtTA/IRES/Puro
backbone (pCLSTRRIP). To selectively target BMI-1 for
knockdown, a miR-30-based shRNA expression cassette was
cloned by primer-extension PCR using the following primers:
TGACAGTGAGCG 39, miR30 rev: 59-CTAAAGTAGCCC-
CTTGAATTCCGAGGCAGTAGGCA-39 and BMI1-shRNA:
sequence, underlined, is flanked by miR-30 sequence). The
resulting PCR product was cut with XhoI and EcoRI and ligated
into pCLSTRRIP. All PCRs were carried out using Phusion High
Fidelity Polymerase (New England Biolabs, Ipswich, MA) and the
resulting vectors were sequenced verified. Inert non-silencing
shRNA sequence was excised from control pTRIPZ vector (Open
Biosystems, Huntsville, AL) and cloned into pCLSTRRIP using
EcoRI and XhoI restriction sites.To activate BMI-1 knockdown
100 ng/ml doxycyline was added to the culture media of stably
To generate viral supernatant, 293FT cells were transfected
with pCLS-CV or pCLS-EF, the packaging plasmid pCD/NL-
BH*DDD (Addgene plasmid 17531) and pMD2.G (VSV-G;
Addgene plasmid 12259) using Polyethylenimine (1 mg/ml,
Sigma-Aldrich). The day after transfection, cells were cultivated
for 6–8 hours with sodium butyrate (10 mM) and viral superna-
tant collected after 48 hrs. Freshly isolated hNCSC were
transduced with concentrated EWS-FLI1 or EGFP virus at a
multiplicity of infection of 2. Transduction efficiencies were
reproducibly around 80%. Infected cells were isolated by FACS-
sorting for EGFP expression. For BMI-1 knockdown studies 293T
cells were transfected with packaging plasmids as above along with
inducible shBMI1 or shNS constructs. Viral supernatant was
collected and EF-NC cells were transduced with shBMI1 or shNS
virus and then selected in puromycin (2 ug/ml) for 24 hrs prior to
treatment with doxycyline. Cells were cultured in puromycin and
doxycyline for one week.
Gene expression studies
Total RNA was isolated using the miRNeasy kit with in-column
DNAse I treatment (Qiagen). cDNA generated with iScript
Figure 1. hNCSC have mesenchymal differentiation potential. (A) Within 2 days of transfer to adherent plates p75+ hNCSC initiated a change
from small, NCSC-like to larger MSC-like cells. By 5 days all cells were mesenchymal in appearance (In all images scale bars=100 mm). (B) Oil-Red O-
and Alizarin Red staining after 3 weeks in defined media confirmed that hNCSC can be induced to differentiate into adipocytes and osteoblasts,
respectively, albeit less robustly than BM-MSC. Simliarly stained BM-MSC that had been exposed to standard growth media are shown as negative
controls. (C) RT-PCR showed that hNCSC express the mesenchymal marker VIM (vimentin) as well as NCSC genes B3GAT1 (HNK-1), NGFR (p75), and NES
(nestin). In contrast, BM-MSC (3 different lines as detailed in methods) do not express NCSC genes. BM-MSC: bone marrow-derived MSC; hNCSC: hESC-
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(Bio-Rad, Hercules, CA) was amplified by PCR using standard
conditions (Primer information provided in Table S4). Quantita-
tive reverse transcriptase PCR (RT-PCR) was performed using
validated TaqMan Assays (Applied Biosystems) on an Applied
Biosystems 7900HT PCR system and expression normalized
relative to GAPDH as described . For whole genome
expression assays, total RNA was processed and hybridized to
Affymetrix HuEx 1.0 arrays in the CHLA/USC Genome Core
according to Affymetrix protocols. RNA from primary tumors was
obtained from Children’s Oncology Group and CHLA tumor
banks. HuEx cel files from normal human tissues were
downloaded from the Affymetrix Netaffx website (www.netaffx.
com). Cell line data were kindly provided by Dr. T. Triche.
Normalization and statistical analysis of microarray data were
performed as detailed below. Gene expression data are available in
the GEO repository (GSE21511).
Affymetrix HuEx data analysis
Data for core probeset regions were quantile normalized using
robust multi-chip averaging in the Partek Genomics Suite software
platform (Partek, St. Louis, Mo). Transcript level data were
derived from normalized exon data using median summarization
and all further analyses performed on transcript-summarized data.
To identify EWS-FLI1 targets, data from control vector-
transduced and EWS-FLI1-transduced samples from 3 indepen-
dent experiments were normalized as above and EWS-FLI1
targets identified by 2-way ANOVA, controlling for the effects of
experimental batch. Analysis of gene ontology was performed
using DAVID bioinformatics resources .
hNCSC possess mesenchymal differentiation potential
Developmental models have recently established that small
numbers of MSC are derived from the neural crest [13,14,25].
Given the neural crest phenotype and putative MSC origin of
ESFT, these cells are attractive candidate cells of origin. We have
previously shown that multipotent, self-renewing hNCSC can be
isolated from hESC and that these cells can be differentiated in vitro
and in vivo into neural crest progeny . To more fully define
their mesenchymal potential hNCSC were generated as previously
described [7,17,26] and then cultured in adherent conditions on
poly-l-ornithine, laminin and fibronectin coated plates. After 2
days in adherent conditions small cuboidal hNCSC were observed
to adopt a larger more mesenchymal morphology (Fig. 1A) and
exposure of these cells to defined media induced differentiation
Figure 2. Flow cytometry confirms MSC markers in adherent hNCSC. hNCSC were isolated from in vitro differentiating hESC using p75-FACS.
Nearly 50% of the cells also expressed the MSC marker CD73 on Day 0. After 2 days in adherent conditions the morphology of isolated cells changed
from small cuboidal cells to larger mesenchymal cells (see Fig. 1A). Consistent with this morphologic change flow cytometric analysis shows that after
5 days in culture most cells continue to express p75 but, in addition, nearly all cells express the MSC-associated markers CD73, CD105, CD90, CD29,
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into adipogenic and osteogenic progeny (Fig. 1B). Importantly,
however, despite rapid adoption of a phenotype reminiscent of
BM-MSC, freshly isolated hNCSC cells were distinct from BM-
MSC in their expression of NCSC-associated genes. (see Fig. 1C
and discussion of microarray data below). Flow cytometric
analyses confirmed that after several days in adherent culture
these hNCSC-derived MSC (hereafter termed hNC-MSC)
expressed high levels of both p75 as well as MSC markers
CD73, CD44, CD29, CD105 and CD90 (Fig. 2). In addition,
hNC-MSC retained the ability to form peripherin-positive
neurons thus demonstrating their multi-potent neuro-mesenchy-
Undifferentiated hNCSC and hNC-MSC are permissive for
To establish whether hNCSC are permissive for EWS-FLI1,
freshly sorted p75+ cells were transduced using an EWS-FLI1
lentiviral vector (Fig. 3A) and transferred to neurosphere
conditions within 24 hrs. Both EWS-FLI1 (EF-NC) and control
vector-transduced (CV-NC) cells rapidly generated EGFP+
spheres that continued to grow in size confirming both successful
transduction and retention of the ability to proliferate as neuro-
spheres (Fig. 3B). RT-PCR confirmed expression of EWS-FLI1 as
well as induction of known downstream target genes NKX2-2 and
ID2 (Fig. 3C). Similarly, when transferred to adherent conditions
and passaged as hNC-MSC, cells continued to expand and no
substantial differences in cell proliferation or death were observed
between control and EWS-FLI1+ cells (Fig. 3D).
EWS-FLI1 modulates expression of previously
characterized as well as novel target genes in hNC-MSC
To characterize the effects of EWS-FLI1 on gene expression in
hNC-MSC, transduced cells were expanded in self-renewal media
for 5 days in adherent conditions and then profiled using
Affymetrix arrays. Over 800 transcripts were significantly
modulated by EWS-FLI1 (FDR,0.1 and fold change .1.5;
Table S1A). Comparison of EWS-FLI1 targets in hNC-MSC to
previously published studies of EWS-FLI1-transduced BM-MSC
[7,26] revealed moderate overlap with pediatric BM-MSC but
only minimal overlap with adult BM-MSC (306 vs. 154 genes in
common) (Table S1B). Overlap between EWS-FLI1 target genes
in hNC-MSC and ESFT cell lines  was also minimal with only
68 genes being commonly regulated in both model systems (Table
S1B). This analysis reaffirms the cell-context dependency of many
EWS-FLI1 targets but also identifies a core set of genes that are
regulated by EWS-FLI1 irrespective of cell type (Table 1).
ESFT are genetically similar to hNCSC and EWS-FLI1
initiates reprogramming of hNC-MSC to a more primitive
Malignant tumors often share genetic and phenotypic charac-
teristics of the normal cells from which they arose. In attempt to
Figure 3. hNCSC and hNC-MSC tolerate expression of EWS-FLI1. (A) pCLS-EWS-FLI1-V5-2A-EGFP lentiviral expression vector. (B) hNCSC
transduced with either control (CV)- or EWS-FLI1-vectors (EF) formed neurospheres in non-adherent culture (phase contrast (top) and GFP-filters
(bottom) 1006). (C) RT-PCR confirmed expression of EWS-FLI1 in EF cells and also induction of known EWS-FLI1 target genes NKX2.2 and ID2. (Results
are shown for 3 independent experiments). (D) Cell cycle was assessed in adherent EGFP+ hNC-MSC 6 weeks after lentiviral transduction. Western
blot (left) confirmed continued robust expression of the EWS-FLI1-V5 protein and flow cytometric analysis of DNA content (right) showed continued
proliferation and minimal cell death in both EF and CV cells in 2 independent experiments.
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define the cell of origin of ESFT prior studies have used gene
expression profiling to compare established tumors to different cell
and tissue types [7,11,27,28]. Although these studies collectively
favor either an MSC or neural crest stem or progenitor cell of
origin, to date no direct comparison between ESFT and NCSC
has been reported. We therefore performed unsupervised
clustering of normalized expression array data from 10 primary
ESFT, 10 ESFT cell lines, hNCSC, hNC-MSC, BM-MSC and 11
different normal adult tissues. As shown, consistent with earlier
studies, the gene expression signature of ESFT was found to be
more similar to stem cells than to differentiated tissues (Fig. 4A).
Additionally, at a whole genome level, ESFT were more related to
hNCSC than to BM-MSC. To address whether histogenesis or
EWS-FLI1 effects are primarily responsible for the NCSC-like
state of ESFT cells we repeated unsupervised clustering of the
aforementioned samples after excluding published [26,27,29] as
well as novel (from the current study) EWS-FLI1 target genes. As
shown, even in the absence of 1,676 EWS-FLI1-modulated
transcripts, ESFT clustered with stem cells and retained close
identity with hNCSC (Fig. 4B).
EWS-FLI1 can partially activate a NCSC signature and trigger
an ESFT initiation program in heterologous cells [7,26,30]. To
determine if EWS-FLI1 initiated reactivation of the hNCSC
program in hNC-MSC, we first defined differences between these
two cell populations. Consistent with phenotypic observations
(Fig. 1 & 2), the molecular signature of hNC-MSC after 5 days in
adherent culture was fundamentally altered compared to undiffer-
entiated hNCSC. In particular, hNC-MSC showed significant up-
regulation of mesenchymal gene expression and down-regulation of
neuroectodermal genes and at a whole genome level these cells
clustered more closely with BM-MSC than their hNCSC ancestors
(Tables S2A & S2B and Fig. 4). Importantly, however, after only 5
days EWS-FLI1 reactivated expression of 81 hNCSC-associated
genes and inhibited expression of 126 hNC-MSC-associated genes
(Table S3A). Gene ontologic analysis of these EWS-FLI1-
modulated targets confirms significant reactivation of neuroecto-
dermal and concomitant repression of mesodermal differentiation
programs in EWS-FLI1 expressing cells (Table S3B).
Together these gene expression profiling studies suggest that
maintenance of the NCSC genetic program is central to ESFT
pathogenesis and that both the cell of origin and expression of
EWS-FLI1 contribute to this state.
EWS-FLI1 expression in hNCSC induces polycomb
proteins and represses p16
To assess the functional consequences of EWS-FLI1 we
monitored 3 different batches of transduced cells for up to 3
months. Whereas CV-NC cells displayed the aforementioned
large fibroblastic morphology, EF-NC cells were generally smaller
and more NCSC-like in appearance (Fig. 5A). In addition,
expression of the NCSC genes NGFR (p75) and B3GAT1 (HNK-1)
was down regulated in CV-NC cells but remained high in EF-NC
Polycomb-mediated gene silencing is an essential feature of stem
cell maintenance and is frequently deregulated in cancer . The
polycomb proteins BMI-1 and EZH2 are over-expressed by and
function as oncogenes in ESFT [7,23,32]. Therefore, we evaluated
whether the observed maintenance of NCSC features in EF-NC
cells was associated with up regulation of these proteins. FACS-
sorted, EGFP+ EF-NC and CV-NC were analyzed by western blot
after 6 weeks in culture. EF-NC cells reproducibly displayed a
marked over-expression of both BMI-1 and EZH2 proteins
(Fig. 5C). Interestingly, although the EZH2 transcript was also
significantly induced, transcriptional induction of BMI-1 was
minimal (data not shown) suggesting that post-transcriptional
regulation might have mediated BMI-1 protein over-expression.
Significantly, up regulation of polycomb proteins was accompa-
nied by silencing of p16 (Fig. 5C) and expression of all 3 proteins in
EF-NC cells was equivalent to freshly isolated hNCSC (Fig. 5D).
Together these findings corroborate the gene expression data that
showed reactivation of a NCSC-like state in EWS-FLI1-trans-
Inhibition of BMI-1 leads to de-repression of p16 and
cellular senescence in EWS-FLI1-expressing neural crest
Although both CV-NC and EF-NC cells continued to expand
equivalently in 2 of 3 experimental populations, in one experiment
CV-NC cells ceased to proliferate after 10 weeks in culture and by
3 months had undergone cellular senescence. In contrast, EF-NC
cells continued to proliferate normally (Fig. 6A) and showed no
evidence of a secondary loss of p53 function (Fig. 6B). Moreover,
these EWS-FLI1+ cells retained the ability to generate neuro-
spheres in anchorage-independent conditions in both defined
self-renewal and serum-containing media further supporting
Table 1. Core target genes significantly regulated by EWS-
FLI1 in BM-MSC*, ESFT cell lines**, and hNC-MSC.
ACTN2 INDMAP2K6 IND
AKAP7 INDMAPT IND
ALG6IND MKKS IND
C13orf18 INDPCSK2 IND
CADPS2 INDPFTK1 IND
DCLRE1BIND RHOH IND
EZH2 IND SORDIND
FCGRT IND STEAP3IND
FOS INDSYNE2 IND
HOOK1 INDCDC42EP3 REP
IL1RAP INDIL6ST REP
LBH INDPALLD REP
*Riggi et al. 2010,
**Kauer et al. 2008.
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Figure 4. Whole genome expression profiling confirms that ESFT cell lines and tumors are most closely related to hNCSC.
(A) Unsupervised 3D principal components analysis (PCA) of freshly isolated hNCSC (N=3), hNC-MSC (N=3), adult bone marrow-derived MSC (BM-
MSC; N=3), 10 primary ESFT, 10 ESFT cell lines and 11 adult tissues (each in triplicate) shows that ESFT cluster more closely with stem cells than any
adult tissue and are most similar to undifferentiated hNCSC. Notably testes and cerebellar tissues cluster separately from other adult tissues (breast,
heart, kidney, liver, muscle, pancreas, prostate, spleen, thyroid). All 17,881 core transcripts represented on the array were included for this analysis.
(B) Unsupervised clustering of samples in (A) was repeated after exclusion of 1,676 EWS-FLI1 target genes. ESFT continue to cluster with hNCSC.
Figure 5. EWS-FLI1 induces a NCSC phenotype in hNC-MSC. (A) EWS-FLI1 (EF-NC) and control vector (CV-NC) transduced hNC-MSC were
passaged in serum-containing media for 3 weeks and morphology imaged by immunocytochemistry. EF-NC cells were smaller and less mesenchymal
in appearance than CV-NC cells (scale bar=100 mm). (B) RT-PCR confirms down regulation of NCSC genes B3GAT1 (HNK-1) and NGFR (p75) in CV-NC
cells after 2 weeks. In contrast, NCSC and EWS-FLI1 target gene expression remained high in EF-NC cells (results for 3 independent experiments are
shown). (C) Transduced, EGFP+ cells were isolated 6 weeks after transduction and analyzed by western blot. Data from 3 independent experiments
show consistent up regulation of BMI-1 and EZH2 and repression of p16 in EF-NC cells. (D) After 6 weeks BMI-1, EZH2 and p16 expression in EF-NC
cells were equivalent to freshly isolated hNCSC. In contrast, CV-NC cells down regulated polycomb proteins and up regulated p16.
EWS-FLI1 Expression in Human Neural Crest Cells
PLoS ONE | www.plosone.org7 April 2011 | Volume 6 | Issue 4 | e19305
maintenance of a stem-like state (Fig. 6C). Silencing of p16 is an
initiating event in malignant transformation of primary mammary
epithelial cells . To determine if epigenetic silencing of p16
was necessary for continued proliferation and senescence avoid-
ance in EF-NC cells we transduced actively proliferating cells with
an inducible BMI-1 shRNA lentiviral construct (Fig. 7A).
Exposure of transduced cells to doxycyline led to induction of
BMI-1-targeted and inert shRNA sequences as indicated by the
appearance of the red fluorescence protein marker (Fig. 7B).
Consistent with BMI-1-mediated silencing of p16, knockdown of
BMI-1 resulted in de-repression of p16 and, unexpectedly, down
regulation of EZH2 (Fig. 7C). Further studies are now required to
elucidate the mechanism of this BMI-1/EZH2 interaction and to
determine if continued high-level expression of BMI-1 contributes,
either directly or indirectly, to maintenance of EZH2 over-
expression. Importantly, BMI-1 knockdown cells ceased to
proliferate within a few days of doxycycline treatment and cyclin
D1 levels decreased (Fig. 7D). One week after initiation of
doxycyline the majority of BMI-1 knockdown cells had undergone
cellular senescence while control cells continued to proliferate
These studies for the first time demonstrate that, like BM-MSC,
neural crest-derived stem cells tolerate EWS-FLI1. Moreover
functional studies suggest that the mechanism of oncogene
tolerance in these cells is mechanistically linked, at least in part,
to up regulation of BMI-1 and epigenetic repression of p16. In
addition, gene expression profiling studies reveal a high degree of
similarity between ESFT and hNCSC, confirming that activation
and maintenance of the NCSC genetic program is an integral
feature of ESFT pathogenesis.
Importantly, although tolerance of EWS-FLI1 and up regula-
tion of polycomb proteins was found to be universal in all hNCSC-
derived populations, some EWS-FLI1+ cells differentially avoided
cellular senescence. These senescence-avoiding cells retained the
ability to form neurospheres in non-adherent culture but did not
form colonies in soft-agar nor subcutaneous tumors in immune
deficient mice (not shown) again demonstrating that like primary
human BM-MSC [7,11,27,28], other epigenetic and/or genetic
events are necessary to achieve full malignant transformation of
human neural crest cells. Technical limitations currently preclude
expansion and study of EWS-FLI1 transduced hNCSC at the level
of single cells. Therefore, although known to be of neural crest
origin, the precise nature of the derivative cells that avoided
senescence downstream of EWS-FLI1 activation remains un-
known. Nevertheless, gene expression and functional studies
support a neuro-mesenchymal stem cell phenotype.
EWS-FLI1 induces cell cycle arrest and death in primary human
fibroblasts , and initiation of malignant transformation in human
BM-MSC [6,7]. This diversity in functional outcome is reflected in a
Figure 6. EWS-FLI1 transduced human neural crest cells avoid
cellular senescence. (A) Senescence-associated beta-galactosidase
staining 11 weeks post-transduction shows that CV-NC but not EF-NC
cells had undergone senescence (scale bar=100 mm) (B) Functional p53
was confirmed in EF-NC cells by induction of p53 and p21 proteins
following gamma irradiation. (C) Senescence-resistant EF-NC retain the
ability to form neurospheres (scale bar=100 mm).
Figure 7. Inhibition of BMI-1 in EF-NC cells restores p16 expression and leads to cellular senescence. (A) Inducible BMI-1 knockdown
lentiviral vector. (B) Red fluorescent protein expression following doxycyline treatment confirmed induction of inert non-silencing (NS) and BMI-1-
targeted (BMI1 kd) shRNA in transduced cells (scale bar=100 mm). (C) Western blot analysis confirmed BMI-1 knockdown in BMI1 kd cells
accompanied by de-repression of p16 and concomitant down regulation of EZH2. (D) BMI-1 knockdown cells also expressed reduced levels of Cyclin
D1 compared to control (NS) cells. (E) Senescence-associated beta-galactosidase staining 1 week post-doxycyline induction shows that BMI-1 kd cells
were senescent (scale bar=100 mm).
EWS-FLI1 Expression in Human Neural Crest Cells
PLoS ONE | www.plosone.org8 April 2011 | Volume 6 | Issue 4 | e19305
marked difference in EWS-ETS target genes in disparate cell types
[34,35,36,37], a difference which is again highlighted by our current
study. Importantly, differences in different cellular contexts are
particularly notable when comparing stem cells and established
ESFT cell lines. These findings suggest that EWS-FLI1 targets are
dynamic and may change during the transition from tumor initiation
to tumor maintenance. Continued evolution of transcription factor
targets is fundamental to cMYC-induced tumor progression  and
is no doubt also necessary for successful EWS-FLI1-mediated
malignant transformation of primary cells to ESFT.
The mechanisms of EWS-FLI1-mediated gene repression are
not yet clearly understood but are likely to be indirect . In
normal stem cells, epigenetic repression of developmental genes is
largely regulated by polycomb proteins which act together in
multi-factorial complexes to modify histones, altering chromatin
structure and inhibiting transcriptional activation . Deregula-
tion of polycomb genes is pervasive in human cancer  and
over-expression of BMI-1 and EZH2 contribute to the tumorige-
nicity of established ESFT cells [7,23,32]. In the current study we
have shown that EWS-FLI1 activation leads to a rapid and
profound up regulation of BMI-1 protein in hNC-MSC. Our
observation that protein induction is more profound than
transcriptional induction is consistent with there being a post-
transcriptional component to BMI-1 regulation in these cells.
Studies in glioma have shown that miRNA128 regulates BMI-1
mRNA and protein expression by directly targeting the 39-UTR of
BMI-1 mRNA . Whether miRNA128 contributes to BMI-1
regulation in ESFT remains to be elucidated. In addition, it has
been proposed that in normal stem cells interactive feedback loops
exist between PRC1 and PRC2 polycomb group complexes and
that these feedback loops lead to altered translation and stability of
individual PRC proteins, thereby affecting stem cell function .
EWS-FLI1 induces EZH2 and we have found that knockdown of
BMI-1 in neural crest cells also results in down-regulation of
EZH2 (Fig. 7C). Further studies are needed to establish the
mechanism of BMI-1 upregulation ESFT and its relationship, if
any, to EZH2 induction.
In contrast to BMI-1, EZH2 is a known direct transcriptional
target of EWS-FLI1  and is one of 46 genes induced by EWS-
FLI1 irrespective of cellular context (Table 1). Moreover, JARID2,
a recently characterized member of the Jumonji family that
complexes with EZH2 to regulate polycomb-mediated gene
repression in stem cells , was induced by EWS-FLI1 in both
hNC-MSC and BM-MSC [7,26]. Together these findings suggest
that abnormal induction of polycomb proteins and subsequent
deregulation of polycomb target gene expression might be critical
early events in EWS-FLI1-induced transformation.
Successful transformation of primary human cells requires that
innate tumor suppressor pathways be repressed. In some experi-
mental models, oncogene-induced transformation has been shown
to be dependent on BMI-1-mediated epigenetic repression of the
p16/ARF-encoding CDKN2A [43,44]. In addition, epigenetic
silencing of p16 is an early event in the transformation of primary
human mammary epithelial cells [33,45]. Our data suggest that
epigenetic repression of p16 by BMI-1 may also be an early
initiating event in EWS-FLI1-induced malignant transformation.
This is in contrast to other studies from our lab which showed that,
in established ESFT, BMI-1 contributes to tumorigenicity by means
that are largely independent of p16 repression . Thus, as
discussed above in reference to EWS-FLI1, disparate molecular
mechanisms may contribute to BMI-1’s function as an oncogene
duringtumorinitiationand tumormaintenanceand wehypothesize
that BMI-1 targets change and evolve during ESFT initiation and
progression. Further studies are now required to define and
compare BMI-1-bound promoters in normal stem cells, their
downstream progeny and established tumors.
In summary, we have used a novel model of hESC-derived
NCSC to investigate EWS-FLI1 biology and the cellular origins of
ESFT. Our findings reveal that ESFT are genetically highly
related to NCSC. In addition, neural crest-derived stem cells are
permissive for EWS-FLI1 expression and susceptible to oncogene-
induced immortalization, at least in part as a result of aberrant up
regulation of BMI-1 and epigenetic repression of p16. Together
these data support the hypothesis that at least some ESFT might
arise from malignant transformation of neural crest-derived stem
B. Overlapping EWS-FLI1 target genes in published datasets.
A. Significant EWS-FLI1 modulated transcripts.
ed gene ontologies among NCSC-specific genes.
A. NCSC-specific gene signature. B. Over-represent-
significantly altered by EWS-FLI1 in NC-MSC. B. Over-
represented gene ontologies among NCSC-specific genes that
were also significantly altered by EWS-FLI1 in NC-MSC.
A. Transcripts specific to NCSC gene signature and
RT-PCR primer sequences.
The authors thank the Stem Cell-, Genome- and FACS Cores at CHLA
and Ron Cole for technical assistance. We thank Dr. Timothy Triche and
Daniel Wai for ESFT cell line microarray data. We acknowledge the staff
of the CHLA and Children’s Oncology Group Biorepositories (Nationwide
Children’s Hospital, Columbus, OH) for their tremendous efforts. This
work was presented in part at the 101stAnnual meeting of the AACR,
Washington, D.C., April 2010.
Conceived and designed the experiments: CvL XJ GvL JH-RH ERL.
Performed the experiments: CvL XJ YG GvL LH AC JH-RH. Analyzed
the data: CvL XJ GvL JH-RH ERL. Contributed reagents/materials/
analysis tools: GvL. Wrote the paper: CvL ERL.
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