fus-ddit3 fusion protein driven igf-ir signaling is a therapeutic … · gerhard-domagk-institute...

Trautmann et al.: IGF-II in myxoid liposarcoma

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FUS-DDIT3 fusion protein driven IGF-IR signaling is a therapeutic target

in myxoid liposarcoma

Marcel Trautmann1*, Jasmin Menzel1, Christian Bertling1, Magdalene Cyra1, Ilka Isfort1,

Konrad Steinestel1, Sandra Elges1, Inga Grünewald1, Bianca Altvater2, Claudia Rossig2,

Stefan Fröhling3,4,5, Susanne Hafner6, Thomas Simmet6, Pierre Åman7, Eva

Wardelmann1, Sebastian Huss1, and Wolfgang Hartmann1*

1 Gerhard-Domagk-Institute of Pathology, University Hospital Münster, Münster,

Germany; 2 Department of Pediatric Hematology and Oncology, University Children´s

Hospital Münster, Münster, Germany; 3 Department of Translational Oncology, National

Center for Tumor Diseases (NCT) Heidelberg and German Cancer Research Center

(DKFZ), Heidelberg, Germany; 4 Section for Personalized Oncology, Heidelberg

University Hospital, Heidelberg, Germany; 5 German Cancer Consortium (DKTK),

Heidelberg, Germany; 6 Institute of Pharmacology of Natural Products & Clinical

Pharmacology, Ulm University, Ulm, Germany; 7 Sahlgrenska Cancer Center, University

of Gothenburg, Gothenburg, Sweden

Running title: IGF-II in myxoid liposarcoma

Keywords: myxoid liposarcoma, FUS-DDIT3, IGF-II, IGF-IR, PI3K/Akt

* Correspondence should be addressed to:

Marcel Trautmann & Wolfgang Hartmann; Gerhard-Domagk-Institute of Pathology,

University Hospital Münster, 48149 Münster, Germany

Phone: +49 (0) 251-83-55440; Fax: +49 (0) 251-83-57559

e-mail: [email protected]; [email protected]

The authors declare no potential conflicts of interest.

Financial support: This study was supported in part by grants from “Innovative Medical

Research“ of the University of Münster Medical School (#HU121421) to MT and SH and

the Deutsche Forschungsgemeinschaft (DFG) (#STE 2467/1-1).

Research. on March 23, 2020. © 2017 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on June 21, 2017; DOI: 10.1158/1078-0432.CCR-17-0130

http://clincancerres.aacrjournals.org/


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STATEMENT OF TRANSLATIONAL RELEVANCE

IGF-IR overexpression has been shown to be associated with an unfavorable clinical

course in myxoid liposarcomas. However, the molecular contribution of IGF-IR to the

pathogenesis of myxoid liposarcoma as well as its specific mechanism of activation has

not been understood so far. We here provide substantial evidence of a specific, to date

unknown molecularly based mechanism of IGF-IR/PI3K/Akt cascade activation in

myxoid liposarcoma through FUS-DDIT3-dependent IGF2 induction. We provide a

rational proof of a cell-autonomous stimulation of myxoid liposarcoma cells involving an

IGF-II/IGF-IR transactivation loop and demonstrate high efficacy of a IGF-IR-directed

therapeutic approach in vitro and in vivo for myxoid liposarcoma cancer therapy. Our

preclinical evaluation substantially contributes to the understanding of myxoid

liposarcoma pathogenesis underlining the molecular and clinical relevance of actionable

tyrosine kinase signals, either based on activating PIK3CA mutations or transmitted via

the IGF-IR as induced by the specific FUS-DDIT3 fusion protein.





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ABSTRACT

Purpose: Myxoid liposarcoma is an aggressive disease with particular propensity to

develop hematogenic metastases. Over 90% of myxoid liposarcoma are characterized

by a reciprocal t(12;16)(q13;p11) translocation. The resulting chimeric FUS-DDIT3

fusion protein plays a crucial role in myxoid liposarcoma pathogenesis; however, its

specific impact on oncogenic signaling pathways remains to be substantiated. We here

investigate the functional role of FUS-DDIT3 in IGF-IR/PI3K/Akt signaling driving myxoid

liposarcoma pathogenesis.

Experimental Design: Immunohistochemical evaluation of key effectors of the

IGF-IR/PI3K/Akt signaling axis was performed in a comprehensive cohort of myxoid

liposarcoma specimens. FUS-DDIT3 dependency and biological function of the

IGF-IR/PI3K/Akt signaling cascade were analyzed using a HT1080 fibrosarcoma-based

myxoid liposarcoma tumor model and multiple tumor-derived myxoid liposarcoma cell

lines. An established myxoid liposarcoma avian chorioallantoic membrane model was

employed for in vivo confirmation of the preclinical in vitro results.

Results: A comprehensive subset of myxoid liposarcoma specimens showed elevated

expression and phosphorylation levels of various IGF-IR/PI3K/Akt signaling effectors. In

HT1080 fibrosarcoma cells, overexpression of FUS-DDIT3 induced aberrant

IGF-IR/PI3K/Akt pathway activity, which was dependent on transcriptional induction of

the IGF2 gene. Conversely, RNAi-mediated FUS-DDIT3 knockdown in myxoid

liposarcoma cells led to an inactivation of IGF-IR/PI3K/Akt signaling associated with

diminished IGF2 mRNA expression. Treatment of myxoid liposarcoma cell lines with

several IGF-IR inhibitors resulted in significant growth inhibition in vitro and in vivo.

Conclusions: Our preclinical study substantiates the fundamental role of the

IGF-IR/PI3K/Akt signaling pathway in myxoid liposarcoma pathogenesis and provides a

mechanism-based rationale for molecular targeted approaches in myxoid liposarcoma

cancer therapy.





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INTRODUCTION

Accounting for 5-10% of all soft tissue sarcomas, myxoid liposarcoma (MLS) represent

20% of all malignant adipocytic tumors (1). In the majority of cases, MLS arise in

younger adults, defining the most frequent liposarcoma subtype in patients <20 years of

age. Clinically, MLS are characterized by a high rate of local recurrences and

development of metastases affecting in total 40% of patients (2). Morphologically, MLS

comprise a large spectrum ranging from paucicellular myxoid tumors to hypercellular

round cell sarcomas associated with a more aggressive clinical course (3). Genetically,

the vast majority of MLS is characterized by a chromosomal t(12;16)(q13;p11)

translocation, juxtaposing the FUS and DDIT3 genes. About 5% of all MLS display an

alternative chromosomal t(12;22) rearrangement leading to an EWSR1-DDIT3 gene

fusion (4). The resulting FUS-DDIT3 and EWSR1-DDIT3 fusion proteins are thought to

play an essential role in MLS pathogenesis, acting as transcriptional (dys-) regulators (5-

8); however, the functional details and the specific impact of the chimeric fusion protein

on oncogenic signaling pathways known to be activated in MLS is incompletely

understood.

It has been shown that MLS are characterized by EGFR, PDGFRB, RET, MET as well

as VEGFR1 activation sustained by autocrine/paracrine loops and receptor tyrosine

kinase (RTK) cross-talk, resulting in activation of the downstream PI3K/Akt signaling

pathway (9,10). PI3K/Akt signaling is a central hub in the transduction of different RTK

inputs involving diverse growth-controlling effectors such as GSK-3, p70 S6 kinase

(p70S6K), ribosomal S6 protein (11-13) and the cell cycle regulator Cyclin D1 (14-16). In

line with the data presented by Negri et al. (9), Barretina and colleagues (17) did not

detect somatic RTK mutations in MLS; however, they were the first to describe a

relatively high frequency (18% of cases) of activating point mutations in the PIK3CA

gene encoding the catalytic PI3K subunit which was associated with shorter

disease-specific survival. Pointing to alternative activation mechanisms of the PI3K/Akt

signaling pathway, Demicco et al. reported loss of PTEN or strong overexpression of the

Insulin-like growth factor-I receptor (IGF-IR) in subsets of MLS which were shown to be

mutually exclusive or to occur only very rarely and simultaneously with PIK3CA

mutations (18). Results previously presented by Cheng and colleagues (19) indicate that





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overexpression of IGF-IR (which was predominantly detected in the prognostically

unfavorable round cell component) is associated with an aggressive clinical course in

MLS.

Current therapeutic approaches in high-grade MLS complement radical surgery with

radiotherapy and/or conventional chemotherapy, conventionally based on

anthracyclines/ifosfamide and recently supplemented with novel agents such as

Trabectedin or Eribulin (20-22). However, though MLS display higher chemotherapy

sensitivity than other liposarcoma subtypes, the high rate of recurrences and metastases

in MLS underlines the urgent need of novel therapeutic options.

Overall, previously published data suggest a particular importance of IGF-IR/PI3K/Akt

signaling in the pathogenesis and progression of MLS (18,19). While the biological

impact of PIK3CA and PTEN alterations is intuitive, it remains open in which way IGF-IR

contributes to MLS oncogenesis and whether pharmacologic IGF-IR inhibition might

result in favorable therapeutic effects. The current study was performed to explore the

functional relevance of IGF-IR/PI3K/Akt signaling in MLS, including its molecular

dependence on the pathognomonic FUS-DDIT3 fusion protein, and to test a molecularly

targeted approach employing a small molecule IGF-IR inhibitor in a preclinical setting.





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MATERIALS AND METHODS

Patients, tumor specimens and tissue microarray (TMA)

In summary, 60 myxoid liposarcoma tumor specimens were included (25 women,

35 men; median age at diagnosis was 48 years, range 24-78 years of age). Median

tumor size was 10 cm (range 1.5-29 cm). According to the current WHO classification of

tumours of Soft Tissue and Bone (23) all diagnoses were reviewed by two experienced

pathologists based on clinical information, morphological criteria, and DDIT3 break-apart

fluorescence in situ hybridization (FISH) or reverse transcriptional PCR (RT-PCR)

analysis, demonstrating the pathognomonic translocations as previously described (24).

Clinicopathological characteristics of the cohort are summarized in Table 1. MLS tissue

microarrays (TMA) were prepared from formalin-fixed, paraffin-embedded (FFPE; with

two representative 1 mm cores) tissue specimens selected from the archival files of the

Gerhard-Domagk-Institute of Pathology, University Hospital Münster. Two areas within

each tumor were selected by two experienced pathologists for the TMA in order to

represent potential heterogeneity, e.g. with regard to the round cell content.

Occasionally occurring necrobiotic areas and their neighborhood were excluded from

TMA sampling to avoid the detection of secondary (e.g. hypoxia-induced) alterations.

The study was approved by the Ethical Committee of the University of Münster

(2015-548-f-S) and conducted in accordance with current ethical standards (Declaration

of Helsinki, 1975).

Cell culture and cell lines

The MLS cell lines MLS402-91 (FUS-DDIT3 type 1; exon 7-2) and MLS1765-92

(FUS-DDIT3 type 8; exon 13-2) were contributed by Pierre Åman (25). For the purpose

of cell line authentication, presence of the pathognomonic t(12;16) translocation was

confirmed by RT-PCR and Sanger sequencing using specific primers for the

translocation subtypes. All monolayer cell cultures were grown under standard

incubation condition (37°C, humidified atmosphere, 5% CO2) and maintained in Roswell

Park Memorial Institute medium 1640 (RPMI; MLS402-91 and MLS1765-92), Dulbecco's

Modified Eagles' medium (DMEM; A673 and HT1080) or Iscove's Modified Dulbecco's

medium (IMDM: Capan-1), supplemented with 10% fetal bovine serum (FBS;





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Life Technologies, Carlsbad, CA, USA). Mycoplasma testing was performed quarterly by

standardized PCR and cells were passaged for a maximum of 25-35 culturing cycles

between thawing and use in the described experiments. To study the effects of

increasing concentrations (0.125-2 µM) of NVP-AEW541 (26,27), BMS-754807 (28,29)

and Picropodophyllin (PPP) (30,31), MLS cells were grown in medium supplemented

with 2% FBS. Cell lysis, protein extraction and immunoblotting were performed 15-72 h

after treatment as previously described (32).

Immunohistochemistry (IHC)

IGF-IR (polyclonal rabbit, 1:100, #3027), phospho-Akt (S473, monoclonal rabbit, clone

D9E, 1:50, #4060), phospho-GSK-3β (S21/9, polyclonal rabbit, 1:50, #9331),

phospho-S6 (S240/244, monoclonal rabbit, clone D68F8, 1:100, #5364),

phospho-p44/42 MAPK (T202/Y204, monoclonal rabbit, clone D13.14.4E, 1:150, #4370)

and Cyclin D1 (monoclonal rabbit, clone 92G2, 1:50, #2978) antibodies were purchased

from Cell Signaling Technology (Danvers, MA, USA), IGF-II (monoclonal mouse, 1:50,

clone S1F2, #05-166) from Merck Millipore (Darmstadt, Germany), MIB-1/Ki-67

(monoclonal rabbit, clone 30-9, #790-4286) from Roche (Basel, Switzerland) and PTEN

(monoclonal rabbit, clone SP218, 1:50, #M5180) from Spring Bioscience.

Immunohistochemical staining was performed with a BenchMark ULTRA Autostainer

(VENTANA/Roche, Basel, Switzerland) on 3 μm MLS TMA sections. In brief, the

staining procedure included: i) heat-induced epitope retrieval (HIER) pretreatment using

Tris-Borate-EDTA buffer (pH 8.4; 95-100°C, 32-72 min) followed by ii) incubation with

respective primary antibodies for 16-120 min and iii) employment of the OptiView DAB

IHC Detection Kit (VENTANA/Roche, Basel, Switzerland) according to the

manufacturer's instructions. Loss of PTEN protein was assessed by

immunohistochemical studies according to a standardized algorithm previously

described (18). Positive and negative control stainings using an appropriate IgG subtype

(DCS) were included. Immunoreactivity was assessed using a semi-quantitative score

(0, negative; 1, weak; 2, moderate; and 3, strong) defining the staining intensity in the

positive control (invasive breast cancer, NST) as strong. Only TMA tissue cores with at

least moderate staining (semi-quantitative score ≥2) were considered positive for the

purposes of the study. The IHC readers were blinded to outcome data, the score





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cutpoint (positive = semi-quantitative score ≥2) was prespecified without prior analyses

of the clinical course.

FUS-DDIT3 fusion protein overexpression in HT1080 cells

Generation of expression plasmids for FUS-DDIT3, FUS, and DDIT3 was previously

described (5). Human HT1080 fibrosarcoma cells were grown in 6-well plates and

transfected with 2.5 µg of plasmid DNA using Lipofectamine 2000 (Life Technologies,

Carlsbad, CA, USA) according to the manufacturer’s instructions. Transient

vector-based expression was confirmed 24 h after transfection by immunoblotting. As

control, HT1080 cells were transfected with the peGFP-N1 control plasmid

(Clontech Laboratories/Takara Bio Inc., Kusatsu, Japan).

Promoter-specific IGF2 RT-PCR

Promoter-specific transcription of IGF2 was determined using a competitive RT-PCR

assay as previously described (33). Briefly, total RNA was extracted from eGFP,

FUS-DDIT3, FUS or DDIT3-transfected HT1080 cells (RNeasy Plus Kit; Qiagen, Hilden,

Germany), reverse-transcribed (SuperScript IV First-Strand Synthesis Super Mix;

Life Technologies, Carlsbad, CA, USA), and PCR-amplified (FastStart Taq Polymerase

dNTP Pack; Roche, Basel, Switzerland) with specific primer sets for the different IGF2

transcripts (P1, P2, P3 and P4). Primer sequences and PCR conditions were previously

published (34). Ribosomal 28S rRNA transcript levels were used as reference (35).

Cell viability assay (MTT)

The MTT cell proliferation kit (Roche, Basel, Switzerland) was applied according to the

manufacturer’s instructions. In brief, MLS402-91 (2x103), MLS1765-92 (1.5x103) and

control cells (A673: Ewing´s sarcoma and Capan-1: pancreatic ductal adenocarcinoma)

(28,36,37) were seeded in 96-well plates (100 µl of medium supplemented with

2% FBS) and exposed to increasing concentrations of NVP-AEW541, BMS-754807 and

PPP (0.125-2 µM) for 72 h. An appropriate DMSO control was included. At least three

independent experiments were performed (each in quintuplicates) and results were

calculated as mean ± SEM.





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RNA interference (RNAi)

To target the constant DDIT3 portion of FUS-DDIT3 by RNAi, a set of pre-validated

duplex oligos was employed: VHS40607 (siRNA#1), VHS40605 (siRNA#2)

(Life Technologies, Carlsbad, CA, USA) and siRNA#3

(5’-GGAAGUGUAUCUUCAUACAdTdT-3’), previously published as TLS-CHOP siRNA

(38). Non-targeting negative control siRNA (BLOCK-iT Alexa Fluor Red Fluorescent

Control) was purchased from Life Technologies (Carlsbad, CA, USA). MLS402-91 and

MLS1765-92 cells were cultured in 25 cm2 cell culture flasks (medium supplemented

with 2% FBS) and transfected with indicated siRNA (25 pmol; cell density of 50%) using

Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA). After incubation for

72 h, siRNA-transfected cells were lysed and knockdown efficiency was documented by

immunoblotting and/or RT-PCR.

Immunoblot analysis

Following primary antibodies were used according to the manufacturer’s instructions:

IGF-IR, phospho-IGF-IR (Tyr1135/1136), Akt, phospho-Akt (Ser473), GSK-3β,

phospho-GSK-3β (Ser21/9), mTOR, phospho-mTOR (Ser2448), p70S6K,

phospho-p70S6K (Thr389), S6, phospho-S6 (Ser235/236 and Ser240/244), p44/42

MAPK, phospho-p44/42 MAPK (Thr202/Tyr204) and Cyclin D1 (all obtained from Cell

Signaling Technology, Danvers, MA, USA); β-actin (Sigma-Aldrich, St Louis, MO, USA);

DDIT3/GADD153, and FUS/TLS (both obtained from Santa Cruz Technology, Dallas,

Texas, USA). The FUS-DDIT3 fusion protein was detected with an antibody targeting

the N-terminus of DDIT3 (which is retained in the FUS-DDIT3 fusion oncoprotein).

Secondary antibody labeling (Bio-Rad Laboratories, Hercules, CA, USA) as well as

immunoblot development was performed using an enhanced chemiluminescence

detection kit (SignalFire ECL Reagent; Cell Signaling Technology, Danvers, MA, USA)

and the Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad

Laboratories, Hercules, CA, USA).

Flow cytometry

Effects of NVP-AEW541 and PPP on MLS apoptotic and mitotic rates were assessed by

flow cytometric analyses. Briefly, MLS cells were grown in 175 cm2 cell culture flasks





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(medium supplemented with 2% FBS) and treated with NVP-AEW541 and PPP

(0.75-1.5 µM; 72 h). Adherent cells were detached using 0.025% Trypsin

(Life Technologies, Carlsbad, CA, USA), fixed in 2% paraformaldehyde (10 min on ice),

washed in PBS, collected by centrifugation and incubated in ice-cold PBS

(supplemented with 0.25% Triton X-100) for 5 min on ice. After an additional washing

step, cells were re-suspended in PBS containing following antibodies: cleaved

Poly-(ADP-ribose)-polymerase (PARP) (Asp214) (BD Biosciences, San Jose, CA, USA;

phycoerythrin-labelled) and phospho-histone H3 (Ser10) (Cell Signaling Technology;

Alexa Fluor 647-labelled) followed by incubation for 60 min at room temperature.

Fluorescence intensity was measured using a FACSCanto II analytical flow cytometer

and cytometric data were analyzed using the FACSDiva software (both BD Biosciences,

San Jose, CA, USA). Each experiment was performed at least in duplicates.

Next-generation sequencing (NGS)

A customized GeneRead DNAseq Mix-n-Match V2 panel (Qiagen, Hilden, Germany)

was used to amplify the exonic region of PIK3CA. Target enrichment was processed by

means of the GeneRead DNAseq Panel PCR V2 Kit (Qiagen), following the

manufacturer’s instructions. All purification and size selection steps were performed

utilizing Agencourt AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). End

repair, A-addition and ligation to NEXTflex-96 DNA barcodes (Bioo Scientific, Austin,

Texas, USA) were carried out using the GeneRead DNA Library I Core Kit (Qiagen).

Amplification of adapter-ligated DNA was conducted using NEXTflex primers (Bioo

Scientific) and the HiFi PCR Master Mix (GeneRead DNA I Amp Kit, Qiagen).

Next-generation sequencing was performed applying 12.5 pM library pools (2% PhiX V3

control) and the MiSeq Reagent v2 chemistry (Illumina, Inc., San Diego, CA, USA). NGS

data analysis was performed by means of the CLC Biomedical Genomics Workbench

software (CLC bio, Qiagen) as described before (39). Validation by Sanger sequencing

was conducted according to standard procedures using the BigDye Terminator v3.1

Cycle Sequencing Kit (Life Technologies, Carlsbad, CA, USA).





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In vivo efficacy of NVP-AEW541 and PPP in MLS chicken chorioallantoic

membrane (CAM) studies

For in vivo experiments, CAM assays were performed as previously described (40). In

brief, MLS402-91 and MLS1765-92 cells (1x106 cells/egg; dissolved in medium/Matrigel

1:1, v/v) were xenografted onto the egg CAM (7 days after fertilization) and incubated at

37°C with 60% relative humidity. On day 8 of incubation, NVP-AEW541, PPP (1 µM) or

control (0.2% DMSO in NaCl 0.9%) were applied topically. The identical treatment

protocol was recapitulated on two consecutive days. Three days after treatment

initiation, tumor-containing CAM xenografts were explanted, fixed in 5% PFA, and

processed for histopathological examination. Tumor volume (mm3) was calculated

according to the formula: TV= length (mm) x width² (mm) x π/6 (41). All in vivo studies

were performed in accordance with the standards of the National and European Union

guidelines.

Compounds

The IGF-IR kinase inhibitors NVP-AEW541 (hydrochloride; C27H29N5O • 2HCl; CAS#:

475489-16-8, IGF-IR ATP antagonist), BMS-754807 (C23H24FN9O;

CAS#: 1001350-96-4, IGF-IR/IR ATP antagonist) and Picropodophyllin (PPP; C22H22O8;

CAS#: 477-47-4, IGHF-IR non-ATP antagonist) (26-31) were purchased from Biomol

(Hamburg, Germany) and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich,

St Louis, MO, USA). The final DMSO concentration did not exceed 0.1% (v/v) for all

in vitro and in vivo applications.

Statistical analysis

Immunohistochemical staining results and Kaplan-Meier survival/event free correlations

were statistically analyzed by Gehan-Breslow-Wilcoxon test (GraphPad Software,

La Jolla, CA, USA). Two-group comparisons were analyzed by unpaired Student’s t-test

(GraphPad Software, La Jolla, CA, USA). Experimental results of MTT assays and flow

cytometric analyses are represented as mean ± SEM (standard error of the mean) from

n independent experiments (n≥3). Statistical differences were considered significant at

p<0.05 (*). The concentration of NVP-AEW541, BMS-754807 and PPP required for





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50% growth inhibition (IC50 value), was calculated by non-linear regression analysis

using the GraphPad Prism (GraphPad Software, La Jolla, CA, USA).





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RESULTS

Expression of IGF-IR and PI3K/Akt/GSK-3β signaling components in human MLS

tumor tissues and MLS cell lines

To determine the involvement of IGF-IR- and PI3K/Akt/GSK-3β-mediated signal

transduction in myxoid liposarcoma tumorigenesis, expression of IGF-IR, IGF-II, Akt

(Ser473), GSK-3β (Ser21/9), S6 (Ser240/244), Cyclin D1 and PTEN were examined in a

comprehensive set of 60 MLS specimens using immunohistochemistry. In addition,

p44/42 MAPK (Thr202/Tyr204) was analyzed as an indicator of activated RAS/RAF/ERK

signaling (summarized in Supplementary Table S1). Positive staining for IGF-IR and

IGF-II was detected in 49.1% and 70.2% of all cases, respectively (Figure 1A-B), while

6 out of 60 specimens (10%) showed no membranous IGF-IR immunoreactivity.

Consistently, several phosphorylated signaling components were highly expressed in

MLS including Akt (Ser473), GSK-3β (Ser21/9), S6 (Ser240/244), and Cyclin D1; p44/42

MAPK (Thr202/Tyr204) was also detected at relevant levels (Figure 1C-G). Loss of

PTEN protein expression was detected in 9% of all studied MLS cases. In total, 26.3%

of MLS specimens displayed moderate to strong phosphorylation levels of Akt (Ser473),

34.5% for GSK-3β (Ser21/9), 34.8% for S6 (Ser240/244) and 53.4% for p44/42 MAPK

(Thr202/Tyr204). Strong Cyclin D1 expression levels were detected in 10.3% of tumors,

while 36.2% displayed moderate and 41.4% showed weak Cyclin D1 expression

(summarized in Figure 2A). Moderate to strong staining for MIB-1 was detected in 21.1%

of all cases (Figure 1H). An overlap of positive IGF-IR/IGF-II immunoreactivity and

phosphorylation of Akt (Ser473), GSK-3β (Ser21/9) and S6 (Ser240/244) was detected

in 33% of MLS specimens (Figure 2B). Expression of IGF-IR and PI3K/Akt/GSK-3β

signaling components did not correlate with the patients’ age, gender, translocation

subtype and/or tumor size. No statistically significant differences in overall/event free

correlations were detected for IGF-IR (p=0.305), IGF-II (p=0.971), p-Akt (Ser473)

(p=0.162), p-GSK-3β (Ser21/9) (p=0.607) or Cyclin D1 (p=0.269) IHC-positive

subgroups. In accordance with the immunohistochemical results in MLS tissue

specimens, elevated protein expression and phosphorylation levels were demonstrated

in total protein extracts (Figure 2C) and immunostainings of MLS cell lines

(Supplementary Figure S1). As oncogenic mutations in the PIK3CA gene might be





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responsible for constitutive activation of Akt signaling, we analyzed the entire PIK3CA

coding region by targeted next-generation sequencing. In 9 out of 60 (15%) MLS

specimens, SNVs in the PIK3CA gene could be detected (summarized in

Supplementary Table S1) whereas no PIK3CA alterations were identified in both MLS

cell lines (Supplementary Figure S2). Tumors with PIK3CA mutations displayed a

statistically significant (p=0.0003) worse overall (OS) and disease-free survival (DFS).

Activation of IGF-IR and PI3K/Akt/GSK-3β signaling is induced by the chimeric

FUS-DDIT3 fusion protein

To evaluate whether signal transduction via IGF-IR and PI3K/Akt/GSK-3β activation is

dependent on the expression of FUS-DDIT3, HT1080 cells were transiently transfected

with FUS-DDIT3, FUS, DDIT3 or eGFP expression vectors. First, we determined the

molecular regulation of promoter-specific IGF2 expression, demonstrating induction of

P2-dependent IGF2 transcripts upon FUS-DDIT3 expression (Figure 3A; upper panel).

The stimulated P2 promoter-dependent IGF2 transcript levels in FUS-DDIT3 expressing

HT1080 cells were comparable to levels in MLS402-91 and MLS1765-92 MLS cell lines

(Figure 3A; lower panel). Upon vector-based FUS-DDIT3 expression, immunoblot

analyses showed significantly increased phosphorylation levels of IGF-IR

(Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9) and mTOR (Ser2448) compared to

cells overexpressing FUS, DDIT3 or eGFP (Figure 3B). No relevant changes in baseline

protein levels were detected, confirming FUS-DDIT3-triggered stimulation of the IGF-IR

and PI3K/Akt/GSK-3β pathway activation as indicated by enhanced phosphorylation

levels. Stimulation of MLS402-91 and MLS1765-92 cells with recombinant human IGF-II

protein (200 ng/ml; 15 min) was associated with enhanced phosphorylation levels of

IGF-IR (Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9) and mTOR (Ser2448),

suggesting IGF-IR mediated signals as a functionally relevant mechanism leading to

PI3K/Akt/GSK-3β activation (Figure 3C). As shown in Supplementary Figure S3, IGF-II

stimulation was able to rescue PI3K/Akt/GSK-3β pathway activation in FUS-DDIT3

depleted myxoid liposarcoma cells. A minor induction of phosphorylation was observed

upon eGFP control expression; however, this activation was not associated with IGF2

transcriptional induction (Figure 3A; upper panel) or elevated expression levels of

Cyclin D1 (Figure 3B).





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FUS-DDIT3 knockdown affects IGF2 mRNA transcription and IGF-IR/Akt/GSK-3β

phosphorylation levels in MLS cell lines

To further analyze the functional contribution of FUS-DDIT3 fusion protein to oncogenic

IGF-IR and PI3K/Akt/GSK-3β mediated signaling by a non-pharmacological approach,

MLS402-91 and MLS1765-92 cells were transfected with siRNA. Consistently,

knockdown of FUS-DDIT3 significantly reduced levels of (I) P2 promoter-dependent

IGF2 transcripts, (II) phosphorylation of IGF-IR (Tyr1135/1136), Akt (Ser473),

GSK-3β (Ser21/9) and mTOR (Ser2448), combined with (III) reduced Cyclin D1

expression (Figure 3D and Supplementary Figure S4). The knockdown efficiency for a

set of pre-validated and published siRNA duplex oligos (38) targeting the DDIT3 portion

of the chimeric FUS-DDIT3 fusion gene (which is retained in the FUS-DDIT3 fusion

oncoprotein) was validated on protein level (Supplementary Figure S4). These results

confirmed that the FUS-DDIT3 fusion protein is involved in the regulation of IGF-IR and

PI3K/Akt/GSK-3β signaling activity through modulation of IGF2 mRNA expression.

Consistent with elevated phosphorylation levels of IGF-IR, Akt, GSK-3β and mTOR

upon transient FUS-DDIT3 expression in HT1080 cells (Figure 3A), phosphorylation and

activation was inversely suppressed compared to non-targeting negative control siRNA

(Figure 3D and Supplementary Figure S4).

NVP-AEW541, BMS-754807 and PPP reduce cell viability of MLS cell lines in vitro

To investigate the biological effects of pharmacological inhibition of IGF-IR, MLS and

control cell lines (A673 and Capan-1; Supplementary Figure S5) were exposed to

increasing concentrations (0.125-2 µM) of small molecule (I) IGF-IR ATP antagonists

(NVP-AEW541 and BMS-754807) and (II) the IGF-IR non-ATP antagonist PPP. In MTT

assays, all three IGF-IR inhibitors were effective in suppressing MLS and A673 cell

viability with IC50 values ranging from 0.36 to 1.35 µM, showing a dose-dependent mode

of action. MLS402-91 (fusion type 1; exon 7-2) cells were more sensitive to IGF-IR

inhibition compared to MLS1765-92 (fusion type 8; exon 13-2) cells. Capan-1 control

cells expressing low levels of IGF-IR showed only minor responses (Figure 4A,

Supplementary Figure S5 and Table 2).





- 16 -

NVP-AEW541 and PPP inhibits IGF-IR and PI3K/Akt/GSK-3β signal transduction

activity

Suppressive effects of treatment with the IGF-IR inhibitors NVP-AEW541 and PPP on

signal transduction activity in MLS cell lines were assessed by immunoblotting. In two

MLS cell lines, significant dose-dependent reduction of FUS-DDIT3-induced

phosphorylation levels were demonstrated for IGF-IR (Tyr1135/1136), Akt (Ser473),

GSK-3β (Ser21/9), p70S6K (Thr389) and S6 (Ser235/236 and Ser240/244) (Figure 4B

and Supplementary Figure S6). Cyclin D1 showed a dose- (0.5-1 µM) and

time-dependent downregulation in MLS402-91 and MLS1765-92 cells (Supplementary

Figure S7).

NVP-AEW541 and PPP reduce cell viability by inducing apoptosis and decreasing

mitotic activity in MLS cell lines

Performing flow cytometric analyses, Poly-adenosine diphosphate (ADP)-ribose

polymerase (PARP; Asp214) cleavage was used as a marker for apoptosis and

phospho-histone H3 (Ser10) was employed as a marker for mitotic activity. After

treatment with increasing concentrations of NVP-AEW541 or PPP (0.75-1.5 µM; 72 h),

MLS402-91 cells showed a significantly increased rate of apoptosis, accompanied by a

decrease of the mitotic fraction (Figure 4C and Supplementary Figure S6). Similar

results were observed in MLS1765-92 cells (Supplementary Figure S8).

In vivo efficacy of NVP-AEW541 and PPP in a CAM model of MLS

To verify the efficacy of IGF-IR inhibition on tumor growth and progression in an in vivo

model of human MLS, we xenografted MLS402-91 and MLS1765-92 cells onto a chick

CAM to initiate MLS tumor formation. Topical NVP-AEW541 and PPP administration

(1 µM) resulted in a significant reduction of tumor volume compared to the DMSO

vehicle control group (Figure 4D and Supplementary Figure S6; *P<0.05).

Representative H&E stainings of CAM tumor specimens are included in Supplementary

Figure S9.





- 17 -

DISCUSSION

Myxoid liposarcoma is a malignant lipogenic soft tissue neoplasia with a particular

propensity to develop distant metastases. High histological grade, generally defined as

>5% round cell component is the major predictor of an unfavorable outcome (3,42).

While there is an established role for conventional radiotherapy and cytotoxic therapies

in MLS (20), molecularly targeted therapeutic approaches are still missing. The vast

majority of MLS are characterized by the FUS-DDIT3 gene fusion encoding an aberrant

transcriptional regulator that has the potential to transform mesenchymal stem cells to

form MLS in mice (8). As in other sarcomas driven by specific gene fusions, MLS

characteristically display only few additional genetic alterations; however, 14%-18% of

MLS were reported to carry activating mutations in the PIK3CA gene encoding the

catalytic PI3K subunit which occur predominantly in the more aggressive round cell

variant of MLS. As alternative mechanisms of PI3K/Akt signaling pathway activation,

rare biallelic losses of PTEN and overexpression of the IGF-IR (in 25% of the cases

and occurring only very rarely simultaneously with PI3KCA mutations) have been

described (17,18). As reported, high prevalence of IGF-IR overexpression in the round

cell variant of MLS (18) fits well with previous data showing that IGF-IR overexpression

in MLS is associated with a poor metastasis-free survival (19). Since therapeutic

targeting of the chimeric fusion protein represents a particular challenge, it appears

reasonable to therapeutically address a signaling pathway which is known to be

significantly associated with a more aggressive MLS phenotype. We therefore set out to

analyze in detail IGF-IR-related signals mediated through the PI3K/Akt signaling

cascade, putting a particular focus on the functional dependency on the chimeric

FUS-DDIT3 fusion protein.

In accordance with previous studies (18,19), we detected moderate to strong expression

levels of IGF-IR and IGF-II in a large subset of MLS including the major sub-fraction of

tumors with a significant round cell component. In immunohistochemical analyses,

strong IGF-IR/IGF-II expression was associated with phosphorylation of Akt, GSK-3,

and/or S6 in 33% of the cases, indicating downstream activation of PI3K/Akt signals

(Figures 1 and 2). In contrast to what was previously reported by Cheng and colleagues

in a series of 32 MLS, we were unable to confirm a significant prognostic impact of

IGF-IR overexpression in our cohort of MLS (19). However, in line with data presented





- 18 -

by Barretina et al. (17), we found PIK3CA mutations to be associated with a negative

prognostic impact (summarized in Supplementary Table S1). Confirming the essential

biological role of IGF-IR dependent signals in MLS lacking PIK3CA or PTEN alterations,

the employed MLS cells (all wildtype for PIK3CA; Supplementary Figure S2) responded

to IGF-II stimulation with a significant induction of phosphorylation of IGF-IR and

PI3K/Akt downstream effectors (Figure 3C and Supplementary Figure S3). To

comprehensively understand the oncogenic mechanisms leading to aberrant activation

of IGF-IR signaling in MLS in detail, we explored the molecular dependence of IGF-IR

signals on the pathognomonic FUS-DDIT3 fusion protein. In response to FUS-DDIT3

knockdown, MLS cells showed loss of phosphorylation of IGF-IR, Akt, GSK-3 and a

significant reduction of total Cyclin D1 protein levels (Figure 3D and Supplementary

Figure S4). These alterations were associated with decreased expression levels of IGF2

promoter P2-dependent transcripts (Figure 3D). To evaluate the functional role of the

chimeric fusion protein in a MLS-independent cell context, we overexpressed the

FUS-DDIT3 fusion protein in HT1080 fibrosarcoma cells and thus confirmed specific

regulation of IGF2 promoter P2 transcription and subsequent activation of downstream

PI3K/Akt effectors through the chimeric oncogenic driver of MLS (Figure 3A and B). Our

finding of a functional connection of the MLS-specific FUS-DDIT3 gene fusion and the

activation of IGF signaling is an essential contribution to the understanding of the role of

IGF-IR in MLS and makes strong proof for the presence of a cell-autonomous

stimulation of MLS cells. Thus, our finding provides a missing link in the concept of MLS

pathogenesis in which a functional connection between the chimeric transcriptional

(dys-) regulator FUS-DDIT3 and the activation of IGF-IR-dependent PI3K/Akt signals

(occurring in a large subset of tumors) is not known. Based on our findings, the IGF-IR

signaling cascade now qualifies as a specific molecularly based target for therapeutic

approaches in MLS. The pattern of activated IGF signals in MLS resembles findings

reported for other fusion-driven sarcomas. We and others reported transcriptional

regulation of IGF2 through the oncogenic SS18-SSX fusion protein and subsequent

activation of the PI3K/Akt signaling cascade in synovial sarcoma (43,44), and the

PAX3-FKHR fusion protein was shown to induce IGF2 in in alveolar rhabdomyosarcoma

(45). In Ewing´s sarcoma, the EWSR1-FLI1 oncoprotein was shown to activate IGF-IR

signals through the transcriptional induction of IGF1 (46,47), and this finding led to the





- 19 -

successful exploration of IGF-IR targeted therapeutic approaches in Ewing´s sarcoma

(48,49). The high prevalence of IGF-IR transmitted signals in different fusion-driven

sarcomas implies a particular importance of inputs transmitted through this cascade for

these soft tissue tumors carrying only few mutations apart from the pathognomonic gene

fusions. From a molecular diagnostic point of view, fusion-driven sarcomas therefore

represent challenging paradigmatic malignancies in which pure high-throughput

genomics can often not provide elementary mutations qualifying as therapeutic targets

but in which functional precision oncology approaches are needed.

To investigate if IGF-IR directed approaches might be of therapeutic benefit in MLS, we

treated MLS and control cells with different small molecule IGF-IR inhibitors (26-31). Our

data show a significant dose-dependent reduction of MLS proliferation and viability

(Figure 4A), associated with the expected decrease in phosphorylation of PI3K/Akt

effectors (Figure 4B and Supplementary Figure S6). Consistent with these in vitro

results, administration of NVP-AEW541 and PPP to xenografted MLS402-91 and

MLS1765-92 cells led to an inhibition of tumor growth in vivo (Figure 4D and

Supplementary Figure S6 and 9). The panel of different substances available for

inhibition of the IGF signaling system has considerably increased during the recent

years and now includes, apart from small molecule tyrosine kinase inhibitors, different

monoclonal antibodies to the IGF-IR, and antibodies to IGF-I and IGF-II (50). In contrast

to relatively disappointing results in early clinical trials with other solid tumors, sustained

success of IGF-IR-directed therapies was observed in defined subsets of sarcomas

(48,49). However, the major challenge of therapeutic approaches addressing the IGF

system remains the identification of appropriate predictive biomarkers. In MLS, (absence

of a) PIK3CA mutation and IGF-II overexpression as well as IGF-IR phosphorylation

might be tested as such predictive indicators.

In conclusion, the results of the current study imply that activation of the IGF-IR/PI3K/Akt

signaling system is a common pattern in MLS which appears to be transcriptionally

controlled, at least in part by induction of IGF2 gene transcription in a

FUS-DDIT3-dependent manner. Disruption of IGF-IR mediated signals via a small

molecule inhibitor may provide an effective therapeutic approach for advanced MLS.

The present preclinical testing of an IGF-IR directed targeted approach shows potent





- 20 -

effects both in vitro and in vivo, qualifying the IGF-IR/PI3K/Akt signaling pathway as a

specific therapeutic target in MLS.

ACKNOWLEDGEMENTS

The authors thank Charlotte Sohlbach and Inka Buchroth for excellent technical support.

This study was supported by the fund “Innovative Medical Research“ of the University of

Münster Medical School (#HU121421).





- 21 -

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TABLES

Table 1. Clinicopathological characteristics of myxoid liposarcoma patients (n=60)

Age (years)

mean (±SD) 48.5 (±12.5)

median (range) 48 (24-78)

<48 28 (46.7%)

≥48 32 (53.3%)

Type

primary tumor 37 (61.7%)

metastasis 9 (15%)

recrudescence 9 (15%)

ND 5 (8.3%)

Morphology

myxoid 37 (61.7%)

round cell 23 (38.3%)

Size (cm)

mean (±SD) 10.3 (±5.6)

median (range) 10 (1.5-29)

<10 26 (43.3%)

≥10 21 (35%)

ND 13 (21.7%)

Sex

female 25 (41.7%)

male 35 (58.3%)

FISH

DDIT3 positive 59 (98.3%)

ND 1 (1.7%)

t(12;16) translocation type

FUS-DDIT3

(type 1; exon 7-2) 13 (21.7%)

FUS-DDIT3

(type 2; exon 5-2) 27 (45%)

ND 20 (33.3%)

SD, standard deviation; ND, not determined; FISH, fluorescence in situ hybridization.





- 27 -

Table 2. IC50

values for IGF-IR inhibitors in myxoid liposarcoma, Ewing´s sarcoma and

pancreatic ductal adenocarcinoma cell lines

Compound IC

50 (µM)

MLS402-91 MLS1765-92 A673 Capan-1

NVP-AEW541 1.16 1.35 0.70 ND

BMS-754807 0.36 0.50 0.21 ND

Picropodophyllin (PPP) 0.66 0.75 0.57 1.38

Cytotoxic effects on myxoid liposarcoma (MLS402-91 and MLS1765-92), Ewing´s sarcoma (A673) and pancreatic adenocarcinoma (Capan-1) cell viability were assessed in MTT assays (72 h). Results are represented as mean of at least three separate experiments performed in quintuplicates (ND, not determined).





- 28 -

FIGURE LEGENDS

Figure 1. Activation of the IGF-IR and PI3K/Akt/GSK-3β signaling axis in representative

cases of myxoid liposarcoma. (A-B) Immunohistochemical staining shows strong

expression of IGF-IR and IGF-II. (C-F) Elevated phosphorylation levels of Akt (S473),

GSK-3β (S21/9) and S6 (S240/244) indicate PI3K/Akt signaling pathway activity with

p44/42 MAPK (T202/Y204) being activated as well. (G-H) Elevated expression levels of

Cyclin D1 and MIB-1 (original magnification: x10, inset x20).

Figure 2. Activation of IGF-IR and PI3K/Akt/GSK-3β signaling in myxoid liposarcoma

tissue specimens and cell lines. (A) Immunohistochemical spectrum of tumor tissue

specimens summarized as bar chart. (B) Venn diagram indicating the concordance of

positive IGF-IR/IGF-II immunoreactivity and phosphorylation of Akt (S473),

S6 (S240/244) and/or GSK-3β (Ser21/9) in 33% of tumor specimens. In total, 5 cases

were quintuple-negative for IGF-IR, IGF-II, Akt (S473), S6 (S240/244) or GSK-3β

(S21/9) expression. (C) Immunoblotting results demonstrate elevated expression and

phosphorylation levels of IGF-IR and PI3K/Akt/GSK-3β signaling components in total

protein extracts of MLS402-91 and MLS1765-92 cells (β-actin was used as loading

reference). Detection of t(12;16) FUS-DDIT3 fusion gene transcripts in MLS402-91

(type 1; exon 7-2) and MLS1765-92 (type 8; exon 13-2) cells by RT-PCR (28S rRNA

was used as loading reference).

Figure 3. Myxoid liposarcoma associated FUS-DDIT3 fusion protein stimulates IGF-IR

and PI3K/Akt/GSK-3β pathway signal transduction. (A) Induction of promoter

P2-dependent IGF2 transcripts in FUS-DDIT3 expressing HT1080 cells (upper panel);

comparable IGF2 levels in FUS-DDIT3-tranfected HT1080 and MLS cell lines (lower

panel). (B) HT1080 cells were transfected with indicated FUS-DDIT3, FUS, DDIT3 or

eGFP control expression vectors to study IGF-IR and PI3K/Akt/GSK-3β mediated signal

transduction. FUS-DDIT3 expression significantly increased phosphorylation of IGF-IR

(Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9) and mTOR (Ser2448), confirming

pathway induction and activity. Elevated target protein levels of Cyclin D1 in HT1080

cells expressing the FUS-DDIT3 fusion protein. (C) Enhanced phosphorylation of IGF-IR





- 29 -

(Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9) and mTOR (Ser2448) upon stimulation

with IGF-II (200 ng/ml; 15 min). (D) In MLS402-91 and MLS1765-92 cells,

siRNA-mediated knockdown of FUS-DDIT3 significantly reduced levels of

P2-promoter-dependent IGF2 transcripts (RT-PCR) and phosphorylation of IGF-IR

(Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9) and mTOR (Ser2448). β-actin and

28S rRNA were used as loading references (NTC, no template control).

Figure 4. In vitro and in vivo evaluation of NVP-AEW541, BMS-754807 and PPP in two

myxoid liposarcoma cell lines. (A) Cell viability of MLS402-91 and MLS1765-92 cells

was significantly reduced by treatment with increasing concentrations of NVP-AEW541,

BMS-754807 and PPP in MTT assays. A673 (Ewing´s sarcoma) and Capan-1

(pancreatic ductal adenocarcinoma) cells were included as sensitive and/or resistant

controls to IGF-IR inhibition, respectively. At least three independent experiments were

performed (each in quintuplicates); results are expressed as mean ± SEM. (B)

NVP-AEW541 suppressed phosphorylation levels of IGF-IR (Tyr1135/1136), Akt

(Ser473), GSK-3β (Ser21/9), p70S6K (Thr389) and S6 (Ser235/236 and Ser240/244) in

both MLS cell lines. Changes in Cyclin D1 expression levels were determined by

immunoblotting. (C) In flow cytometric analyses, significantly increased rates of

apoptosis (cleaved PARP) and decreased mitotic fractions (phospho-histone H3) were

detected upon treatment with NVP-AEW541 (0.75-1.5 µM; DMSO was employed as

control). (D) MLS cells were xenografted on the CAM of chick eggs. Tumor-bearing eggs

were randomized and treated with NVP-AEW541 (1 µM) or control (0.2% DMSO in NaCl

0.9%). Significantly reduced tumor volumes ± SEM (NVP-AEW541-treated; *P<0.05)

and representative explants are shown (H&E staining of CAM tumor specimens are

included in Supplementary Figure S9).




Trautmann et al.:

FUS-DDIT3 fusion protein driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma

Figure 1. Activation of the IGF-IR and PI3K/Akt/GSK-3β signaling axis in representative

cases of myxoid liposarcoma. (A-B) Immunohistochemical staining shows strong expression

of IGF-IR and IGF-II. (C-F) Elevated phosphorylation levels of Akt (S473), GSK-3β (S21/9)

and S6 (S240/244) indicate PI3K/Akt signaling pathway activity with p44/42 MAPK

(T202/Y204) being activated as well. (G-H) Elevated expression levels of Cyclin D1 and

MIB-1 (original magnification: x10, inset x20).




MLS402-91

MLS1765-92

Akt

p-Akt(Ser473)

GSK-3β

p-GSK-3β(Ser21/9)

IGF-IR

p-IGF-IR(Tyr1135/1136)

p70S6K

p-p70S6K (Thr389)

S6

p-S6(Ser235/236)

p-S6(Ser240/244)

β-actin

Cyclin D1

mTOR

p-mTOR(Ser2448)

FUS-DDIT3 (type 8)165 bp

FUS-DDIT3 (type 1)197 bp

28S rRNA130 bp

MLS402-91

MLS1765-92

p44/42 MAPK

p-p44/42 MAPK(Thr202/Tyr204)

Figure 2.

A C

B




Trautmann et al.:


Figure 2. Activation of IGF-IR and PI3K/Akt/GSK-3β signaling in myxoid liposarcoma tissue

specimens and cell lines. (A) Immunohistochemical spectrum of tumor tissue specimens

summarized as bar chart. (B) Venn diagram indicating the concordance of positive

IGF-IR/IGF-II immunoreactivity and phosphorylation of Akt (S473), S6 (S240/244) and/or

GSK-3β (Ser21/9) in 33% of tumor specimens. In total, 5 cases were quintuple-negative for

IGF-IR, IGF-II, Akt (S473), S6 (S240/244) or GSK-3β (S21/9) expression. (C) Immunoblotting

results demonstrate elevated expression and phosphorylation levels of IGF-IR and

PI3K/Akt/GSK-3β signaling components in total protein extracts of MLS402-91 and

MLS1765-92 cells (β-actin was used as loading reference). Detection of t(12;16) FUS-DDIT3

fusion gene transcripts in MLS402-91 (type 1; exon 7-2) and MLS1765-92 (type 8; exon 13-2)

cells by RT-PCR (28S rRNA was used as loading reference).




Figure 3.

A

C

B

D




Trautmann et al.:


Figure 3. Myxoid liposarcoma associated FUS-DDIT3 fusion protein stimulates IGF-IR and

PI3K/Akt/GSK-3β pathway signal transduction. (A) Induction of promoter P2-dependent IGF2

transcripts in FUS-DDIT3 expressing HT1080 cells (upper panel); comparable IGF2 levels in

FUS-DDIT3-tranfected HT1080 and MLS cell lines (lower panel). (B) HT1080 cells were

transfected with indicated FUS-DDIT3, FUS, DDIT3 or eGFP control expression vectors to

study IGF-IR and PI3K/Akt/GSK-3β mediated signal transduction. FUS-DDIT3 expression

significantly increased phosphorylation of IGF-IR (Tyr1135/1136), Akt (Ser473),

GSK-3β (Ser21/9) and mTOR (Ser2448), confirming pathway induction and activity. Elevated

target protein levels of Cyclin D1 in HT1080 cells expressing the FUS-DDIT3 fusion protein.

(C) Enhanced phosphorylation of IGF-IR (Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9)

and mTOR (Ser2448) upon stimulation with IGF-II (200 ng/ml; 15 min). (D) In MLS402-91 and

MLS1765-92 cells, siRNA-mediated knockdown of FUS-DDIT3 significantly reduced levels of

P2-promoter-dependent IGF2 transcripts (RT-PCR) and phosphorylation of IGF-IR

(Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9) and mTOR (Ser2448). β-actin and

28S rRNA were used as loading references (NTC, no template control).




Figure 4.

A

B C

D




Trautmann et al.:


Figure 4. In vitro and in vivo evaluation of NVP-AEW541, BMS-754807 and PPP in two

myxoid liposarcoma cell lines. (A) Cell viability of MLS402-91 and MLS1765-92 cells was

significantly reduced by treatment with increasing concentrations of NVP-AEW541,

BMS-754807 and PPP in MTT assays. A673 (Ewing´s sarcoma) and Capan-1 (pancreatic

ductal adenocarcinoma) cells were included as sensitive and/or resistant controls to IGF-IR

inhibition, respectively. At least three independent experiments were performed (each in

quintuplicates); results are expressed as mean ± SEM. (B) NVP-AEW541 suppressed

phosphorylation levels of IGF-IR (Tyr1135/1136), Akt (Ser473), GSK-3β (Ser21/9), p70S6K

(Thr389) and S6 (Ser235/236 and Ser240/244) in both MLS cell lines. Changes in Cyclin D1

expression levels were determined by immunoblotting. (C) In flow cytometric analyses,

significantly increased rates of apoptosis (cleaved PARP) and decreased mitotic fractions

(phospho-histone H3) were detected upon treatment with NVP-AEW541 (0.75-1.5 µM; DMSO

was employed as control). (D) MLS cells were xenografted on the CAM of chick eggs. Tumor-

bearing eggs were randomized and treated with NVP-AEW541 (1 µM) or control (0.2%

DMSO in NaCl 0.9%). Significantly reduced tumor volumes ± SEM (NVP-AEW541-treated;

*P<0.05) and representative explants are shown (H&E staining of CAM tumor specimens are

included in Supplementary Figure S8).




Published OnlineFirst June 21, 2017.Clin Cancer Res Marcel Trautmann, Jasmin Menzel, Christian Bertling, et al. therapeutic target in myxoid liposarcomaFUS-DDIT3 fusion protein driven IGF-IR signaling is a

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