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TITLE PAGE Phosphoproteomic Profiling Reveals IL6-mediated Paracrine Signaling within the Ewing Sarcoma Family of Tumors Jennifer L. Anderson1,2, Björn Titz3,4,5, Ryan Akiyama2, Evangelia Komisopoulou3,4,5, Ann Park2, William D. Tap6, Thomas G. Graeber3,4,5,7,8, and Christopher T. Denny1,2,4,7 1Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, 90095 2Division of Hematology/Oncology, Department of Pediatrics, Gwynne Hazen Cherry Memorial Laboratories, University of California, Los Angeles, Los Angeles, California, 90095 3Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, California, 90095 4Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California, 90095 5Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, 90095 6Sarcoma Medical Oncology Service, Division of Solid Tumors, Department of Medicine, Memorial Sloan Kettering Cancer Center and Department of Medicine, Weill Cornell Medical College, New York, NY 10065 7California NanoSystems Institute, University of California, Los Angeles, Los Angeles, California, 90095 8UCLA Metabolomics Center, University of California, Los Angeles, Los Angeles, California, 90095 Running title: Cellular signaling in Ewing sarcoma family tumors Key Words: phosphoproteomics, paracrine signaling, STAT3, sarcoma Support: J. L. A. received support from the Ruth L. Kirschstein National Research Service Award GM07185 and a UCLA Graduate Division Dissertation Year Fellowship. This work was also supported by NIH grant CA087771 (C. T. D.). T.G.G. is the recipient of a Research Scholar Award from the American Cancer Society (RSG-12-257-01-TBE) and an Established Investigator Award from the Melanoma Research Alliance (20120279), and is supported by NIH/National Center for Advancing Translational Science (NCATS) UCLA CTSI Grant Number UL1TR000124. Corresponding author: Christopher T. Denny, M.D. 650 Charles E. Young Drive South Factor 10-240 Los Angeles, CA 90095 cdenny@ucla.edu phone (310) 825-0704 fax (310) 267-2848 Conflict of interest: The authors declare no conflict of interest. Word count: 6,115 Total number of figures: 6
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ABSTRACT Members of the Ewing sarcoma family of tumors (EFST) contain tumor-associated translocations that
give rise to oncogenic transcription factors, most commonly EWS/FLI1. EWS/FLI1 plays a dominant role
in tumor progression by modulating the expression of hundreds of target genes. Here, the impact of
EWS/FLI1 inhibition, by RNAi-mediated knockdown, on cellular signaling was investigated using mass
spectrometry-based phosphoproteomics to quantify global changes in phosphorylation. This unbiased
approach identified hundreds of unique phosphopeptides enriched in processes such as regulation of cell
cycle and cytoskeleton organization. In particular, phosphotyrosine profiling revealed a large upregulation
of STAT3 phosphorylation upon EWS/FLI1 knockdown. However, single cell analysis demonstrated that
this was not a cell-autonomous effect of EWS/FLI1 deficiency, but rather a signaling effect occurring in
cells in which knockdown does not occur. Conditioned media from knockdown cells was sufficient to
induce STAT3 phosphorylation in control cells, verifying the presence of a soluble factor that can activate
STAT3. Cytokine analysis and ligand/receptor inhibition experiments determined that this activation
occurred, in part, though an IL6-dependent mechanism. Taken together, the data support a model in
which EWS/FLI1 deficiency results in the secretion of soluble factors such as IL6 which activate STAT
signaling in bystander cells that maintain EWS/FLI1 expression. Furthermore, these soluble factors were
shown to protect against apoptosis.
Implications:
EWS/FLI1 inhibition results in a novel adaptive response and suggests that targeting the IL6/STAT3
signaling pathway may increase the efficacy of ESFT therapies.
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INTRODUCTION Advancements in the understanding of the molecular mechanisms of oncogenesis have led to the
development of targeted therapeutics. For example, activating mutations in kinases such as EGFR in lung
cancer or B-RAF in melanoma have been inhibited by specific small molecules to increase therapeutic
efficacy. However, striking initial responses are rarely sustained due to innate and acquired resistance
mechanisms (1, 2). In the case of melanoma, initial suppression of the MAPK pathway by B-RAF
inhibitors is followed by reactivation that occurs through relief of a negative feedback loop (3). In other
systems, activation of redundant pathways can occur through cell autonomous mechanisms or be
mediated by stromal secretion of growth factors into the tumor microenvironment (4). These adaptive
responses by tumor cells to evade the effects of targeted therapeutics present a challenge to single agent
therapy.
Targeted therapy has also been utilized for the treatment of the Ewing sarcoma family of tumors
(ESFT). As opposed to activating kinase mutations, ESFT pathogenesis is primarily driven by what
appears to be an aberrant transcription factor generated by a chromosomal translocation. In most tumors,
this translocation fuses the EWS gene to the ETS transcription factor FLI1 (5). The fusion protein
EWS/FLI1 retains domains that facilitate interaction with transcriptional regulators and DNA binding,
which provides the ability to alter gene expression (6). EWS/FLI1 is capable of oncogenic transformation
and maintenance of expression is required for ESFT cell growth, indicating a dominant role in
tumorigenesis (6, 7).
Since EWS/FLI1 presents an ideal therapeutic target, several strategies have been employed to
identify a compound that inhibits its function. Initial small molecule screens identified compounds that
inhibited EWS/FLI1 modulation of gene expression including cytarabine (8), mithramycin (9), and
midostaurin (10). Other screens have been utilized to find molecules that bind to EWS/FLI1 or disrupt its
ability to bind DNA. YK-4-279, a derivative of a compound found to bind to EWS/FLI1, was demonstrated
to decrease EWS/FLI1 activity by blocking its interaction with the transcriptional co-activator RHA (11).
Additionally, low concentrations of actinomycin D were found to selectively inhibit EWS/FLI1 binding to
DNA (12). Trabectidin, evaluated based on its ability to inhibit a similar fusion in myxoid liposarcoma, was
also shown to inhibit EWS/FLI1 activity and induce apoptosis in ESFT cell lines (13).
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Unfortunately, the in vitro efficacy of these compounds thus far has not translated to the clinic.
Phase II trials of cytarabine and trabectidin did not demonstrate potent single agent activity and stable
disease was observed in only a minority of patients (14, 15). Modest single agent activity was also
observed with other targeted therapeutics evaluated in ESFT, including drugs directed against the insulin-
like growth factor receptor. These low clinical response rates highlight the adaptive responses of ESFT
when exposed to single agent therapy. As additional molecularly targeted compounds are being
evaluated in clinical trials, increased understanding of ESFT cellular signaling is needed to address
mechanisms of drug resistance and optimize therapeutic efficacy. Therefore, we chose to investigate
changes in protein phosphorylation upon inhibition of EWS/FLI1 in ESFT. We utilized shRNA-mediated
knock down as a model of EWS/FLI1 inhibition since reduction of expression encapsulates the multiple
mechanisms employed by various small molecules. Mass spectrometry-based phosphoproteomics was
used to quantitate global changes in phosphorylation levels after EWS/FLI1 knock down. Our results
revealed a paracrine signaling mechanism that induces cytokine secretion in EWS/FLI1 targeted cells and
subsequent STAT3 activation in bystander cells. This novel adaptive response suggests combination
therapy with STAT3 inhibitors may increase the efficacy of targeted therapeutics in ESFT.
MATERIALS AND METHODS
Cell culture. ESFT cell lines (RDES, TC-174, SK-N-MC, SKES, A4573, A673, and 6647) were cultured in
Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal bovine serum (FBS). ESFT cell lines
were either purchased from ATCC or were a gift from Timothy J. Triche, MD, PhD at the Saban Research
Institute, Children’s Hospital Los Angeles. Cell lines from ATCC undergo authentication via morphology
check by microscopy, growth curve analysis, isoenzymology, short tandem repeat analysis, and
mycoplasm detection. All cell lines underwent the following authentication process at UCLA: mycoplasm
detection, morphology check and documentation with microscopy and digital photography, growth curve
analysis, mitochondrial DNA analysis (in which the cell line identity is confirmed by mitochondrial DNA
comparative analysis of the highly variable regions I/II modified Cambridge sequence), and extensive
characterization including analysis for the EWS translocation and potential mutations (PTEN, PI3K,
CDKN2A) by RT-PCR. 293T cells used for virus production were cultured in Dulbecco’s modified Eagle
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medium (DMEM) containing 10% fetal calf serum and supplemented with L-glutamine (2 mM) and
penicillin-streptomycin (50 IU/ml and 50 μg/ml, respectively).
EWS/FLI1 818 and EF4 shRNA constructs were cloned into the CSCG lentiviral vector as
previously described (16, 17). The dominant negative STAT3 construct, in which tyrosine 705 is mutated
to phenylalanine, was cloned from pRc/CMV STAT3 Y705F Flag (Addgene plasmid 8709) (18) into the
SRα-MSV-TK Neo retroviral vector (19). Lentiviral and retroviral stocks were generated as previously
described (17).
Reagents. IGF1 was provided by Pinchas Cohen (UCLA). Stattic (STAT3 Inhibitor V) was obtained from
Santa Cruz Biotechnology. Human recombinant IL-6, GM-CSF, and CXCL1 were obtained from R&D
Systems. Doxorubicin HCl was obtained from Shandong Tianyu Fine Chemical Co., Ltd. NVP-AEW541
was obtained from Cayman Chemical.
Quantitative Real Time PCR. RNA was harvested using the RNeasy Mini Kit (Qiagen) or PureLink RNA
Mini Kit (Invitrogen). cDNA was synthesized from approximately 2 μg of RNA using the SuperScript III
First-Strand Synthesis System (Invitrogen). For real time PCR, a 1:10 dilution of cDNA was combined
with forward and reverse primers and master mix containing SYBR green, Taq, and dNTPs (Applied
Biosystems). Reactions were run at 95°C for 10 min, followed by 40 cycles at 95°C for 10 s, 60°C for 30 s,
and 72°C for 20 s on a DNA Engine Opticon 2 Real-Time Cycle (MJ Research/Bio-rad). Results were
analyzed with Opticon Monitor software (MJ Research/Bio-Rad). Primers used to quantify cellular
transcript levels are as follows: GAPDH: 5’-ATGTTCGTCATGGGTGTGAA-3’ and 5’-
CCAGGGGTGCTAAGCAGTT-3; EWS/FLI1: 5’-GCCAAGCTCCAAGTCAATATAGC-3’ and 5’-
GAGGCCAGAATTCATGTTATTGC-3’; and IL-6: 5’-AGCCACTCACCTCTTCAGAACGAA-3’ and 5’-
AGTGCCTCTTTGCTGCTTTCACAC-3’. EWS/FLI1 primers were originally described by Tirode et. al. (20).
IL-6 primers were originally described by Inda et. al. (21).
Immunoblot. Cells were incubated for approximately one hour on ice in lysis buffer (50 mM Tris pH 7.6,
0.5% NP-40, 10% glycerol, 30 mM NaCl, 1 mM EDTA) supplemented with Complete Mini EDTA-free
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protease inhibitor cocktail (Roche), 1 mM Na3VO4, and 1 mM NaF. Lysates were combined with 6X
protein sample buffer (0.35 M Tris pH 6.8, 10% SDS, 30% glycerol, 0.6 M DTT, 0.012% bromophenol
blue) and boiled for 5-10 minutes prior to loading on an 8% or 4-15% gradient polyacrylamide gel. The
primary antibodies used for these studies were rabbit anti-phospho-STAT3 (Tyr705), rabbit anti-phospho-
STAT3 (Ser727), mouse anti-STAT3, rabbit anti-gp130, and rabbit anti-cleaved PARP from Cell Signaling
Technology; mouse anti-FLAG M2 and mouse anti-β-actin from Sigma; mouse anti-FLI1 from BD
Biosciences; and mouse anti-phosphotyrosine (clone 4G10, HRP conjugate) from Millipore. Secondary
antibodies conjugated to HRP were sheep anti-mouse IgG from GE Healthcare; bovine anti-goat IgG and
goat anti-rabbit IgG from Santa Cruz Biotechnology. Secondary antibodies conjugated to infrared dyes
were IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG from LI-COR
Biosciences. Fluorescent westerns were imaged using the Odyssey Infrared Imaging System (LI-COR
Biosciences). Signals were quantified by measuring the integrated intensity values of each band using
Odyssey software (LI-COR Biosciences).
Phosphopeptide enrichment. Cells were incubated for approximately one hour on ice in lysis buffer
supplemented with protease inhibitor and 1 mM Na3VO4. For serine/threonine enrichment, 1 mM NaF was
added to the lysis buffer. Lysates were centrifuged at 1000 g for 5 min and supernatant was saved. Four
volumes of ice-cold (-20°C) acetone were added and mixture was vortexed and incubated at -20°C for 1-2
hours. Precipitated proteins were pelleted by centrifuging at 6,000 g for 15 min at 0°C. The pellet was
washed once with 10 ml of ice-cold acetone to remove any residual NP-40, then resuspended in 8M urea,
50 mM Tris pH 7.5, and 1 mM Na3VO4 (and 1 mM NaF for phosphoserine/threonine enrichment) by
incubating overnight at 4°C with rotation. Phosphotyrosine peptides were enriched by
immunoprecipitation with a pan-specific anti-phosphotyrosine antibody (clone 4G10, Millipore) from 25-33
mg of total protein as previously described (22, 23). Phosphoserine/threonine peptides were purified from
9-10 mg of total protein by a combination of strong cation exchange chromatography and titanium dioxide
(TiO2) enrichment as previously described (24), except that peptides were concentrated and desalted
using ZipTip C18-based solid phase extraction (twice).
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Mass spectrometry and phosphopeptide quantitation. Mass spectrometry was performed using a
quantitative, label-free approach that has been demonstrated to show high concordance in quantitation
and standard error to a label-based approach (SILAC) (22). Phosphorylated peptides were analyzed by
LC-MS/MS with an Eksigent autosampler coupled with a Nano2DLC pump (Eksigent) and LTQ-Orbitrap
(Thermo Fisher Scientific) as previously described (25). MS/MS fragmentation spectra were searched
with SEQUEST (Version v.27, rev. 12, Thermo Fisher Scientific) against a database containing the
combined human-mouse International Protein Index (IPI) protein database (downloaded December 2006
from ftp.ebi.ac.uk) for peptides enriched for phosphotyrosine or against a human IPI database (version
3.71) for peptides enriched for phosphoserine/threonine. Search parameters were as previously
described (25), except that dynamic modifications also included phosphorylated serine and threonine.
To identify phosphopeptide peaks sequenced in some samples but not others, the chromatogram
elution profiles are aligned using a dynamic time warping algorithm (26). Further explanation of this
protocol can be found in the supporting information of Zimman et. al. (24) and Rubbi et. al. (22). Relative
amounts of the same phosphopeptide across samples run together were determined using custom
software to integrate the area under the unfragmented (MS1) monoisotopic peptide peak (23, 24). All
peaks corresponding to phosphopeptides were visually inspected and manually corrected if necessary.
The number of unique phosphorylation sites identified in our experiments was determined by
collapsing multiple phosphopeptide ions representing the same phosphorylation site. Phosphosites with
multiple detections (e.g. different ion charge state, modification) were quantified by summing the MS1
integration values for each phosphopeptide ion. Additionally, for the phosphoserine/threonine analysis,
phosphosites that were detected in multiple SCX fractions were quantified by summing the MS1
integration values for each fraction. The residue numbers listed for phosphosites correspond to the
indicated IPI accession number.
Cell viability and growth assays. The numbers of viable cells were determined indirectly by MTT assay.
Cells were seeded in 96-well plates, with each cell type or treatment condition performed in triplicate, and
incubated overnight. After drug treatment or growth period, 10 μl of 5 mg/ml MTT (3-(4,5-
dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) in PBS was added to cells and allowed to incubate
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for 2-4 hours at 37°C. Cells were then lysed with 100 μl of 15% SDS in 15 mM HCl and incubated
overnight at room temperature in the dark. Plate absorbance was read at 595 nm using a Bio-rad
microplate reader. Percent viability was calculated by normalizing absorbance values to those from cells
grown in media without drug after background subtraction. IC50 values were calculated by fitting dose-
response curves to a four-parameter, variable slope sigmoid dose-response model (Prism Software,
GraphPad). Synergistic, additive, or antagonistic effects of Stattic combination treatment were determined
based on combination indices and isobologram plots generated with CompuSyn software (ComboSyn,
Inc., Paramus, NJ) using the method of Chou and Talalay (27). Relative growth was calculated by
normalizing absorbance values to those from day 0 after background subtraction.
Immunofluorescence. Cells were grown on 4- or 8-well chamber slides, then fixed in 3.7% formaldehyde
in PBS for 15 minutes at room temperature and permeabilized with 100% methanol for 10 min at -20°C.
After blocking for one hour with Protein Block (Dako) diluted 1:10 in PBS, cells were incubated with
primary antibody overnight at 4°C and secondary antibody for one hour at room temperature. The primary
antibodies used were rabbit anti-phospho-STAT3 Tyr705 and mouse anti-STAT3 from Cell Signaling
Technology and anti-STAT3 from Abcam. The rabbit and mouse secondary antibodies used were
conjugated to Alexa Fluor 594 (Invitrogen). After antibody incubation, cover slips were mounted with
medium containing DAPI (VECTASHIELD Mounting Medium with DAPI, Vector Laboratories). Slides were
analyzed by fluorescent microscopy with a Zeiss AxioImager microscope (Carl Zeiss).
Flow cytometry. Cells were fixed in 1.5% formaldehyde for 10 min at room temperature, then
permeabilized with 100% ice-cold methanol for 20 min at 4°C. Cells were then washed twice with staining
media (0.5% BSA in PBS, pH 7.4) and incubated with primary antibody for one hour at room temperature.
After incubation, cells were again washed twice with staining media, then resuspended in PBS and
analyzed using a Becton Dickinson modified FACScan analytic flow cytometer. 10,000 live cell events
were recorded after gating cells using forward scatter and side scatter to remove debris and dead cells.
Median fluorescence intensity was used to quantify changes in phospho-STAT3 signal. The primary
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antibody used for this analysis was rabbit anti-phospho-STAT3 (Tyr705) XP Alexa Fluor 647 conjugate
from Cell Signaling Technology. Flow cytometry data was analyzed using FlowJo (Tree Star, Inc.).
Conditioned media. ESFT cells were grown in 10% FBS in IMDM serum for 72 hours in 10 cm plates,
between two and five days post lentiviral transduction. Alternatively, ESFT cells were transferred to serum
free IMDM or IMDM containing 1% serum four days post transduction and cells were grown for 48 hours.
Conditioned media was centrifuged at 2000 rpm for 5 min in a swinging bucket rotor to pellet any cell
debris.
Cytokine array, ELISA, and neutralizing antibodies. The RayBio Human Cytokine Antibody Array C-
Series 2000 kit (RayBiotech, Inc.) was used according to the manufacturer’s instructions. The
concentration of IL-6 in conditioned media was quantified using a human IL-6 Quantikine ELISA Kit (R&D
Systems). Phospho-STAT3 levels in ESFT cells expressing EWS/FLI1 were quantified using a PathScan
Phospho-STAT3 (Tyr705) Sandwich ELISA kit (Cell Signaling Technology). IL-6 and gp130 neutralizing
antibodies were obtained from R&D Systems.
RESULTS
Phosphoproteomic profiling identifies phosphopeptides modulated by EWS/FLI1
Genes modulated by EWS/FLI1 include members of signal transduction pathways, such as insulin-like
growth factor binding protein 3 (28), the mitotic kinases Aurora A and B (29), and caveolin-1 (30).
Therefore, we hypothesized that EWS/FLI1 inhibition could lead to changes in the activity of critical
signaling components in ESFT. Phosphotyrosine immunoblot analysis showed an overall decrease in
protein phosphorylation upon EWS/FLI1 knock down (Supplemental Figure 1A), indicating the fusion
protein plays a role in cellular signaling. To identify modulated phosphoproteins in an unbiased fashion,
we applied a quantitative, label-free mass spectrometry-based approach (23, 26). The ESFT cell line
A673 was transduced with an EWS/FLI1 shRNA construct or empty vector control. Phosphotyrosine or
phosphoserine/threonine enrichment followed by tandem mass spectrometry was used to quantitate
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relative phosphopeptide levels. Global changes in phosphorylation levels upon EWS/FLI1 knock down
were calculated by determining the phosphopeptide ratio between EWS/FLI1 shRNA and control cells.
Analysis of serine/threonine phosphopeptides detected 571 unique phosphopeptides
corresponding to 336 proteins and phosphotyrosine profiling identified 16 phosphopeptides
(Supplemental Tables 1-5). The phosphoserine/threonine data set was filtered for peptides that were
modulated upon EWS/FLI1 knock down. This generated a list of 210 phosphopeptides, 86 of which that
showed an increase in phosphorylation and 124 of which that displayed a decrease (Figure 1A). Since
change in phosphorylation could be due to change in total protein level, we compared this list to known
genes that are regulated by EWS/FLI1 (31). Only 23 out of 210 phosphopeptides were associated with
genes that are modulated by EWS/FLI1 (Supplemental Figure 1B,C), suggesting the majority of
phosphopeptide modulation is not solely due to EWS/FLI1 transcriptional regulation. DAVID (32, 33) was
used to determine pathways and biological processes that were enriched in response to EWS/FLI1
inhibition (Figure 1B,C, Supplemental Tables 6-9). Phosphopeptides whose levels were increased after
EWS/FLI1 knock down were associated with adhesion and cytoskeletal organization (Figure 1B). This
agrees with a recent study that demonstrated that ESFT cells display increased adhesion and migration
upon EWS/FLI1 knock down (34). The phosphopeptides that displayed a decrease in phosphorylation
were mainly associated with cell cycle regulation (Figure 1B,C). Since A673 cells with diminished
EWS/FLI1 expression proliferate at a reduced rate, this likely contributes to the enrichment of cell cycle
associated terms observed after EWS/FLI1 knock down.
Phosphotyrosine and phosphoserine/threonine peptides were rank ordered based on the sum of
the log fold change in phosphopeptide levels between EWS/FLI1 knock down and control samples
(Figure 1A, 2A). The most down regulated phosphoprotein was IRS2 (insulin receptor substrate 2) (Figure
1A), an adapter protein that transmits signals from insulin and insulin-like growth factor receptors. This
result is consistent with previous studies that have shown that EWS/FLI1 modulates components of the
IGF1 pathway (28, 35). We also observed a large decrease in phosphorylation of PRKCB (protein kinase
C beta) (Figure 1A), which was recently described to be overexpressed in ESFT. PRKCB has been
demonstrated to be a direct target of EWS/FLI1 and inhibition of the protein reduces ESFT growth in vitro
and in vivo (36). Phosphotyrosine-based rank ordering revealed the most differentially regulated
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phosphopeptide corresponded to an increase in phosphorylation of STAT3 at tyrosine 705 (Figure 2A),
with an average fold change of 11.5 between phosphopeptide levels in EWS/FLI1 knock down and
control cells. STAT3 phosphorylation was confirmed with a phospho-specific antibody (Figure 2B).
Quantitative immunoblot analysis detected low levels of phospho-STAT3 in control cells that increased an
average of 15.7-fold upon EWS/FLI1 knock down (Figure 2C). This response was also observed with the
use of a second shRNA construct (EF4) (Figure 2B).
Phospho-STAT3 up regulation primarily occurs in a subset of cells untransduced by lentiviral
shRNA
To measure STAT3 activation as a result of phosphorylation, STAT3 immunofluorescence was performed
to visualize localization before and after EWS/FLI1 knock down in A673 cells. Phosphorylation at tyrosine
705 allows STAT3 to dimerize, then translocate to the nucleus where it acts as a transcription factor (37).
STAT3 immunostaining showed largely nuclear signals in both knock down and control cells, with slightly
more intense cytoplasmic staining in the control cells (Figure 3A). A similar pattern was observed with a
distinct STAT3 antibody and at higher magnification (Supplemental Figure 2). The nuclear localization
suggests active STAT3 signaling also occurs in cells that express EWS/FLI1.
The similarity in STAT3 staining between control and EWS/FLI1 knock down cells led us to
perform phospho-specific staining (Figure 3B). A673 control cells displayed a low level of phospho-
STAT3 while a subset of cells in the knock down sample showed prominent staining. In general, this up
regulation trend was expected based our immunoblot results. However, the subset of EWS/FLI1 knock
down cells that displayed high levels of phospho-STAT3 showed almost no overlap with the GFP positive
population marking cells transduced with the lentiviral shRNA. These data suggest a paracrine
mechanism in which cells with successful EWS/FLI1 knock down cause the activation of STAT signaling
in cells that maintain EWS/FLI1 expression.
To quantitate phospho-STAT3 levels in ESFT control and EWS/FLI1 shRNA GFP positive and
negative populations, we performed phospho-specific flow cytometry. ESFT cells transduced with empty
vector control or EWS/FLI1 shRNA were divided into two populations based on GFP fluorescence
intensity and phospho-STAT3 levels were measured through the use of a fluorochrome-conjugated
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phospho-specific antibody. EWS/FLI1 transcript levels in EWS/FLI1 shRNA GFP negative populations
were similar to those of control cells while levels in GFP positive cells were reduced by approximately
85% (Supplemental Figure 3D). When comparing all A673 control and EWS/FLI1 shRNA cells, those
transduced with EWS/FLI1 shRNA displayed an increase in median fluorescence intensity (MFI) of 3.17
(Supplemental Figure 3A). However, when this comparison was performed on GFP negative and positive
populations, the GFP negative cells showed a nearly 4-fold increase in MFI for cells transduced with
EWS/FLI1 shRNA while GFP positive cells showed only a 1.9-fold increase (Figure 3C). Similar effects
were observed using A4573 cells (Supplemental Figure 3B). While the magnitude of the fold change was
smaller in A4573 cells, it was reproducible and statistically significant (Supplemental Figure 3C). These
results support the concept that the up regulation of phospho-STAT3 after EWS/FLI1 knock down occurs
primarily in a population untransduced by the lentiviral shRNA and is thus uninfluenced cell autonomously
by EWS/FLI1 knock down.
Soluble factors secreted upon EWS/FLI1 knock down are sufficient to induce STAT3
phosphorylation
The evidence that phospho-STAT3 up regulation and EWS/FLI1 knock down occur in separate subsets of
cells suggested these populations could be communicating with each other either though direct cell-to-cell
contact or through secretion of soluble factors. To test for the presence of soluble factors, conditioned
media from ESFT cells transduced with either vector control (CT) or EWS/FLI1 shRNA (818) was added
to control cells and STAT3 phosphorylation was assayed by immunoblot. Conditioned media from
EWS/FLI1 shRNA but not control cells was able to stimulate phospho-STAT3 (Figure 4A,B). This
response occurs quickly and is maintained for at least 24 hours (Figure 4B). STAT3 phosphorylation was
also induced with conditioned media from cells transduced with a second EWS/FLI1 shRNA construct
(EF4) and with serum-free conditioned media (Figure 4A). This indicates a soluble factor secreted upon
EWS/FLI1 knock down is responsible for STAT3 activation.
Two of the four ESFT cell lines assayed for the ability of conditioned media from EWS/FLI1
shRNA transduced cells to stimulate phospho-STAT3, TC-174 and SK-N-MC, displayed only a weak
response. To determine if this is due to a lack of secreted factor in the conditioned media or expression of
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the appropriate receptor on the cell surface, we added A673 EWS/FLI1 shRNA conditioned media to
these cells (Figure 4B). Both cells lines showed a large increase in phospho-STAT3 upon conditioned
media exposure, indicating they express the appropriate receptors, but do not secrete as much of the
soluble factor upon EWS/FLI1 knock down as the A673 cells. This may be due to a combination of lower
viral transduction rates when compared to the A673 cells (Supplemental Figure 4) and that ESFT cell
lines other than A673 undergo growth arrest after EWS/FLI1 knock down (16).
In order to determine which soluble factor(s) were responsible for the increase in phospho-STAT3,
we used an antibody array to simultaneously measure 174 cytokines and growth factors in serum free
conditioned media from A673 cells transduced with empty vector control or EWS/FLI1 shRNA. A few
cytokines displayed a dramatic increase in signal intensity while the majority of the factors showed little or
no change upon EWS/FLI1 knock down (Figure 4C). The status of all 174 cytokines is included in
Supplemental Table 10. In particular, IL-6, GM-CSF, and CXCL1 (GRO-α) were present in much higher
levels in the EWS/FLI1 knock down conditioned media compared to that of control (Figure 4C,D). To test
which of these factors is able to activate STAT3, purified, recombinant proteins were added to ESFT cells
and phospho-STAT3 levels were compared to cells treated with conditioned media from EWS/FLI1
shRNA (818) transduced cells. Only IL-6 was able to stimulate STAT3 phosphorylation, though not to the
level of the conditioned media (Figure 4E). Since IL-6 is a known activator of STAT3, we chose to further
investigate its role in ESFT signaling.
STAT3 phosphorylation is induced primarily through an IL-6 dependent mechanism
ELISA analysis was performed to validate the results of the cytokine array and quantitate the levels of IL-
6 secreted by ESFT cells. Conditioned media from A4573 and A673 EWS/FLI1 knock down cells
contained elevated levels of IL-6 (Figure 5A). Quantitation of IL-6 transcript levels also demonstrated an
increase in IL-6 RNA upon EWS/FLI1 knock down (Figure 5B).
To determine if IL-6 is necessary for STAT3 activation, we inhibited either the ligand or its
receptor gp130. First, an IL-6 neutralization antibody was added to EWS/FLI1 knock down (818)
conditioned media and subsequently immunoprecipitated. Immunodepletion of IL-6 was confirmed by
ELISA (Figure 5C,D) and the media was added to untransduced cells. Resulting phospho-STAT3 levels
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were compared to cells treated with control or EWS/FLI1 knock down conditioned media (Figure 5E,F).
Especially in A673 cells, removing IL-6 prevents phosphorylation of STAT3. Conditioned media in which
IL-6 is only partially immunodepleted (Figure 5D) retains the ability to stimulate STAT3 phosphorylation
(Figure 5F). An analogous experiment was performed in which ESFT cells were pre-incubated with a
gp130 neutralization antibody. EWS/FLI1 knock down conditioned media was added to these cells as well
as those that were not pre-treated with the antibody. Evaluation of STAT3 phosphorylation revealed that
blocking gp130 also inhibited up-regulation of phospho-STAT3 (Figure 5G). These results provide
evidence that STAT3 is being activated through an IL-6 dependent mechanism (Figure 5I).
We next utilized phospho-specific immunoblot analysis to examine the activity of STAT3 and
potential upstream kinases upon EWS/FLI1 knock down. While we did not observe a difference in STAT3
phosphorylation at serine 727 between control and unsorted EWS/FLI1 knock down cells, examination of
sorted populations revealed GFP negative cells possess increased levels of phospho-S727 compared to
GFP positive cells with reduced EWS/FLI1 expression (Figure 5H). This demonstrates that paracrine
activation of STAT3 results in increased phosphorylation at both tyrosine 705 and serine 727.
Examination of GFP positive and negative populations of cells transduced with EWS/FLI1 shRNA also
showed that both JAK2 and SRC family kinases (SFK) displayed reduced phosphorylation at sites within
the activation loop of the kinase domain after EWS/FLI1 knock down (Figure 5H). GFP positive cells also
displayed a modest decrease in gp130 levels, which may contribute to the decrease in kinase JAK2 and
SFK activity. Furthermore, GFP positive cells with reduced EWS/FLI1 expression appear to possess less
total STAT3 than GFP negative cells. This decrease in total protein and diminished activity of upstream
kinases could contribute to the decreased paracrine STAT3 activation observed in cells with reduced
levels of EWS/FLI1. Further experiments are warranted to fully elucidate this mechanism.
STAT3 plays a complex role in ESFT growth and survival
Since activation of STAT3 promotes tumorigenesis through up regulation of cell survival and proliferation
factors (38), we sought to investigate its effects on ESFT cell growth. The paracrine activation of STAT3
that occurs upon EWS/FLI1 knock down suggests these cells might display increased proliferation rates
or sensitivity to STAT3 inhibition. Additionally, the observed nuclear localization and basal
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phosphorylation of STAT3 indicate a possible dependence on STAT3 signaling in cells that maintain
EWS/FLI1 expression. To investigate the role of STAT3 in each of these populations, we used a small
molecule inhibitor, Stattic (39), and dominant negative construct (18) to inhibit STAT3 phosphorylation
and measured subsequent effects on ESFT cell proliferation.
STAT3 phosphorylation at tyrosine 705 was validated by ELISA in control ESFT cells in which
paracrine STAT3 activation has not been induced by EWS/FLI1 knock down (Supplemental Figure 5B).
Stattic treatment revealed these cells are sensitive to STAT3 inhibition, with a half maximal inhibitory
concentration (IC50) of approximately 2 μM (Supplemental Figure 5C). Increasing concentrations of
Stattic were demonstrated to inhibit STAT3 phosphorylation in EWS/FLI1 knock down and control cells
(Supplemental Figure 5A,B) compared to DMSO treated controls. Stattic also inhibited the proliferation of
ESFT cells regardless of EWS/FLI1 expression, though greater inhibitory effects were observed in
EWS/FLI1 knock down cells treated with Stattic (Figure 6A). EWS/FLI1 knock down reduced ESFT cell
proliferation to degrees that varied based on knock down efficiency. A larger growth inhibitory effect was
observed in A673 cells due to more potent reduction of EWS/FLI1 expression (Supplemental Figure
4A,B). Additional growth assays using a dominant negative construct also demonstrated that STAT3
inhibition diminishes ESFT cell growth (Figure 6B). Furthermore, dominant negative STAT3 hinders
EWS/FLI1-mediated STAT3 phosphorylation (Supplemental Figure 5D) and combined inhibition of STAT3
and EWS/FLI1 has an increased effect compared to targeting EWS/FLI1 alone (Figure 6B).
Given the role of STAT3 in ESFT growth, we next asked if the paracrine activation of STAT3 that
occurs upon EWS/FLI1 knock down results in increased in cellular proliferation. In some instances, ESFT
cells treated with conditioned media derived from cells transduced with EWS/FLI1 shRNA displayed
increased growth compared to cells treated with conditioned media from control cells. However, these
results were not consistent across various anchorage dependent and independent assays (data not
shown). As a result, we focused on the role of factors secreted upon EWS/FLI1 knock down to promote
cell survival.
ESFT cell lines were treated with conditioned media containing reduced serum in order to induce
apoptosis. After 24 hours of treatment, we observed an increased amount of cleaved PARP compared to
untreated controls. ESFT cells treated with conditioned media derived from EWS/FLI1 knock down cells
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displayed significantly less PARP cleavage than those treated with conditioned media from control cells
(Figure 6C,D). This effect is mediated in part by IL-6. Adding IL-6 to control conditioned media reduced
PARP cleavage and immunodepleting IL-6 from knock down conditioned media increased PARP
cleavage (Figure 6E). These results demonstrate that soluble factors secreted upon EWS/FLI1 knock
down confer protection against apoptosis.
The paracrine STAT3 activation that occurs upon EWS/FLI1 knock down implies targeting this
pathway could sensitize ESFT cells to EWS/FLI1-directed therapy. Therefore, we evaluated the effects of
combining a STAT3 inhibitor with cytotoxic agents that inhibit EWS/FLI1 function. When Stattic was
combined with either cytarabine or mithramycin, mostly additive effects were observed. Synergy was only
observed at the highest dose levels tested (data not shown). However, since cytarabine disrupts DNA
synthesis by acting as a nucleoside analog and mithramycin inhibits RNA synthesis by binding to the
minor groove of DNA, neither of these agents specifically targets EWS/FLI1. Additional targets hit by
these drugs may obscure the effects of Stattic inhibition.
Since specific small molecule inhibitors of EWS/FLI1 are not available, we explored the effects of
combining STAT3 inhibition with other ESFT therapies. We first tested if IL-6-mediated paracrine STAT3
activation occurs as a response to stresses other than EWS/FLI1 knock down. Treatment of ESFT cells
with the chemotherapeutic agent doxorubicin resulted in a dose dependent increase in IL-6 secretion.
However, the levels of IL-6 in conditioned media from doxorubicin treated ESFT cells were 2 to 3 orders
of magnitude less than those observed for media from EWS/FLI1 knock down cells. Additionally, this
conditioned media was able to increase STAT3 phosphorylation in ESFT cells, but not to the extent of
knock down conditioned media (data not shown). While less robust than the effects observed upon
EWS/FLI1 knock down, doxorubicin-induced paracrine STAT3 activation provides a rationale for
combining STAT3 inhibition with therapeutics other than those that inhibit EWS/FLI1. Therefore, we
evaluated the effects of combining Stattic with conventional and targeted therapies utilized for the
treatment of ESFT.
When Stattic was combined with the IGF1R small molecular inhibitor NVP-AEW541, synergy was
observed in both A673 and A4573 cells (Figure 6F,G, Supplemental Figure 6A,B). In A673 cells,
combining Stattic with doxorubicin displayed synergistic effects (Figure 6H,I). In A4573 cells, while the
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effective doses of the combination treatment lied below the linear additive isoboles (Supplemental Figure
6D), the combination indices for four out of five dose levels were approximately 1, indicating additivity
(Supplemental Figure 6C). These data indicate that STAT3 inhibition could increase the efficacy of ESFT
therapies.
DISCUSSION
Less focus has been placed on the role of signal transduction in ESFT since tumor progression is
primarily driven by EWS/FLI1-mediated regulation of gene expression. However, low response rates for
clinical testing of targeted therapeutics in ESFT emphasize the necessity for better understanding of
cellular signaling. Our studies aimed to generate a global, unbiased view of changes in cellular signaling
upon EWS/FLI1 inhibition to gain further insight into potential mechanisms of drug resistance. Our results
included novel phosphoproteins modulated by EWS/FLI1 as well as the elucidation of a paracrine
signaling pathway. Tyrosine phosphoprofiling revealed STAT3 phosphorylation to be up regulated upon
EWS/FLI1 knock down. Single cell analysis demonstrated this does not occur through direct regulation,
but through a paracrine mechanism mediated in strong part by IL-6 secretion. STAT3 inhibition reduced
ESFT cell growth alone or in combination with EWS/FLI1 knock down and enhanced the effects of
chemotherapeutics and targeted agents in ESFT. Furthermore, IL-6 containing conditioned media from
EWS/FLI1 knock down cells was demonstrated to have anti-apoptotic effects.
STAT3 is persistently activated in multiple malignancies and promotes tumorigenesis by up
regulating cellular proliferation and survival factors as well as those that promote immunosuppression
(40). This activation can occur through IL-6 secretion by tumor cells or stromal cells within the tumor
microenvironment. In ESFT, STAT3 is phosphorylated in approximately 50% of tumor samples in addition
to multiple cell lines (41, 42). Previous studies have also demonstrated the role of STAT3 in ESFT
proliferation. Treatment with a specific STAT3 inhibitor reduced the growth of ESFT cell lines in vitro (41).
Additionally, targeting JAK1/2 blocked both endogenous and IL-6 mediated STAT3 activation in ESFT
and inhibited cell growth in vitro and in vivo (43). Our own independent assessment with a distinct STAT3
inhibitor and dominant negative construct corroborates these results. Our work further expands upon the
role of IL-6/JAK/STAT3 signaling in ESFT by characterizing the induction of STAT3 activity that occurs
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upon EWS/FLI1 inhibition. We also demonstrated the benefits of combining a STAT3 inhibitor with other
agents.
Elevated IL-6 levels in the tumor microenvironment have been shown to promote tumor cell
proliferation and induce drug resistance. Lower drug efficacy due to cytokine secretion has been
observed in HER2-positive breast cancer, where trastuzumab resistance is mediated by IL-6 secretion
that leads to the expansion of a stem cell subpopulation (44). In lung cancer, IL-6 production by stromal
fibroblasts or tumor cells harboring EGFR mutations led to STAT3 activation and resistance to the
irreversible EGFR inhibitor afatinib (45). Additionally, paracrine IL-6 production protected both
neuroblastoma and osteosarcoma cells from drug-induced apoptosis and increased the proliferation and
migration of osteosarcoma cells (46, 47). Since IL-6 is secreted upon EWS/FLI1 knock down, we
hypothesized that soluble factors could also play a role in ESFT pathogenesis. The anti-apoptotic effects
of IL-6 containing conditioned media and synergistic effects from combining a STAT3 inhibitor with
existing ESFT therapeutics that we observed indicate secreted IL-6 may also promote drug resistance in
ESFT. Additionally, analysis of serum levels of patients with bone tumors including Ewing sarcoma
demonstrated significantly elevated IL-6 levels, which correlated with poor overall survival (48). This study
supports our data that factors secreted in the tumor microenvironment enhance tumor cell survival. While
our initial observation for a role of STAT3 signaling in ESFT cell survival involved a paracrine signaling
event between unaffected and EWS/FLI1 knock down cells, our co-treatment synergy results
demonstrate that STAT3 signaling does play a complex survival role in EWS/FLI1 expressing cells.
Further interactions between tumor cells and their microenvironment, such as stromal cell secretion of
STAT3-inducing ligands, will also need to be characterized.
While we have demonstrated that increased STAT3 phosphorylation that occurs upon EWS/FLI1
knock down is mediated in strong part by IL-6 secretion, more work is needed to fully elucidate this
mechanism. Our data indicate that IL-6 is the predominant factor, but blocking IL-6 or gp130 did not
completely abrogate induction of STAT3 phosphorylation. This argues that other secreted factors also
contribute to STAT3 activation. Our cytokine array results revealed multiple growth factors and cytokines
that were up regulated upon EWS/FLI1 knock down, including IL-8, GM-CSF, and CXCL1. Ewing
sarcoma patient serum also contained additional elevated cytokines such as IL-8, IL-1ra, and M-CSF (48),
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suggesting a combination of soluble factors cooperate with IL-6 to mediate its effect. Additionally, it is
unclear how IL-6 production is increased upon EWS/FLI1 knock down. IL-6 is one of several pro-
inflammatory cytokines whose expression is mediated by the transcription factor NF-κB (49). If EWS/FLI1
represses NF-κB, release of this inhibition upon EWS/FLI1 knock down is one possible explanation for an
increase in IL-6 levels. Furthermore, STAT3 activation and IL-6 production can be propagated by a
feedforward loop, so a small initial increase in IL-6 may result in a larger, sustained response (40).
In summary, our investigation uncovered a novel paracrine signaling pathway that expanded
upon the role of STAT3 signaling in ESFT pathogenesis. This provides a rationale for combining inhibitors
of this pathway with other agents to enhance the efficacy of ESFT therapies. Several agents targeting
components of the IL-6/JAK/STAT3 pathway have been evaluated in the clinical setting, including JAK
inhibitors and monoclonal antibodies that block IL-6 or the IL-6 receptor (50). Additionally, dasatinib,
which inhibits tyrosine kinases including SRC, is currently being evaluated in phase I/II trials for sarcoma
both as a single agent and in combination with other therapies (ClincialTrials.gov). These studies taken
together with our results suggest the use of additional agents directed against members of the IL-
6/JAK/STAT3 pathway could be utilized to improve clinical responses in ESFT.
ACKNOWLEDGEMENTS
Flow cytometry was performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center
for AIDS Research Flow Cytometry Core Facility that is supported by National Institutes of Health awards
CA-16042 and AI-28697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of
Medicine at UCLA. We thank Matteo Pellegrini (UCLA) for helpful discussions and providing
bioinformatics assistance.
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FIGURE LEGENDS Figure 1. EWS/FLI1 modulates phosphorylation of proteins involved in cell cycle, cell adhesion,
and cytoskeletal organization. (A) Rank analysis of phosphopeptides modulated by EWS/FLI1
identified through serine/threonine phosphoprofiling of A673 cells transduced with shRNA (818) targeting
EWS/FLI1. The heatmap displays the log2 fold change between knock down and control cells. Columns
represent biological replicates. Phosphopeptides that were modulated by greater than 1.2 fold in each
replicate were included in the heatmap. This includes 210 phosphopeptides, 86 of which that displayed
an increase in phosphorylation and 124 that displayed a decrease upon EWS/FLI1 knock down. Red
indicates positive and green indicates negative log ratios. Peptides were ranked based on the sum of the
log fold change across samples (rank score). The top and bottom 20 phosphopeptides based on the rank
analysis are enlarged. Cell lysates used for phosphoprofiling were harvested five days post lentiviral
transduction. (B) Top five over represented Gene Ontology biological processes for phosphopeptides that
displayed an increase or decrease in phosphorylation upon EWS/FLI1 knock down. (C) Top over
represented pathways for phosphopeptides that displayed an increase or decrease in phosphorylation
upon EWS/FLI1 knock down. (B,C) P-value is from a modified Fisher’s exact test to determine if the
percentage of submitted genes is statistically enriched compared to the percentage of genes in the
human genome. Benjamini multiple testing correction technique was performed to globally correct
enrichment p-value to control family-wide false discovery rate. **Benjamini p-value < 0.001, *Benjamini p-
value < 0.1.
Figure 2. STAT3 phosphorylation at residue 705 is up regulated upon EWS/FLI1 knock down. (A)
Rank analysis of phosphopeptides identified through tyrosine phosphoprofiling of ESFT cells transduced
with shRNA (818) targeting EWS/FLI1. The heatmap displays the log2 fold change between knock down
and control cells. Columns represent biological replicates. Red indicates positive and green indicates
negative log ratios. Gray indicates missing data. Peptides were ranked based on the sum of the log fold
change across all samples. Cell lysates used for phosphoprofiling were harvested five to eight days post
lentiviral transduction. (B) Immunoblot analysis of phospho-STAT3 (Y705) and total STAT3 levels in A673
cells transduced with EWS/FLI1 shRNA (818 or EF4) and corresponding vector controls (CT). Lysates
were harvested from cells transduced with 818 shRNA eight days post transduction and from cells
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transduced with EF4 shRNA five days post transduction. Phospho-STAT3 levels were quantitated based
on Odyssey software integrated intensity values. Values are listed below each band. (C) Quantitation of
phospho-STAT3 immunoblot signals from EWS/FLI1 knock down (818) and control samples based on
Odyssey software integrated intensity values. Phospho-STAT3 levels were normalized to total STAT3
levels. 818 values were normalized to vector control. The data plotted is an average of three biological
replicates. Error bars represent standard deviation.
Figure 3. STAT3 phosphorylation and EWS/FLI1 knock down occur in different populations of
ESFT cells. (A) STAT3 immunostaining of A673 cells transduced with vector control or EWS/FLI1 shRNA.
Transduced cells are GFP positive as the lentiviral vector contains a GFP marker. Cell nuclei were
visualized using DAPI. Pictures were taken at 40X magnification. (B) Phospho-STAT3 (Y705)
immunostaining of A673 cells transduced with vector control and EWS/FLI1 shRNA. Pictures were taken
at 40X magnification. (C) Flow cytometric analysis of phospho-STAT3 (Y705) and GFP levels in A673
cells transduced with vector control or EWS/FLI1 shRNA. Phospho-STAT3 fluorescence intensity levels
are plotted for GFP negative and positive populations. The ratio of median fluorescence intensity (MFI)
between cells transduced with EWS/FLI1 shRNA and control cells is indicated on the graphs.
Figure 4. Elevated cytokines present within conditioned media derived from EWS/FLI1 knock
down cells are able to induce STAT3 phosphorylation. (A,B) Immunoblot analysis of phospho-STAT3
(Y705) and total STAT3 in ESFT cells transduced with EWS/FLI1 shRNA (818 or EF4) or corresponding
vector controls (CT) five days post lentiviral transduction, or ESFT cells transduced with vector controls
that were stimulated with conditioned media from knock down or control cells for one hour (A) or one and
24 hours (B). (C) Cytokine array analysis of 174 growth factor and cytokines in conditioned media from
A673 control and EWS/FLI1 knock down cells. Cells were transferred to serum-free media four days post
lentiviral transduction with empty vector or EWS/FLI1 shRNA (818) and conditioned media was collected
two days later. Selected growth factors that are up regulated upon EWS/FLI1 knock down or are present
in high levels are circled and labeled in red. (D) ImageJ was used to measure the integrated density
values of selected cytokines and positive controls from (C). Relative intensity was calculated by dividing
cytokine values by control values. (E) Immunoblot analysis of phospho-STAT3 (Y705) and total STAT3 in
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A673 and A4573 cells treated with conditioned media from cells transduced with EWS/FLI1 shRNA (818
CM) or 100 ng/mL of human recombinant IL-6, GM-CSF, or CXCL1 individually or all three recombinant
proteins for one hour.
Figure 5. Increased STAT3 phosphorylation upon EWS/FLI1 knock down is partially dependent on
IL-6/gp130 signaling. (A) ELISA analysis of IL-6 levels in conditioned media from control or EWS/FLI1
knock down ESFT cells. Data plotted is the average of at least three biological replicates. Error bars
indicate standard deviation. (B) Fold change in IL-6 transcript levels after EWS/FLI1 knock down.
Quantitative real time PCR was used to determine IL-6 and GADPH copy number based on standard
dilutions. IL-6 copy numbers were normalized to those of GAPDH, then values from EWS/FLI1 knock
down samples were divided by values from control samples to determine fold change. Columns represent
the average of three biological replicates, error bars represent standard deviation. (C) ELISA analysis of
IL-6 levels in conditioned media samples used to treat cells in E. (D) ELISA analysis of IL-6 levels in
knock down conditioned media before (818) and after (1-4) IL-6 immunodepletion used to treat cells in F.
(E) Immunoblot analysis of phospho-STAT3 (Y705) and total STAT3 in A4573 and A673 cells treated with
conditioned media from cells transduced with vector control (CT CM) or EWS/FLI1 shRNA (818 CM), or
818 CM in which IL-6 has been removed by immunoprecipitation by 2.5 μg/mL of IL-6 neutralizing
antibody. A673 818 CM was diluted 1:4 for this experiment. (F) Phospho-STAT3 (Y705) and total STAT3
immunoblot analysis of A673 cells treated with conditioned media from EWS/FLI1 knock down (818) or
control (CT) A673 cells, or four different samples of knock down conditioned media in which IL-6 has
been immunodepleted (1-4). Immunodepletion was performed as in E. (G) Immunoblot analysis of
phospho-STAT3 (Y705) and total STAT3 in A4573 and A673 cells treated with conditioned media from
cells transduced with vector control (CT CM) or EWS/FLI1 shRNA (818 CM), or treated with 818 CM after
a one hour incubation with 10 μg/ml gp130 blocking antibody. A673 818 CM was diluted 1:10 for this
experiment. (H) Immunoblot analysis of gp130, phospho-STAT3 (Y705, S727), total STAT3, phospho-
JAK2 (Y1007/1008), total JAK2, and phospho-SRC family kinase (SFK) (Y416) in A673 cells transduced
with vector control (CT) or EWS/FLI1 shRNA (818, unsorted and sorted GFP negative and positive
populations). (I) Model of IL-6-mediated paracrine signaling. EWS/FLI1 knock down results in the
secretion of IL-6, which binds to the gp130/IL-6R receptor. In cells that maintain EWS/FLI1 expression
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(blue), receptor activation leads to downstream phosphorylation of STAT3. Cells with reduced EWS/FLI1
levels (green) demonstrate a diminished capacity for IL-6-mediated STAT3 activation.
Figure 6. STAT3 signaling affects growth and survival of ESFT cells. (A) Growth curves for ESFT
cells transduced with EWS/FLI1 shRNA (818) or empty vector control (CT) treated with Stattic for 0 to 3
days. A4573 cells were treated with 10 µM Stattic and A673 cells were treated with 7.5 µM Stattic. Cell
viability was measured using an MTT assay. Absorbance values for each treatment condition were
normalized to day 0 and plotted against time to assess relative growth. Data plotted is from the average
of five experiments. Days 0 to 3 correspond to 3 to 6 days post lentiviral transduction. By day 3 (6 days
post EWS/FLI1 knock down), A4573 818 and control cells display similar growth rates due to an
increased proportion of untranduced cells in the A4573 818 population. (B) Growth curves for A673 cells
transduced with EWS/FLI1 shRNA (818), dominant negative STAT3 (STAT3 Y705F), or empty vector
controls (U6, Tk Neo). Cell viability was measured using an MTT assay. Absorbance values for each
treatment condition were normalized to day 0 and plotted against time to assess relative growth. Data
plotted is from the average of five experiments. Two-way ANOVA was used to compare Tk Neo 818 and
STAT3 Y705F 818 growth curves. *P = 0.028. (C) Immunoblot analysis of cleaved PARP levels from ESFT cells treated with conditioned media containing 1% FBS from A673 cells transduced with vector
control (CT CM) or EWS/FLI1 shRNA (818 CM) for 24 hours. Cleaved PARP levels were quantitated
based on Odyssey software integrated intensity values. Values are listed below each band. (D) A4573
and SK-N-MC cells were treated with conditioned media containing 1% FBS from A673 cells transduced
with vector control (CT CM) or EWS/FLI1 shRNA (818 CM) for 24 hours. Cleaved PARP levels were
quantitated based on Odyssey software integrated intensity values from immunoblot. Columns represent
the average of six biological replicates, error bars represent SEM. A paired t test was used to
demonstrate significant difference in PARP cleavage between cells treated with control and EWS/FLI1
shRNA conditioned media. (E) Immunoblot analysis of cleaved PARP levels from SK-N-MC cells treated
with conditioned media containing 1% FBS from A673 cells transduced with vector control (CT CM) or
EWS/FLI1 shRNA (818 CM), CT CM supplemented with 100 ng/mL IL-6, or 818 CM in which IL-6 has
been immunodepleted for 24 hours. Cleaved PARP levels were quantitated based on Odyssey software
integrated intensity values. Values are listed below each band. (F) A673 cells were exposed to a series of
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1.5-fold dilutions of Stattic and NVP-AEW541 alone or in combination at a constant ratio of 1:2 for 72
hours, then cell viability was determined by MTT assay. (G) Isobologram plot of the effect of Stattic
combined with NVP-AEW541. The effective doses of NVP-AEW541 and Stattic are plotted on the x- and
y-axis with lines of linear additivity connecting the ED50, ED75, and E90 for individual treatments. (H) A673
cells were exposed to a series of 1.5-fold dilutions of Stattic (72h) and doxorubicin (4h) alone or in
combination at a constant ratio of 10:1. For combination treatment, cells were treated with doxorubicin for
4 hours, then Stattic for 72 hours. Cell viability was determined by MTT assay after 72 hours. (I)
Isobologram plot of the effect of Stattic combined with doxorubicin. The effective doses (ED) of
doxorubicin and Stattic are plotted on the x- and y-axis with lines of linear additivity connecting the ED50,
ED75, and E90 for individual treatments. (F,H) Columns represent the average of three independent
experiments, error bars represent standard deviation. Combination index values greater than 1, equal to 1,
or less than one indicate antagonism, additivity, or synergy. (G,I) Points for combination treatment above,
on, or below the lines indicate antagonism, additivity, or synergy, respectively.
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Published OnlineFirst August 4, 2014.Mol Cancer Res Jennifer L Anderson, Bjorn Titz, Ryan Akiyama, et al. Signaling within the Ewing Sarcoma Family of TumorsPhosphoproteomic Profiling Reveals IL6-mediated Paracrine
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