TITLE PAGE - Molecular Cancer Research · 2014. 8. 2. · subsequent STAT3 activation in bystander cells. This novel adaptive response suggests combination therapy with STAT3 inhibitors

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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 [email protected] 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




15

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




16

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




17

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




18

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),




19

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




25

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




26

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




27

(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




28

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.




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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TITLE PAGE - Molecular Cancer Research · 2014. 8. 2. · subsequent STAT3 activation in bystander cells. This novel adaptive response suggests combination therapy with STAT3 inhibitors