View
14
Download
0
Category
Preview:
Citation preview
EWS-WT1 Oncoprotein Activates Neuronal Reprogramming Factor ASCL1 and Promotes
Neural Differentiation
Hong-Jun Kang1, Jun Hong Park
1, WeiPing Chen
2, Soo Im Kang
1,4, Krzysztof Moroz
1, Marc Ladanyi
3
and Sean Bong Lee1
Affiliations: 1Tulane University School of Medicine, Department of Pathology and Laboratory
Medicine, 1430 Tulane Ave., New Orleans, LA 70112, USA; 2Genomics Core Facility, National
Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville
Pike, Bethesda, MD 20892, USA; 3Department of Pathology, Memorial Sloan-Kettering Cancer Center,
New York, NY 10065, USA.
4Present address: Institute for Cancer Genetics, Department of Pathology and Cell Biology, Columbia
University Medical Center, New York, NY 10032, USA.
Running title: EWS-WT1 activates ASCL1 and induces neural differentiation
Key Words: EWS-WT1, DSRCT, ASCL1, neural gene expression, neural reprogramming
Financial support: This research was supported in part by the Intramural Research Program of the
NIH, NIDDK (S.B.L.), and by the Tulane Startup Fund (S.B.L.).
Corresponding author: Sean Bong Lee, Ph.D., Tulane University School of Medicine, Department of
Pathology and Laboratory Medicine, 1700 Tulane Ave. Room 808, New Orleans, LA 70112; Tel: (504)
988-1331; Fax: (504) 988-7389; E-mail: slee30@tulane.edu
Conflicts of interest: The authors declare no potential conflicts of interest.
Word count: 5,062 words (4,840 words excluding the title page)
Total number of figures and tables: 6 main figures, 6 supplement figures and 2 supplement tables
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Abstract
The oncogenic fusion gene EWS-WT1 is the defining chromosomal translocation in desmoplastic small
round cell tumors (DSRCT), a rare but aggressive soft tissue sarcoma with a high rate of mortality.
EWS-WT1 functions as an aberrant transcription factor that drives tumorigenesis, but the mechanistic
basis for its pathogenic activity is not well understood. To address this question, we created a transgenic
mouse strain that permits physiologic expression of EWS-WT1 under the native murine Ews promoter.
EWS-WT1 expression led to a dramatic induction of many neuronal genes in embryonic fibroblasts and
primary DSRCT, most notably the neural reprogramming factor ASCL1. Mechanistic analyses
demonstrated that EWS-WT1 directly bound the proximal promoter of ASCL1, activating its
transcription through multiple WT1-responsive elements. Conversely, EWS-WT1 silencing in DSRCT
cells reduced ASCL1 expression and cell viability. Notably, exposure of DSRCT cells to neuronal
induction media increased neural gene expression and induced neurite-like projections, both of which
were abrogated by silencing EWS-WT1. Taken together, our findings reveal that EWS-WT1 can
activate neural gene expression and direct partial neural differentiation via ASCL1, suggesting agents
that promote neural differentiation might offer a novel therapeutic approach to treat DSRCT.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Introduction
Desmoplastic small round cell tumor (DSRCT) is a rare but aggressive tumor occurring predominantly
in adolescents and young adults (1). DSRCT is poorly understood and highly lethal, resulting in 85%
mortality within 5 years despite aggressive multimodal therapy (2, 3). The majority of tumors are found
in serous membrane of abdominal or pelvic cavity without an apparent involvement of any organ
systems and form multiple nests of malignant cells embedded in dense desmoplastic stroma (1). A
distinct immunophenotypic feature of DSRCT is a multi-lineage expression of epithelial, mesenchymal,
neuronal and muscle markers (3). Despite the presence of these multi-lineage markers, the tumor cells
appear poorly differentiated. To date, there is no molecular rationale for the multi-lineage expression
and the tumor cell of origin remains undefined.
DSRCT is caused by a balanced chromosomal translocation t(11;22)(p13;q12) that results in a fusion of
the N-terminal domain (NTD) of Ewing sarcoma gene, EWSR1 (termed EWS) to the C-terminal domain
(CTD) of Wilms Tumor gene, WT1 (4, 5). EWS encodes a RNA/ssDNA binding protein that play
multiple roles in diverse cellular processes, such as maturation of pre B lymphocytes, meiosis,
hypersensitivity to DNA damage, prevention of premature senescence of fibroblasts (6) and
hematopoietic stem cells (7), mitosis (8), cell-fate determination of classical brown fat (9), regulation of
microRNAs (10) and regulation of genotoxic-induced alternative splicing (11, 12). The NTD of EWS
contains multiple degenerate repeats of SYGQQS motif that functions as a potent transcriptional
activation domain (13, 14). WT1 is inactivated in 10-15% of Wilms tumors, a childhood kidney cancer
(15). WT1 encodes a transcription factor with four Cys2-His2 zinc fingers at the C-terminus, and
undergoes an alternative splicing involving only three amino acids (Lys, Thr and Ser, termed KTS)
between the zinc fingers 3 and 4 (16). This splicing produces two isoforms that either lacks (-) or
contains (+) the KTS which alters the DNA binding specificity. In DSRCT, only the last three zinc
fingers of WT1 are fused to EWS and the KTS splicing is preserved (5), resulting in the production of
two isoforms: EWS-WT1-KTS (herein termed E-KTS) and EWS-WT1+KTS (E+KTS).
The presence of EWS-WT1 translocation in all DSRCT points to the fusion products as the initiator of
this tumor. Interestingly, the two isoforms differ in their oncogenic activities as only the E-KTS, but
not E+KTS, has the potential to transform NIH3T3 cells in vitro (17). Therefore, the majority of studies
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
have focused on identifying the target genes of E-KTS (18-22), while only two genes have been
identified as direct targets of E+KTS (22, 23). These studies also revealed different DNA recognition
sequences of each isoform: E-KTS binds to either a GC-rich, 5’-GXG(T/G)GGGXG-3’ (X is any base)
(17, 20), or TCCn-repeats (n>3) (18), while E+KTS recognizes 5’-GGAGG(A/G)-3’ (23). Despite these
findings, how EWS-WT1 drives oncogenesis remains poorly understood; consequently, the prognosis
of DSRCT remains grim and the development of effective therapy is urgently needed.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Materials and Methods
Cell culture and reagents
HEK293 (CRL-1573) and U2OS (HTB-96) cells were purchased from American Type Culture
Collection and MEFs were generated as described (6). These cells were grown in DMEM with 10%
FBS, 100U/ml penicillin and 100g/ml streptomycin (Invitrogen). JN-DSRCT-1 cells (24),
authenticated by the presence of EWS-WT1 translocation and free of mycoplasma, were grown in
DMEM/F-12 media with 10% fetal bovine serum (FBS). Tamoxifen, 4-hydroxytamoxifen (4-HT) and
G418 (Sigma), and N2 supplement (Invitrogen) were purchased.
Generation of EWS-WT1 knock-in mice and animal care
To generate a conditional EWS-WT1 mouse, we followed the strategy that was used to generate a
conditional EWS-FLI1 mouse (25). Briefly, we inserted a loxP-flanked transcriptional ‘STOP’ cassette
(26) in an antisense direction into Ews intron 6 (Figure 1A). A human WT1(-KTS) cDNA (exons 8-10)
was fused to Ews exon 7 to create an EWS/WT1(-KTS) ‘knock-in’ allele. The targeting construct was
used to generate correctly targeted mouse ES cells (TC-1 (27)) as determined by Southern blot analysis
(25). Positive ES clones were injected into C57BL6 blastocysts (NIDDK Mouse Knockout Core) and
the resulting chimeras were crossed to C57BL6 females to achieve germ-line transmission. A
conditional EWS-WT1(+KTS) knock-in mouse was generated previously (6). A transgenic mouse
constitutively expressing the CreER allele (B6.Cg-Tg(CAG-cre/Esr1)5Amc/J) was purchased (Jackson
Laboratory). The primers for genotyping EWS-WT1 or CreER mice are described in Supplemental
information. All animal procedures were approved and handled according to the guidelines provided by
the Tulane Institutional Animal Care and Use Committee and by the NIH Animal Research Advisory
Committee.
Microarray analysis
MEFs harboring E-KTS/CreER+, E+KTS/CreER
+ or CreER
+ were either untreated or treated with 1
M 4-HT. At 24h, total RNA was prepared using RNeasy Kit (Qiagen). Gene expression profiling with
or without (reference) 4-HT was performed using Affymetrix Mouse Genome 430 2.0 arrays. Three
biological replicates were analyzed for each sample. The data were analyzed using an Affymetrix RMA
algorithm. Genes with greater than 1.5-fold difference and P value <0.05 were selected by ANOVA
using Partek Pro (Partek, St. Charles, MO). The heat maps were generated by using either RMA raw
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
signal values or fold change values from ANOVA lists by Partek. Microarray data have been submitted
to GEO database (GSE53301). GO analysis of primary tumors were performed using DAVID v6.7
(The Database for Annotation, Visualization and Integrated Discovery) (28, 29).
Real-time qRT-PCR
Total RNA was converted to cDNA using SuperScript III Reverse Transcriptase (Invitrogen). Each
sample was analyzed by real-time quantitative RT-PCR (qRT-PCR) using a TaqMan probe for Ascl1
(Mm03058063_m1) (Applied Biosystems). The relative transcript quantity was calculated by
comparative Ct method normalized against Gapdh.
Promoter-reporter assays
A human ASCL1 promoter (-1043 to +233) luciferase reporter construct was purchased (GeneCopoeia)
and subcloned into pGL3Basic vector (Promega). The ASCL1 promoter-luciferase construct, along with
Renilla luciferase plasmid, was transfected into JN-DSRCT-1 and HEK293 cells by Lipofectamine
2000 (Invitrogen). At 48h post-transfection, luciferase activities were measured using Dual Luciferase
Assay Kit (Promega). Site-specific mutations (M1-M3) in the ASCL1 promoter were generated by
DNA synthesis (Blue Heron Biotechnology) and confirmed by sequencing.
Chromatin immunoprecipitation
ChIP assay was performed as described (30) using anti-WT1 (C19, Santa Cruz) or anti-RNA Pol II
(Millipore) antibodies. The primers used to amplify the ASCL1 promoter are listed in Supplemental
information.
Colony formation assay
Cells were transduced with lentiviruses expressing shWT1 (shWT1-2 or shWT1-3), shASCL1
(shASCL1-1 or shASCL1-2) or scrambled control and cultured in presence of puromycin for 2 weeks.
After staining with crystal violet, colonies were counted and photographed. Three independent
experiments were performed in triplicates. The shWT1 and shASCL1 sequences (Sigma) are listed in
Supplemental information.
Immunofluorescence
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.5% Triton X-100 for 10
min, blocked with 5% goat serum (Sigma) and incubated with the following primary antibodies: mouse
Tuj1 (1:1,000) or rabbit anti-MAP2 (1:1,000). Alexa Fluor 488 and 594 (Invitrogen) were used as
secondary antibodies. Confocal microscopy was performed using an inverted laser scanning confocal
microscope (Zeiss Axiovert 200M) and analyzed with the LSM 510 confocal software (Zeiss).
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Results
Generation of mice harboring conditional EWS-WT1 knock-in alleles
To determine the physiological mechanisms underlying EWS-WT1 driven tumorigenesis, we decided to
express EWS-WT1 under the control of native Ews transcriptional network. We fused the WT1(-KTS)
cDNA encoding the last three zinc fingers of WT1 lacking the KTS to the mouse Ews exon 7 (Fig. 1A),
recreating the exact fusion transcript found in DSRCT. Similar to EWS-FLI1 mice (25), the loxP-
flanked transcriptional ‘STOP’ was inserted in the antisense direction, which was essential for the
successful targeting of EWS-WT1(-KTS). The STOP inserted in the sense direction completely
prevented homologous recombination (data not shown), suggesting that, similar to EWS-FLI1, a leaky
EWS-WT1(-KTS) expression impedes ES cell growth. Successfully targeted ES cells were used to
generate a conditional EWS-WT1(-KTS) knock-in mouse. We have previously generated a conditional
EWS-WT1(+KTS) knock-in mouse (6). We herein designate heterozygous mice carrying EWS-WT1(-
KTS) as E-KTS and EWS-WT1(+KTS) as E+KTS. E-KTS and E+KTS mice appeared healthy and were
backcrossed to C57BL6 mice for more than 8 generations.
Inducible expression of E-KTS and E+KTS
We first crossed E-KTS or E+KTS mice with EIIa-Cre transgenic mice (general deleter line), but we
only obtained E+KTS;Cre+ mice (data not shown), suggesting that constitutive expression of E-KTS
caused lethality. Therefore, we crossed E-KTS or E+KTS mice with a transgenic mouse constitutively
expressing Cre recombinase fused to mutated estrogen receptor (CreER), allowing an inducible
expression of EWS-WT1 with tamoxifen (Supplement Fig. 1A). To test the efficiency, we
intraperitoneally (i.p.) administered 3 doses of tamoxifen (3mg/40g body weight, given every 3 days)
and examined CreER-mediated excision of STOP by PCR. We observed varying degrees of
recombination in different tissues, with the highest recombination observed in the kidney and pancreas,
intermediate recombination in the brain, heart and lung, and very little recombination in the liver and
spleen (Supplement Fig. 1B). Tamoxifen did not induce recombination in any of the tissues in E-KTS
mice without CreER. To determine the cytotoxicity of E-KTS or E+KTS, we administered 3 doses of
tamoxifen and monitored the mice daily. Notably, all E-KTS;CreER+ mice (n=4, age 6-8 weeks) died
by 10-11 days from the first tamoxifen injection, while tamoxifen-treated E+KTS;CreER+ (n=4) or E-
KTS;CreER- (n=4) mice lived over 16 months. These observations indicate that global expression of E-
KTS, but not E+KTS, causes lethality in mice.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Expression of E-KTS, but not E+KTS, leads to inhibition of cell growth
To examine the cytotoxic effects of E-KTS in detail, we derived MEFs from E-KTS;CreER+ and
E+KTS;CreER+ embryos. We observed near complete excision of STOP in both MEFs following 4-
hydroxytamoxifen (4-HT) treatment (Fig. 1B). There was no recombination in the absence of 4-HT or
without CreER expression. Similar to EWS-FLI1 MEFs (25), an aberrantly spliced transcript (EWS-
STOP-WT1) containing 147-bp derived from the antisense STOP is present in either MEFs in the
absence of 4-HT (Fig. 1C). Upon 4-HT-mediated excision of STOP, expression of the correctly spliced
E-KTS and E+KTS transcripts (EWS-WT1) emerged at 6h and reached maximal levels at 24h (Fig.
1C). Expression of E-KTS and E+KTS proteins was detectable at 12h and reached maximal levels at
24h, but the expression of E-KTS was substantially lower than E+KTS (Fig. 1D). As previously
demonstrated for EWS-FLI1, no detectable E-KTS protein was generated in the absence of 4-HT (Fig.
1D), likely due to a rapid degradation of the proteins generated from the aberrantly spliced transcripts
(25). Strikingly, expression of E-KTS resulted in a rapid cessation of cell growth while E+KTS
expressing cells grew normally (Fig. 2A). To gain insights into the E-KTS-induced growth arrest, we
examined expression of various cell cycle proteins by immunoblotting. Expression of key cell cycle
regulators, Cyclin A and Cyclin D1, was markedly reduced in cells expressing E-KTS at 48h compared
to CreER MEFs (Fig. 2B). Notably, phosphorylated AKT was absent in E-KTS expressing cells even
though total AKT levels were comparable. However, expression of other cell cycle proteins and various
CDK inhibitors (Fig. 2B, right panels) was unaltered. MEFs expressing E-KTS did not undergo
apoptosis as determined by PARP cleavage (data not shown).
Genome-wide gene expression profiling
To gain insights into the E-KTS- and E+KTS-mediated transcriptional regulation, we performed whole-
genome expression analysis. Interrogation of over 39,000 probe sets with RNA isolated from E-KTS or
E+KTS expressing MEFs revealed that 3,228 transcripts (2,051 induced and 1,177 repressed) showed
significant expression changes (p<0.05 and >1.5-fold change) following E-KTS expression while 1,557
transcripts (780 induced and 777 repressed) showed significant alterations upon E+KTS expression
(Supplement Fig. 2A). About 300 transcripts were significantly altered by 4-HT treatment in CreER
MEFs, demonstrating a relatively small effect of 4-HT. Notably, there was a very little overlap of genes
regulated by the two isoforms, suggesting that each isoform is recruited to distinct promoter/enhancer
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
regions and regulate different genes. Gene Ontology (GO) analysis revealed that a large number of
neural-related genes were induced by E-KTS but not by E+KTS (Supplement Table 1 and Fig. 3A).
This was surprising since EWS-WT1 has never been shown to regulate neural genes. However, it is
well established that most primary DSRCTs express neural markers such as Neuron Specific Enolase
(NSE) and S100 protein (3). GO analysis revealed that expression of E+KTS resulted in a repression of
genes involved in DNA replication and repair pathways (Supplement Table 1).
Neural genes are overexpressed in primary DSRCT
To determine whether neural genes are enriched in primary DSRCT, we analyzed expression profile of
28 primary DSRCT performed previously in comparison to four other tumor types (31). GO analysis of
genes that are up-regulated (>2-fold) in DSRCT compared to either Ewing sarcoma (ES), alveolar
rhabdomyosarcoma (ARMS) or alveolar soft part sarcoma (ASPS) revealed an enrichment of neural
pathways (Supplement Table 2), but not when compared to synovial sarcoma (SS). The enrichment of
neural pathways was not evident by genes that were repressed in DSRCT (data not shown). We next
examined the expression of all the genes listed in the identified GO neural pathways in 137 primary
tumors. Remarkably, a large number of these neural genes were uniquely overexpressed in 28 DSRCT
compared to other tumors (Supplement Fig. 3 and Supplement Table 2). A smaller subset of neural
genes was also uniquely overexpressed in SS, which might explain the failure of our GO analysis to
reveal neural pathway enrichment when DSRCT was compared to SS. Notably, a subset of neural
genes that were highly activated by E-KTS in MEFs was also enriched in primary DSRCT, including
ASCL1, PLXNB1 (Plexin B1) and NTRK3 (Neurotrophic Tyrosine Kinase, Receptor 3) (Fig. 3B). These
results suggest that neural gene expression in DSRCT might be directly regulated by E-KTS.
ASCL1 is directly activated by E-KTS
ASCL1 is one of the neural reprogramming factors that directly converts fibroblasts into neurons (32,
33). Given the enrichment of neural genes in DSRCT, we next determined whether E-KTS directly
activated ASCL1 transcription. 4-HT-mediated expression of E-KTS, but not E+KTS or CreER,
resulted in a massive increase in ASCL1 mRNA (>150-fold) and protein expression in MEFs (Fig. 4A
and B). Expression of E-KTS in a heterologous osteosarcoma cells (U2OS) also resulted in a robust
induction of ASCL1 compared to E+KTS or empty vector control (Supplement Fig. 2B). ASCL1
expression was most abundant in a DSRCT cell line, JN-DSRCT-1, as compared to U2OS or three
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Ewing sarcoma cell lines (Fig. 4C). Depletion of EWS-WT1 in JN-DSRCT-1 cells with lentiviruses
containing shRNAs against the 3’ region of WT1 (shWT1-2 or shWT1-3 but not with shWT1-1)
resulted in a successful knockdown of EWS-WT1 (Fig. 4D). Concomitantly, silencing EWS-WT1 led to
almost complete inhibition of ASCL1 expression, but not in controls. We note that endogenous WT1 is
not expressed at detectable levels in JN-DSRCT-1 cells by immunoblotting (data not shown).
Collectively, these results demonstrate that E-KTS is directly responsible for the ASCL1 transcription in
MEFs and JN-DSRCT-1 cells.
EWS-WT1 or ASCL1 expression is critical for DSRCT cell survival
EWS-WT1 is the defining oncogene in DSRCT but it is not known whether continued expression of
EWS-WT1 is necessary to sustain tumor cell growth. Depletion of EWS-WT1 or ASCL1 by two
independent shRNAs in JN-DSRCT-1 cells resulted in a complete loss of tumor cell growth as revealed
by colony formation assay (Fig. 4E-G). These results suggest that persistent expression of EWS-WT1 is
required for tumor cell growth. Interestingly, expression of ASCL1 was also essential for DSRCT
tumor cell growth.
Identification of E-KTS responsive elements in the ASCL1 promoter
To identify the regulatory sequences responsive to E-KTS, we tested a proximal (1.2-kb) human ASCL1
promoter in a luciferase reporter assay. Expression of E-KTS, but not E+KTS, in HEK293 cells
resulted in a concomitant increase in the ASCL1 promoter-driven luciferase activity in a dose-dependent
manner (Fig. 5A). Transfection of the ASCL1 promoter-reporter in JN-DSRCT-1 cells also resulted in a
modest but significant induction of luciferase reporter compared to U2OS or A4573 cells, while a small
(1.5-fold) increase was observed in CHP100 cells (Supplement Fig. 4A). Inspection of human ASCL1
proximal promoter sequences (-1043 to +233) revealed a presence of 12 potential GC-rich E-KTS
binding sites (E-KTS-BS): two upstream (between -951 to -820) and ten within 400-bp of the
transcriptional start site (Fig. 5B). Chromatin immunoprecipitation (ChIP) analysis of JN-DSRCT-1
cells with an antibody against the C-terminus of WT1 demonstrated that EWS-WT1 is mostly bound to
the proximal (-408 to -56) ASCL1 promoter but not in the region (-765 to -384) harboring no E-KTS-
BS. A weak recruitment was detected in the upstream region (-1078 to -743) (Fig. 5B). Consistent with
this, the D2 promoter (-406 to +233) containing the 10 potential E-KTS-BS was fully responsive to E-
KTS as the 1.2-kb promoter (Fig. 5C). Alignment of the human and mouse ASCL1 proximal promoter
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
regions (-400 to +1) revealed a high conservation of the entire region (86% identity), including the
multiple potential E-KTS-BS (Supplement Fig. 4B).
To determine the precise E-KTS-responsive elements in ASCL1 promoter, we introduced substitution
mutations at 4 distal (M1), 6 proximal (M2) or all 10 (M3) potential E-KTS-BS in the D2 promoter
(Supplement Fig. 4C) and tested them in reporter assays. Destroying the 4 distal sites (M1) resulted in
about 88% reduction in the luciferase activity while abolishing the 6 proximal sites (M2) resulted in
95% inhibition of E-KTS transcription (Fig. 5D). The M3 promoter, which destroyed all E-KTS-BS,
completely (99%) lost the ability to mediate transcription by E-KTS, demonstrating that multiple E-
KTS-BS are necessary for the full activation by E-KTS.
Partial reprogramming of fibroblasts to neuron-like cells by E-KTS
ASCL1, along with POU3F2 (POU domain, class 3, transcription factor 2, also called BRN2) and
MYT1L (Myelin Transcription Factor 1-like), have been shown to directly reprogram fibroblasts into
excitatory neurons (32). Direct reprogramming of fibroblasts into other types of neurons, such as
dopaminergic or motor neuron, can be achieved by a combination of different neural transcription
factors, but ASCL1 is required for nearly all direct neural reprogramming (33). Furthermore, ASCL1,
when expressed alone, has been shown to induce a partial reprogramming of fibroblasts into neuron-
like cells (32). Thus, we tested whether E-KTS, which directly activates ASCL1 expression, could lead
to a partial neural reprogramming of MEFs. Following induction of E-KTS or E+KTS with 4-HT for 2
days, cells were cultured in neural induction media (DMEM/F12 + N2 supplement (1X)) for 10 days
(fresh media given every 2 days). Remarkably, immature neuron-like cells appeared in the E-KTS
expressing MEFs displaying elongated bi- or tri-polar projections that resembled neurites (Fig. 6A).
Immunofluorescence with antibodies against neuron-specific -III-Tubulin (TUBB3 recognized by
Tuj1 antibody) and Microtubule-Associated Protein 2 (MAP2) revealed that about 20% of cells
expressed these neural markers in E-KTS expressing cells (Fig. 6B and Supplement Fig. 5A). There
were less than 5% of Tuj1-positive cells in E-KTS MEFs without 4-HT and no Tuj1-positive cells
appeared in E+KTS MEFs regardless of 4-HT. Extended neural induction period (up to 18 days) did
not increase the number of neuron-like cells nor result in more complex neuronal morphology in E-
KTS MEFs (data not shown). These results demonstrate that E-KTS expression leads to a partial neural
reprogramming of MEFs, likely via ASCL1.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Partial differentiation of JN-DSRCT-1 cells to neuron-like cells
We next examined whether JN-DSRCT-1 cells could be differentiated towards neural lineage by the
endogenous expression of ASCL1. When JN-DSRCT-1 cells were cultured with the neuronal induction
media for 3 days, about 30% of cells displayed short, but occasionally long, bi- or tri-polar Tuj1+
neurite-like projections (Fig. 6C and D). TUBB3 expression was only observed in N2-treated JN-
DSRCT-1 cells but not in the absence of N2 or in U2OS cells with or without N2 (Fig. 6C). Induction
of TUBB3 was observed as early as 24h and continued to increase to 72h (Fig. 6E). Notably, siRNA-
mediated acute depletion of EWS-WT1 led to nearly complete inhibition of Tuj1+ neurite projections
and to marked decrease in TUBB3 expression in JN-DSRCT-1 cells (Supplement Fig. 5B-D),
demonstrating that EWS-WT1 is responsible for the partial neural differentiation.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Discussion
In this study, we report the generation of two conditional EWS-WT1 knockin mice, each expressing a
specific isoform under the control of endogenous EWS transcriptional network. A previous study has
shown that expression of E-KTS, but not E+KTS, was sufficient to transform NIH3T3 cells (17).
Consistent with this, expression of E+KTS in MEFs had no effects on cell growth and mice expressing
E+KTS did not develop any spontaneous tumor (data not shown). However, our study does not
preclude a supporting role for E+KTS in DSRCT tumorigenesis. In contrast, expression of E-KTS
resulted in a rapid cessation of cell growth and rapid death of mice. Though the exact cause of death is
unknown, Ki-67 immunostaining showed that expression of E-KTS caused a significant reduction in
the number of proliferating cells in crypts of intestinal epithelium (Supplement Fig. 6), suggesting a
block in proliferation of gastrointestinal epithelium and malabsorption might be responsible. In support
of this, mice expressing E-KTS lost weight by an average of 3.9g (n=4) in 4 days while the control
mice gained an average of 0.8g (n=3).
Other mouse models expressing oncogenic fusion genes also showed lethal effects (25, 34-39),
suggesting that expression of chimeric oncogenes in primary cells often leads to negative cellular
effects. Therefore, we postulate that in DSRCT, EWS-WT1 translocation must occur in a permissive
cell type(s) or in the context of permissive microenvironment that will allow persistent EWS-WT1
expression. These cells will likely be the tumor cells of origin in DSRCT. An analogous situation exists
in Ewing sarcoma where expression of EWS-FLI1 is toxic to most cell types (25, 40, 41) but its
expression is permitted in mesenchymal stem cells (42, 43) or in neural crest stem cells (44). Therefore,
identifying a cell type that tolerates E-KTS expression is critical to understanding DSRCT
tumorigenesis and for the development of a mouse model.
One of the hallmarks of DSRCT is that tumor cells express neural and other lineage markers (3), but
the rationale for the multilineage expression has not been provided. Our findings demonstrate that E-
KTS induces a number of neural genes including ASCL1, which will likely induce additional neural-
related genes. Thus, this study provides a molecular rationale for the neural gene expression in DSRCT.
Prior to this study, it was not known whether continued expression of EWS-WT1 is required for tumor
cell growth. Our findings showed that persistent EWS-WT1 expression is essential for tumor cell
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
growth, making it an ideal target for therapy. Surprisingly, depletion of ASCL1 also led to inhibition of
JN-DSRCT-1 cell growth, revealing another potential therapeutic target. When JN-DSRCT-1 cells
were stimulated to undergo neural differentiation, however, ASCL1 expression was further induced
along with its target genes (Supplement Fig. 5E) and partial neural differentiation ensued. Thus,
ASCL1 appears to have dual functions: (1) maintaining tumor cell growth under a proliferative signal
and (2) promoting neural differentiation under a neural differentiation signal.
Direct reprogramming of fibroblasts into neurons requires the expression of three factors: ASCL1,
POU3F2 and MYT1L (32). When we attempted to induce full neural reprogramming in JN-DSRCT-1
cells by expressing POU3F2, MYT1L or NEUROD1, either alone or in combination, expression of any
of these factors under neural differentiation condition resulted in a rapid cell death (data not shown),
demonstrating that JN-DSRCT-1 cells are incompatible with full neural reprogramming. However, a
partial neural differentiation was achieved in JN-DSRCT-1 cells, likely due to E-KTS-activated
ASCL1. These findings suggest a possibility that induction of partial neural differentiation in DSRCT
patients might lead to an arrest or delay of aggressive tumor cell growth, providing a potentially novel
and effective therapy against this incurable disease.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Acknowledgments
We thank Cuiling Li and Chuxia Deng (NIDDK Mouse Knockout Core) for the mouse ES cell injection
and Chithra Keembiyehetty (NIDDK Genomics Core) for performing the microarray analysis and Yun-
Ping Wu (NIDDK) for confocal microscopy. This work was supported in part by the NIDDK
Intramural Research Program and by the Tulane Startup Fund (S.B.L.).
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
References
1. Gerald WL, Miller HK, Battifora H, Miettinen M, Silva EG, Rosai J. Intra-abdominal desmoplastic
small round-cell tumor. Report of 19 cases of a distinctive type of high-grade polyphenotypic
malignancy affecting young individuals. Am J Surg Pathol. 1991;15:499-513.
2. Gerald WL, Haber DA. The EWS-WT1 gene fusion in desmoplastic small round cell tumor. Semin
Cancer Biol. 2005;15:197-205.
3. Gerald WL, Ladanyi M, de Alava E, Cuatrecasas M, Kushner BH, LaQuaglia MP, et al. Clinical,
pathologic, and molecular spectrum of tumors associated with t(11;22)(p13;q12): desmoplastic
small round-cell tumor and its variants. J Clin Oncol. 1998;16:3028-36.
4. Ladanyi M, Gerald W. Fusion of the EWS and WT1 genes in the desmoplastic small round cell
tumor. Cancer Res. 1994;54:2837-40.
5. Gerald WL, Rosai J, Ladanyi M. Characterization of the genomic breakpoint and chimeric
transcripts in the EWS-WT1 gene fusion of desmoplastic small round cell tumor. Proc Natl Acad
Sci U S A. 1995;92:1028-32.
6. Li H, Watford W, Li C, Parmelee A, Bryant MA, Deng C, et al. Ewing sarcoma gene EWS is
essential for meiosis and B lymphocyte development. J Clin Invest. 2007;117:1314-23.
7. Cho J, Shen H, Yu H, Li H, Cheng T, Lee SB, et al. Ewing sarcoma gene Ews regulates
hematopoietic stem cell senescence. Blood. 2011;117:1156-66.
8. Azuma M, Embree LJ, Sabaawy H, Hickstein DD. Ewing sarcoma protein ewsr1 maintains mitotic
integrity and proneural cell survival in the zebrafish embryo. PloS one. 2007;2:e979.
9. Park JH, Kang HJ, Kang SI, Lee JE, Hur J, Ge K, et al. A multifunctional protein, EWS, is essential
for early brown fat lineage determination. Dev Cell. 2013;26:393-404.
10. Kim KY, Hwang YJ, Jung MK, Choe J, Kim Y, Kim S, et al. A multifunctional protein EWS
regulates the expression of Drosha and microRNAs. Cell Death Differ. 2013.
11. Dutertre M, Sanchez G, De Cian MC, Barbier J, Dardenne E, Gratadou L, et al. Cotranscriptional
exon skipping in the genotoxic stress response. Nat Struct Mol Biol. 2010;17:1358-66.
12. Paronetto MP, Minana B, Valcarcel J. The Ewing sarcoma protein regulates DNA damage-induced
alternative splicing. Mol Cell. 2011;43:353-68.
13. Ng KP, Potikyan G, Savene RO, Denny CT, Uversky VN, Lee KA. Multiple aromatic side chains
within a disordered structure are critical for transcription and transforming activity of EWS family
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
oncoproteins. Proceedings of the National Academy of Sciences of the United States of America.
2007;104:479-84.
14. May WA, Lessnick SL, Braun BS, Klemsz M, Lewis BC, Lunsford LB, et al. The Ewing's sarcoma
EWS/FLI-1 fusion gene encodes a more potent transcriptional activator and is a more powerful
transforming gene than FLI-1. Mol Cell Biol. 1993;13:7393-8.
15. Lee SB, Haber DA. Wilms tumor and the WT1 gene. Exp Cell Res. 2001;264:74-99.
16. Haber DA, Sohn RL, Buckler AJ, Pelletier J, Call KM, Housman DE. Alternative splicing and
genomic structure of the Wilms tumor gene WT1. Proceedings of the National Academy of
Sciences of the United States of America. 1991;88:9618-22.
17. Kim J, Lee K, Pelletier J. The desmoplastic small round cell tumor t(11;22) translocation produces
EWS/WT1 isoforms with differing oncogenic properties. Oncogene. 1998;16:1973-9.
18. Lee SB, Kolquist KA, Nichols K, Englert C, Maheswaran S, Ladanyi M, et al. The EWS-WT1
translocation product induces PDGFA in desmoplastic small round-cell tumour. Nat Genet.
1997;17:309-13.
19. Wong JC, Lee SB, Bell MD, Reynolds PA, Fiore E, Stamenkovic I, et al. Induction of the
interleukin-2/15 receptor beta-chain by the EWS-WT1 translocation product. Oncogene.
2002;21:2009-19.
20. Palmer RE, Lee SB, Wong JC, Reynolds PA, Zhang H, Truong V, et al. Induction of BAIAP3 by
the EWS-WT1 chimeric fusion implicates regulated exocytosis in tumorigenesis. Cancer Cell.
2002;2:497-505.
21. Ito E, Honma R, Imai J, Azuma S, Kanno T, Mori S, et al. A tetraspanin-family protein, T-cell
acute lymphoblastic leukemia-associated antigen 1, is induced by the Ewing's sarcoma-Wilms'
tumor 1 fusion protein of desmoplastic small round-cell tumor. Am J Pathol. 2003;163:2165-72.
22. Li H, Smolen GA, Beers LF, Xia L, Gerald W, Wang J, et al. Adenosine transporter ENT4 is a
direct target of EWS/WT1 translocation product and is highly expressed in desmoplastic small
round cell tumor. PLoS One. 2008;3:e2353.
23. Reynolds PA, Smolen GA, Palmer RE, Sgroi D, Yajnik V, Gerald WL, et al. Identification of a
DNA-binding site and transcriptional target for the EWS-WT1(+KTS) oncoprotein. Genes Dev.
2003;17:2094-107.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
24. Nishio J, Iwasaki H, Ishiguro M, Ohjimi Y, Fujita C, Yanai F, et al. Establishment and
characterization of a novel human desmoplastic small round cell tumor cell line, JN-DSRCT-1. Lab
Invest. 2002;82:1175-82.
25. Sohn EJ, Li H, Reidy K, Beers LF, Christensen BL, Lee SB. EWS/FLI1 oncogene activates caspase
3 transcription and triggers apoptosis in vivo. Cancer Res. 2010;70:1154-63.
26. Sauer B. Manipulation of transgenes by site-specific recombination: use of Cre recombinase.
Methods Enzymol. 1993;225:890-900.
27. Deng C, Wynshaw-Boris A, Zhou F, Kuo A, Leder P. Fibroblast growth factor receptor 3 is a
negative regulator of bone growth. Cell. 1996;84:911-21.
28. Huang da W, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists
using DAVID bioinformatics resources. Nat Protoc. 2009;4:44-57.
29. Huang da W, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the
comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37:1-13.
30. Kim HS, Kim MS, Hancock AL, Harper JC, Park JY, Poy G, et al. Identification of novel Wilms'
tumor suppressor gene target genes implicated in kidney development. J Biol Chem.
2007;282:16278-87.
31. Filion C, Motoi T, Olshen AB, Lae M, Emnett RJ, Gutmann DH, et al. The EWSR1/NR4A3 fusion
protein of extraskeletal myxoid chondrosarcoma activates the PPARG nuclear receptor gene. J
Pathol. 2009;217:83-93.
32. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M. Direct conversion of
fibroblasts to functional neurons by defined factors. Nature. 2010;463:1035-41.
33. Yang N, Ng YH, Pang ZP, Sudhof TC, Wernig M. Induced neuronal cells: how to make and define
a neuron. Cell Stem Cell. 2011;9:517-25.
34. Codrington R, Pannell R, Forster A, Drynan LF, Daser A, Lobato N, et al. The Ews-ERG fusion
protein can initiate neoplasia from lineage-committed haematopoietic cells. PLoS Biol. 2005;3:e242.
35. Haldar M, Hancock JD, Coffin CM, Lessnick SL, Capecchi MR. A conditional mouse model of
synovial sarcoma: insights into a myogenic origin. Cancer Cell. 2007;11:375-88.
36. Higuchi M, O'Brien D, Kumaravelu P, Lenny N, Yeoh EJ, Downing JR. Expression of a
conditional AML1-ETO oncogene bypasses embryonic lethality and establishes a murine model of
human t(8;21) acute myeloid leukemia. Cancer Cell. 2002;1:63-74.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
37. Keller C, Hansen MS, Coffin CM, Capecchi MR. Pax3:Fkhr interferes with embryonic Pax3 and
Pax7 function: implications for alveolar rhabdomyosarcoma cell of origin. Genes & development.
2004;18:2608-13.
38. Lin PP, Pandey MK, Jin F, Xiong S, Deavers M, Parant JM, et al. EWS-FLI1 induces
developmental abnormalities and accelerates sarcoma formation in a transgenic mouse model.
Cancer Res. 2008;68:8968-75.
39. Lagutina I, Conway SJ, Sublett J, Grosveld GC. Pax3-FKHR knock-in mice show developmental
aberrations but do not develop tumors. Molecular and cellular biology. 2002;22:7204-16.
40. Deneen B, Denny CT. Loss of p16 pathways stabilizes EWS/FLI1 expression and complements
EWS/FLI1 mediated transformation. Oncogene. 2001;20:6731-41.
41. Lessnick SL, Dacwag CS, Golub TR. The Ewing's sarcoma oncoprotein EWS/FLI induces a p53-
dependent growth arrest in primary human fibroblasts. Cancer Cell. 2002;1:393-401.
42. Castillero-Trejo Y, Eliazer S, Xiang L, Richardson JA, Ilaria RL, Jr. Expression of the EWS/FLI-1
oncogene in murine primary bone-derived cells Results in EWS/FLI-1-dependent, ewing sarcoma-
like tumors. Cancer Res. 2005;65:8698-705.
43. Riggi N, Cironi L, Provero P, Suva ML, Kaloulis K, Garcia-Echeverria C, et al. Development of
Ewing's sarcoma from primary bone marrow-derived mesenchymal progenitor cells. Cancer Res.
2005;65:11459-68.
44. von Levetzow C, Jiang X, Gwye Y, von Levetzow G, Hung L, Cooper A, et al. Modeling initiation
of Ewing sarcoma in human neural crest cells. PLoS One. 2011;6:e19305.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Figure Legends
Figure 1. Generation of inducible EWS-WT1 MEFs. (A) Schematics of conditional EWS/WT1
alleles. pA, poly-adenylation signal; Neo, neomycin-resistance gene. Arrows indicate PCR primers. (B)
CreER-mediated recombination analyzed by PCR of genomic DNA isolated from MEFs cultured with
or without 1μM 4-HT. (C) Inducible expression of EWS/WT1 transcripts was analyzed by RT-PCR in
MEFs treated for the indicated times with 4-HT. EWS-STOP-WT1 indicates the aberrantly spliced
transcript. Gapdh was used as a control. (D) MEFs treated as in (C) were immunoblotted with anti-
WT1 or anti-Actin.
Figure 2. E-KTS induces cell growth arrest in MEFs. (A) E-KTS and E+KTS MEFs were grown
with or without 1μM 4-HT and cell number was counted daily. Three independent experiments were
performed in triplicates. (B) Whole cell lysates from CreER and E-KTS MEFs grown with or without
4-HT were analyzed by immunoblotting with the indicated antibodies.
Figure 3. E-KTS activates neural genes in MEFs and in primary tumors. (A) A heat map of
significantly altered genes in neural pathways in E-KTS expressing MEFs. (B) A heat map of some
neural genes that are enriched in primary DSRCT (n=28) compared to other tumors (31). ARMS:
Alveolar Rhabdomyosarcoma (n=23), ES: Ewing sarcoma (n=28), SS: Synovial sarcoma (n=46) and
ASPS: Alveolar Soft Part sarcoma (n=12).
Figure 4. E-KTS activates ASCL1 expression. (A) MEFs cultured with or without 1μM 4-HT for 24h
were analyzed for expression of Ascl1 by qRT-PCR. Three independent experiments were performed in
triplicates. *P<0.05, Student’s t-test. (B) Whole cell lysates from CreER or E-KTS MEFs grown with
or without 4-HT were immunoblotted with anti-ASCL1 or anti-Actin. (C) U2OS, CHP100, A4573,
RD-ES and JN-DSRCT-1 cells were immunoblotted with anti-WT1, anti-ASCL1 or anti-Actin. The
arrow indicates EWS/WT1. *nonspecific band. Note that EWS-WT1 migrates higher (62kDa) in JN-
DSRCT-1 cells due to a different EWS translocation breakpoint (24). (D) JN-DSRCT-1 cells were
transduced with lentivirus carrying three independent shWT1 or control and analyzed by
immunoblotting with anti-EWS, anti-ASCL1 or anti-Actin. (E) JN-DSRCT-1 cells were transduced
with lentiviruses carrying control, two independent shWT1 or two independent shASCL1 and the total
number of colonies was counted and compared to the control (set to 100%). Three independent
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
experiments were performed in triplicate. *P<0.05, Student’s t-test. (F) Representative images from (E)
are shown. (G) JN-DSRCT-1 cells were transduced with lentiviruses carrying scrambled or two
independent shASCL1 and analyzed by immunoblotting with anti-ASCL1 or anti-Actin.
Figure 5. E-KTS activates ASCL1 via multiple E-KTS-responsive elements. (A) HEK293T cells
were transfected with empty vector or increasing amounts (0.1 g, 0.2 g and 0.6 g) of E-KTS or
E+KTS along with the 1.2kb ASCL1 promoter construct and luciferase activity was measured. Three
independent experiments were performed in triplicates. **
P<0.01, ***
P<0.001, Student’s t-test. (B) ChIP
analysis of ASCL1 promoter in JN-DSRCT-1 cells. Crosslinked chromatin was immunoprecipitated
with rabbit IgG or anti-WT1 (C19) antibody and were amplified by PCR with the indicate primers
(arrows). RNA Pol II antibody was used as a positive control. Circles represent putative E-KTS binding
sites. TSS: Transcription start site (+1). (C) ASCL1 promoter-reporter deletion constructs or a pGL3-
Basic (control) were transfected in JN-DSRCT-1 cells and luciferase activity was measured. (D) The
D2 promoter or mutated (M1, M2 and M3) promoter-reporter constructs were transfected in JN-
DSRCT-1 cells and luciferase activity was measured. The X-circles represent mutated E-KTS binding
sites. Three independent experiments were performed in triplicates. ***
P<0.001.
Figure 6. E-KTS induces partial neural reprogramming. (A) E-KTS and E+KTS MEFs cultured
with or without 1μM 4-HT were cultured for 10 days in N2-containing media. Cells were
immunostained with Tuj1 antibody and DAPI. Scale bar=20 m. (B) The total number of Tuj1-positive
cells was counted from 26 randomly selected fields (n>790). (C) Immunostaining of JN-DSRCT-1 and
U2OS cells with Tuj1 antibody following 3 days of culture with or without N2 media. Scale bar=20 m.
(D) The total number of Tuj1-positive JN-DSRCT-1 cells was counted from 26 randomly selected
fields (n>290). (E) The N2-treated JN-DSRCT-1 cells were immunoblotted with Tuj1 or anti-Lamin
A/C.
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Actin
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Published OnlineFirst June 16, 2014.Cancer Res Hong-Jun Kang, Jun Hong Park, WeiPing Chen, et al. ASCL1 and promotes neural differentiationEWS-WT1 oncoprotein activates neural reprogramming factor
Updated version
10.1158/0008-5472.CAN-13-3663doi:
Access the most recent version of this article at:
Material
Supplementary
http://cancerres.aacrjournals.org/content/suppl/2014/06/16/0008-5472.CAN-13-3663.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://cancerres.aacrjournals.org/content/early/2014/06/14/0008-5472.CAN-13-3663To request permission to re-use all or part of this article, use this link
Research. on February 13, 2020. © 2014 American Association for Cancercancerres.aacrjournals.org Downloaded from
Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 16, 2014; DOI: 10.1158/0008-5472.CAN-13-3663
Recommended