fus-ddit3 fusion protein driven igf-ir signaling is a therapeutic … · gerhard-domagk-institute...
TRANSCRIPT
Trautmann et al.: IGF-II in myxoid liposarcoma
- 1 -
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 2 -
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.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 3 -
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.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 4 -
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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 5 -
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.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 6 -
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;
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 7 -
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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 8 -
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.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 9 -
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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 10 -
(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).
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 11 -
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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 12 -
50% growth inhibition (IC50 value), was calculated by non-linear regression analysis
using the GraphPad Prism (GraphPad Software, La Jolla, CA, USA).
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 13 -
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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 14 -
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).
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 15 -
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).
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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).
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 21 -
REFERENCES
1. Fletcher CDM, Unni K, Mertens F. Pathology and Genetics of Tumours of Soft Tissue and Bone. World Health Organization Classification of Tumours 2002:200-4.
2. Dei Tos AP. Liposarcomas: diagnostic pitfalls and new insights. Histopathology 2014;64(1):38-52.
3. Antonescu CR, Tschernyavsky SJ, Decuseara R, Leung DH, Woodruff JM, Brennan MF, et al. Prognostic impact of P53 status, TLS-CHOP fusion transcript structure, and histological grade in myxoid liposarcoma: a molecular and clinicopathologic study of 82 cases. Clin Cancer Res 2001;7(12):3977-87.
4. Panagopoulos I, Hoglund M, Mertens F, Mandahl N, Mitelman F, Aman P. Fusion of the EWS and CHOP genes in myxoid liposarcoma. Oncogene 1996;12(3):489-94.
5. Engstrom K, Willen H, Kabjorn-Gustafsson C, Andersson C, Olsson M, Goransson M, et al. The myxoid/round cell liposarcoma fusion oncogene FUS-DDIT3 and the normal DDIT3 induce a liposarcoma phenotype in transfected human fibrosarcoma cells. Am J Pathol 2006;168(5):1642-53.
6. Kuroda M, Ishida T, Takanashi M, Satoh M, Machinami R, Watanabe T. Oncogenic transformation and inhibition of adipocytic conversion of preadipocytes by TLS/FUS-CHOP type II chimeric protein. Am J Pathol 1997;151(3):735-44.
7. Perez-Losada J, Pintado B, Gutierrez-Adan A, Flores T, Banares-Gonzalez B, del Campo JC, et al. The chimeric FUS/TLS-CHOP fusion protein specifically induces liposarcomas in transgenic mice. Oncogene 2000;19(20):2413-22.
8. Riggi N, Cironi L, Provero P, Suva ML, Stehle JC, Baumer K, et al. Expression of the FUS-CHOP fusion protein in primary mesenchymal progenitor cells gives rise to a model of myxoid liposarcoma. Cancer Res 2006;66(14):7016-23.
9. Negri T, Virdis E, Brich S, Bozzi F, Tamborini E, Tarantino E, et al. Functional mapping of receptor tyrosine kinases in myxoid liposarcoma. Clin Cancer Res 2010;16(14):3581-93.
10. Andersson MK, Goransson M, Olofsson A, Andersson C, Aman P. Nuclear expression of FLT1 and its ligand PGF in FUS-DDIT3 carrying myxoid liposarcomas suggests the existence of an intracrine signaling loop. BMC Cancer 2010;10:249.
11. Martin-Pérez J, Thomas G. Ordered phosphorylation of 40S ribosomal protein S6 after serum stimulation of quiescent 3T3 cells. Proceedings of the National Academy of Sciences 1983;80(4):926-30.
12. Meyuhas O. Chapter Two-Ribosomal Protein S6 Phosphorylation: Four Decades of Research. International review of cell and molecular biology 2015;320:41-73.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 22 -
13. Wettenhall R, Erikson E, Maller J. Ordered multisite phosphorylation of Xenopus ribosomal protein S6 by S6 kinase II. Journal of Biological Chemistry 1992;267(13):9021-27.
14. Diehl JA, Cheng M, Roussel MF, Sherr CJ. Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 1998;12(22):3499-511.
15. Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004;9(6):667-76.
16. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer 2002;2(7):489-501.
17. Barretina J, Taylor BS, Banerji S, Ramos AH, Lagos-Quintana M, Decarolis PL, et al. Subtype-specific genomic alterations define new targets for soft-tissue sarcoma therapy. Nat Genet 2010;42(8):715-21.
18. Demicco EG, Torres KE, Ghadimi MP, Colombo C, Bolshakov S, Hoffman A, et al. Involvement of the PI3K/Akt pathway in myxoid/round cell liposarcoma. Mod Pathol 2012;25(2):212-21.
19. Cheng H, Dodge J, Mehl E, Liu S, Poulin N, van de Rijn M, et al. Validation of immature adipogenic status and identification of prognostic biomarkers in myxoid liposarcoma using tissue microarrays. Hum Pathol 2009;40(9):1244-51.
20. Ratan R, Patel SR. Chemotherapy for soft tissue sarcoma. Cancer 2016;122(19):2952-60.
21. Grosso F, Jones RL, Demetri GD, Judson IR, Blay J-Y, Le Cesne A, et al. Efficacy of trabectedin (ecteinascidin-743) in advanced pretreated myxoid liposarcomas: a retrospective study. The lancet oncology 2007;8(7):595-602.
22. Schöffski P, Chawla S, Maki RG, Italiano A, Gelderblom H, Choy E, et al. Eribulin versus dacarbazine in previously treated patients with advanced liposarcoma or leiomyosarcoma: a randomised, open-label, multicentre, phase 3 trial. The Lancet 2016;387(10028):1629-37.
23. Fletcher CD, Organization WH. WHO classification of tumours of soft tissue and bone:[this book reflects the views of a working group that convened for a consensus and editorial meeting at the University of Zurich, Switzerland, 18-20 April 2012]. Internat. Agency for Research on Cancer; 2013.
24. Powers MP, Wang WL, Hernandez VS, Patel KS, Lev DC, Lazar AJ, et al. Detection of myxoid liposarcoma-associated FUS-DDIT3 rearrangement variants including a newly identified breakpoint using an optimized RT-PCR assay. Mod Pathol 2010;23(10):1307-15.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 23 -
25. Aman P, Ron D, Mandahl N, Fioretos T, Heim S, Arheden K, et al. Rearrangement of the transcription factor gene CHOP in myxoid liposarcomas with t(12;16)(q13;p11). Genes Chromosomes Cancer 1992;5(4):278-85.
26. Garcıa-Echeverrıa C, Pearson MA, Marti A, Meyer T, Mestan J, Zimmermann J, et al. In vivo antitumor activity of NVP-AEW541—a novel, potent, and selective inhibitor of the IGF-IR kinase. Cancer cell 2004;5(3):231-39.
27. Manara MC, Landuzzi L, Nanni P, Nicoletti G, Zambelli D, Lollini PL, et al. Preclinical in vivo study of new insulin-like growth factor-I receptor–specific inhibitor in Ewing's sarcoma. Clinical Cancer Research 2007;13(4):1322-30.
28. Carboni JM, Wittman M, Yang Z, Lee F, Greer A, Hurlburt W, et al. BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR. Molecular cancer therapeutics 2009;8(12):3341-49.
29. Wittman MD, Carboni JM, Yang Z, Lee FY, Antman M, Attar R, et al. Discovery of a 2, 4-disubstituted pyrrolo [1, 2-f][1, 2, 4] triazine inhibitor (BMS-754807) of insulin-like growth factor receptor (IGF-1R) kinase in clinical development. Journal of medicinal chemistry 2009;52(23):7360-63.
30. Girnita A, Girnita L, del Prete F, Bartolazzi A, Larsson O, Axelson M. Cyclolignans as inhibitors of the insulin-like growth factor-1 receptor and malignant cell growth. Cancer research 2004;64(1):236-42.
31. Vasilcanu D, Girnita A, Girnita L, Vasilcanu R, Axelson M, Larsson O. The cyclolignan PPP induces activation loop-specific inhibition of tyrosine phosphorylation of the insulin-like growth factor-1 receptor. Link to the phosphatidyl inositol-3 kinase/Akt apoptotic pathway. Oncogene 2004;23(47):7854-62.
32. Trautmann M, Sievers E, Aretz S, Kindler D, Michels S, Friedrichs N, et al. SS18-SSX fusion protein-induced Wnt/beta-catenin signaling is a therapeutic target in synovial sarcoma. Oncogene 2014;33(42):5006-16.
33. Hartmann W, Waha A, Koch A, Albrecht S, Gray SG, Ekstrom TJ, et al. Promoter-specific transcription of the IGF2 gene: a novel rapid, non-radioactive and highly sensitive protocol for mRNA analysis. Virchows Arch 2001;439(6):803-7.
34. Hartmann W, Waha A, Koch A, Albrecht S, Gray SG, Ekström TJ, et al. Promoter-specific transcription of the IGF2 gene: a novel rapid, non-radioactive and highly sensitive protocol for mRNA analysis. Virchows Archiv 2001;439(6):803-07.
35. Thellin O, Zorzi W, Lakaye B, De Borman B, Coumans B, Hennen G, et al. Housekeeping genes as internal standards: use and limits. J Biotechnol 1999;75(2-3):291-5.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 24 -
36. Martins AS, Ordóñez JL, Amaral AT, Prins F, Floris G, Debiec-Rychter M, et al. IGF1R signaling in Ewing sarcoma is shaped by clathrin-/caveolin-dependent endocytosis. PloS one 2011;6(5):e19846.
37. Patel M, Gomez NC, McFadden AW, Moats-Staats BM, Wu S, Rojas A, et al. PTEN deficiency mediates a reciprocal response to IGFI and mTOR inhibition. Molecular Cancer Research 2014;12(11):1610-20.
38. Oikawa K, Tanaka M, Itoh S, Takanashi M, Ozaki T, Muragaki Y, et al. A novel oncogenic pathway by TLS-CHOP involving repression of MDA-7/IL-24 expression. Br J Cancer 2012;106(12):1976-9.
39. Grünewald I, Trautmann M, Busch A, Bauer L, Huss S, Schweinshaupt P, et al. MDM2 and CDK4 amplifications are rare events in salivary duct carcinomas. Oncotarget 2016.
40. Isachenko V, Mallmann P, Petrunkina AM, Rahimi G, Nawroth F, Hancke K, et al. Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue. PLoS One 2012;7(3):e32549.
41. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer chemotherapy and pharmacology 1989;24(3):148-54.
42. Smith TA, Easley KA, Goldblum JR. Myxoid/round cell liposarcoma of the extremities. A clinicopathologic study of 29 cases with particular attention to extent of round cell liposarcoma. Am J Surg Pathol 1996;20(2):171-80.
43. de Bruijn DR, Allander SV, van Dijk AH, Willemse MP, Thijssen J, van Groningen JJ, et al. The synovial-sarcoma-associated SS18-SSX2 fusion protein induces epigenetic gene (de)regulation. Cancer Res 2006;66(19):9474-82.
44. Michels S, Trautmann M, Sievers E, Kindler D, Huss S, Renner M, et al. SRC signaling is crucial in the growth of synovial sarcoma cells. Cancer research 2013;73(8):2518-28.
45. Khan J, Bittner ML, Saal LH, Teichmann U, Azorsa DO, Gooden GC, et al. cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene. Proc Natl Acad Sci U S A 1999;96(23):13264-9.
46. Cironi L, Riggi N, Provero P, Wolf N, Suva ML, Suva D, et al. IGF1 is a common target gene of Ewing's sarcoma fusion proteins in mesenchymal progenitor cells. PLoS One 2008;3(7):e2634.
47. Herrero-Martin D, Osuna D, Ordonez JL, Sevillano V, Martins AS, Mackintosh C, et al. Stable interference of EWS-FLI1 in an Ewing sarcoma cell line impairs IGF-1/IGF-1R signalling and reveals TOPK as a new target. Br J Cancer 2009;101(1):80-90.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 25 -
48. Juergens H, Daw NC, Geoerger B, Ferrari S, Villarroel M, Aerts I, et al. Preliminary efficacy of the anti-insulin-like growth factor type 1 receptor antibody figitumumab in patients with refractory Ewing sarcoma. J Clin Oncol 2011;29(34):4534-40.
49. Pappo AS, Patel SR, Crowley J, Reinke DK, Kuenkele KP, Chawla SP, et al. R1507, a monoclonal antibody to the insulin-like growth factor 1 receptor, in patients with recurrent or refractory Ewing sarcoma family of tumors: results of a phase II Sarcoma Alliance for Research through Collaboration study. J Clin Oncol 2011;29(34):4541-7.
50. Iams WT, Lovly CM. Molecular Pathways: Clinical Applications and Future Direction of Insulin-like Growth Factor-1 Receptor Pathway Blockade. Clin Cancer Res 2015;21(19):4270-7.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 26 -
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.
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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).
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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
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
Trautmann et al.: IGF-II in myxoid liposarcoma
- 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).
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
Figure 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
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).
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
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
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
Trautmann et al.:
FUS-DDIT3 fusion protein driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma
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).
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
Figure 3.
A
C
B
D
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
Trautmann et al.:
FUS-DDIT3 fusion protein driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma
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).
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
Figure 4.
A
B C
D
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
Trautmann et al.:
FUS-DDIT3 fusion protein driven IGF-IR signaling is a therapeutic target in myxoid liposarcoma
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).
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
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
Updated version
10.1158/1078-0432.CCR-17-0130doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2017/07/28/1078-0432.CCR-17-0130.DC3
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/early/2017/07/28/1078-0432.CCR-17-0130To request permission to re-use all or part of this article, use this link
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