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Therapeutic potential of TAS-115 via c-MET and PDGFRα signal inhibition for synovial sarcoma

  • Shutaro Yamada1,
  • Yoshinori Imura2,
  • Takaaki Nakai1,
  • Sho Nakai1,
  • Naohiro Yasuda1,
  • Keiko Kaneko1,
  • Hidetatsu Outani2,
  • Satoshi Takenaka1,
  • Kenichiro Hamada1,
  • Akira Myoui1,
  • Nobuhito Araki2,
  • Takafumi Ueda3,
  • Kazuyuki Itoh4,
  • Hideki Yoshikawa1 and
  • Norifumi Naka1Email author
BMC Cancer201717:334

DOI: 10.1186/s12885-017-3324-3

Received: 7 June 2016

Accepted: 4 May 2017

Published: 16 May 2017

Abstract

Background

The prognosis of synovial sarcoma (SS), an aggressive soft tissue sarcoma, remains poor. We previously reported that c-MET or platelet-derived growth factor receptor α (PDGFRα) signalling pathway is related to SS progression based upon the findings of phospho-receptor tyrosine kinase (RTK) arrays. TAS-115 is a novel c-MET/ vascular endothelial growth factor receptor-targeting tyrosine kinase inhibitor that has been shown to inhibit multiple RTKs. Here we aimed to investigate the therapeutic potential of TAS-115 against SS.

Methods

We first evaluated which signalling pathway was relevant to the viability of three human SS cell lines: Yamato-SS, SYO-1 and HS-SY-II. Next, we assessed the anticancer activity and mechanism of action of TAS-115 in these SS cell lines. Finally, we compared the ability of TAS-115 to inhibit c-MET and PDGFRα phosphorylation with that of pazopanib.

Results

We classified the SS cell lines as c-MET-dependent or PDGFRα-dependent based upon the differences in the signalling pathway relevant for growth and/or survival. We also found that c-MET and PDGFRα were the primary activators of both phosphatidylinositol 3-kinase/AKT and mitogen-activated protein kinase pathways in c-MET-dependent and PDGFRα-dependent SS cells, respectively. TAS-115 treatment blocked the phosphorylation of PDGFRα as well as that of c-MET and their downstream effectors, leading to marked growth inhibition in both types of SS cell lines in in vitro and in vivo studies. Furthermore, PDGFRα phosphorylation, on at least four representative autophosphorylation sites, was impeded by TAS-115 equivalently to pazopanib.

Conclusions

These experimental results have demonstrated the significance of c-MET and PDGFRα signalling for growth and/or survival of SS tumours. TAS-115 monotherapy may benefit SS patients whose tumours are dependent upon either c-MET or PDGFRα signalling by functioning as a multiple tyrosine kinase inhibitor to suppress c-MET as well as PDGFRα pathways.

Keywords

TAS-115 Synovial sarcoma C-MET PDGFRα Molecular targeted therapy

Background

Synovial sarcoma (SS) is a malignant soft tissue sarcoma characterized by a recurrent chromosomal translocation [t(X;18)(p11;q11)] that forms the fusion protein, SS18-SSX [1]. SS accounts for 5%–10% of all soft tissue sarcomas and mainly affects adolescents and young adults [2]. This disease commonly presents in the extremities (80%) [2] and distant metastases tend to be found mainly in the lungs [3]. Although the mainstay of treatment comprises wide surgical excision, chemotherapy and radiotherapy, the 5-year overall survival rate of SS is only 30%–70% [37]. Therefore, developing novel therapeutic approaches for SS is urgently needed.

We previously reported that c-MET or platelet-derived growth factor receptor α (PDGFRα) signalling pathway was relevant for SS progression, based upon the findings of phospho-receptor tyrosine kinase (RTK) arrays [8]. c-MET, an RTK encoded by the c-met proto-oncogene, is known to be a hepatocyte growth factor (HGF) receptor [9]. Activation of the HGF/c-MET axis in cancer has been reported to be involved in cellular proliferation, survival, migration and angiogenesis [10]. We have found that a selective c-MET inhibitor suppresses the growth of Yamato-SS cells, but fails to suppress the growth of SYO-1 or HS-SY-II cells [11]. PDGFRα and PGDFRβ signalling indirectly promotes tumour development by activating the mesenchymal cells in the tumour microenvironment and directly stimulates the growth of malignant cells [12]. Pazopanib, a PDGFR/ vascular endothelial growth factor receptor (VEGFR)/ c-kit (stem cell factor receptor) inhibitor [13], is the only tyrosine kinase inhibitor approved for advanced soft tissue sarcomas in Japan. Hosaka et al. showed that pazopanib suppressed the growth of SYO-1 and HS-SY-II cells through inhibition of the PDGFRα and phosphatidylinositol 3-kinase (PI3K)/AKT pathways [14]. Based upon these studies, we hypothesize that inhibition of the c-MET or PDGFRα signalling pathway would be a therapeutic strategy for the treatment of SS.

TAS-115, a novel c-MET/VEGFR-targeting tyrosine kinase inhibitor that exerts its effect via ATP antagonism, has been reported to inhibit multiple RTKs [15]. Recently, it was reported that TAS-115 had a favourable tolerability profile and exhibited antitumour activity in human gastric cancer [15, 16] and in human lung cancer [17, 18] via inhibition of c-MET/VEGFR signalling. However, the efficacy of this drug for soft tissue sarcomas remains unclear.

In the present study, we first evaluated the phosphorylation status of RTKs in three human SS cell lines, Yamato-SS, SYO-1 and HS-SY-II, and then investigated which RTK was critical for the viability of each of these cell lines. Next, we tested the antitumour activity and the mechanism of action of TAS-115 in these SS cells. Finally, we compared the inhibitory activity of TAS-115 on the c-MET and PDGFRα pathways with that of pazopanib. On the basis of our observations, we discuss the potential clinical value of TAS-115 monotherapy, via c-MET and PDGFRα signal inhibition, in patients with SS.

Methods

Cell lines

The Yamato-SS cell line was established from surgically resected tumours in our laboratory, as previously described [19]. SYO-1 was kindly supplied by Dr. Ozaki (Okayama University, Okayama, Japan) [20]. HS-SY-II [21] was provided by the RIKEN BRC (Tsukuba, Japan) through the National Bio-Resource Project of the MEXT, Japan. We authenticated Yamato-SS and HS-SY-II through short tandem repeat inspection. SYO-1 was confirmed by the expression of the SS18-SSX2 fusion gene by reverse transcription polymerase chain reaction. Yamato-SS and SYO-1 cells originally derived from biphasic synovial sarcomas, while HS-SY-II originated from a monophasic synovial sarcoma [1921]. These cells were cultured in Dulbecco’s Modified Eagle’s Medium (Life Technologies, Carlsbad, CA, USA) containing 10% foetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C with 5% CO2 and 100% humidity.

Reagents and antibodies

TAS-115 [4-[2-fluoro-4-[[[(2-phenylacetyl)amino]thioxomethyl]amino]-phenoxy]-7-methoxy-N-methyl-6-quinolinecarboxamide] and pazopanib [5-[[4-[(2,3-dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2-methylbenzenesulfonamide] were provided by Taiho Pharmaceutical Co., Ltd. (Tsukuba, Japan) and Novartis Pharma AG (Basel, Switzerland), respectively. According to the manufacturer’s instructions, TAS-115 and pazopanib were suspended in dimethyl sulfoxide (DMSO, Sigma-Aldrich) for in vitro experiments. TAS-115 and pazopanib were diluted to the appropriate concentrations for in vivo experiments, according to the manufacturer’s instruction. Recombinant human (rh) PDGF-BB was obtained from Sigma-Aldrich.

Antibodies against PDGFRα (#7074), p-PDGFRα (Tyr754; #2992, Tyr849; #3170, Tyr1018; #4547), c-MET (#8198), p-MET (Tyr1234/1235; #3077), AKT (#4691), p-AKT (Ser473; #4060), ERK (#4695), p-ERK (Thr202/Tyr204; #4370), PARP (#9542) and β-actin (#4970) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). All of these antibodies were used at 1:1000 dilution for immunoblot analyses. An antibody against p-PDGFRα (Tyr762; AF21141) was purchased from R&D systems (Minneapolis, MN, USA). This antibody was used at a concentration of 0.5 μg/ml for immunoblot analyses. An antibody against PCNA (sc-56) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and used at a concentration of 1:50 for immunohistochemistry. Horseradish peroxidase (HRP)-conjugated secondary antibody was obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA).

Immunoblot analysis

After washing with PBS, cells were lysed in RIPA buffer (Thermo Scientific, Waltham, MA, USA) supplemented with 1% protease/phosphatase inhibitor cocktail (Cell Signaling Technology). Protein concentrations were measured using the bicinchoninic acid method (Thermo Scientific). The cell lysates were separated on 4–12% Bis-Tris gels (Life Technologies) and transferred to polyvinylidene difluoride (PVDF) membranes (Nippon Genetics, Tokyo, Japan). After blocking with 5% skim milk in Tris-buffered saline supplemented with Tween20 (TBS-T) at room temperature, the membranes were incubated with primary antibodies in Can Get Signal solution 1 (Toyobo Life Science, Tokyo, Japan) at 4 °C overnight, followed by incubation with secondary antibodies in Can Get Signal solution 2 (Toyobo Life Science) at room temperature for 1 h. After washing with TBS-T, immunoreactive bands were visualized using chemiluminescent reagents (ECL prime; GE Healthcare Life Sciences and ImmunoStar LD; Wako, Osaka, Japan).

RNA interference

Lipofectamine 2000 (Life Technologies) was used to transfect cells with 20 nM siRNAs, according to the manufacturer’s instruction. Two kinds of siRNAs targeting c-MET (constructs I and II; #6618 and #6622) and a non-targeting siRNA (#6568) were purchased from Cell Signaling Technology, Inc. Two kinds of siRNAs targeting PDGFRα (Hs_PDGFRα_1109, 6393) and a non-targeting siRNA (SIC-001) were obtained from Sigma-Aldrich.

Cell proliferation assay

SS cells were plated in 96-well plates for cell proliferation assays. The cell proliferation rate was measured using the premixed WST-1 cell proliferation assay system (Takara Bio, Inc., Otsu, Japan). The relative cell proliferation rate was calculated by subtracting absorbance measurements obtained from a microplate reader at 690 nm from those obtained at 450 nm.

Cell cycle analysis

SS cells (5 × 105 per dish) were seeded in 10-cm culture dishes and incubated overnight and then treated with TAS-115 or control (DMSO) for 24 h. The cells were harvested and stained with Propidium Iodide (PI) solution (25 μg/ml PI, 0.03% NP-40, 0.02 mg/ml RNase A, 0.1% sodium citrate) for 30 min at room temperature. We analysed the cell cycle using a BD FACSCanto II flow cytometer (Becton Dickinson (BD) Biosciences, San Jose, CA, USA).

In vivo xenograft experiments

The animal studies were performed in accordance with a guideline approved by the Institutional Animal Care and Use Committee of the Osaka University Graduate School of Medicine. We used Yamato-SS and SYO-1 cells because of their consistency in producing tumours in xenograft models. Yamato-SS cells (3 × 107) or SYO-1 cells (1 × 107) were inoculated subcutaneously into the flank of 5-week-old male BALB/c nu/nu mice (SLC, Shizuoka, Japan). Tumour volume (mm3) was defined as (A × B2)/2, where A and B were the longest and the shortest diameter of the tumour, respectively. Oral administration of TAS-115 was initiated after the average size of the established tumours reached around 100 mm3. TAS-115 was administered once daily at a dose of 50 or 200 mg/kg for 4 weeks. Xenograft tumour volume and the body weight of mice were measured once a week. After 4 weeks of treatment, the mice were euthanized and the tumour weight was measured. Resected tumours were used for immunohistochemical studies.

For immunoblot analyses, mice bearing tumours were orally treated with TAS-115 (200 mg/kg) and with pazopanib (100 mg/kg) for consecutive 3 days. Three hours after the last drug administration, the tumours were resected and extracted in Tissue Protein Extraction Reagent (Thermo Scientific). The dose of TAS-115 or pazopanib was determined based upon prior reports in which daily administration of TAS-115 (50–200 mg/kg) or pazopanib (100 mg/kg) resulted in significant growth inhibition of the xenograft tumours [15, 16, 22, 23].

Immunohistochemistry

Xenograft tumours were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections (4 μm) were deparaffinized and dehydrated. After antigen retrieval in 10-mM citrate buffer at 95 °C for 30 min, endogenous peroxidase activity was blocked with methanol containing 3% H2O2 for 10 min. The sections were incubated with primary antibodies at 4 °C overnight, with secondary antibodies for 1 h on the next day, and then stained with 3,3′-diaminobenzidine tetrahydrochloride (DAB; Dako, Glostrup, Denmark). Finally, these sections were counterstained with hematoxylin.

Measurement of PCNA positive rate

PCNA positive rate was assessed by counting >500 cells from 3 random fields of each specimen under × 200 magnification in the best-stained tumour area of each section.

Statistical analysis

We used Student’s t-tests for experiments in vitro and the Mann–Whitney U test for experiments in vivo. Values of p < 0.05 were considered statistically significant.

Results

SS cell lines could be divided into two groups: C-MET-dependent and PDGFRα-dependent SS cells

We first performed immunoblot analyses to evaluate the phosphorylation status of RTKs in the Yamato-SS, SYO-1 and HS-SY-II cell lines. Immunoblot analyses revealed that c-MET was activated in Yamato-SS cells, whereas PDGFRα was activated in all three SS cell lines (Fig. 1a).
Fig. 1

c-MET and PDGFRα signals are crucial for the proliferation of SS cells. a Phosphorylation status of RTKs in 3 SS cell lines. b Yamato-SS (3 × 103), SYO-1 (5 × 103) and HS-SY-II (1 × 104) cells were transfected with siRNAs against c-MET. Transfected cells were cultured for 96 h and relative cell proliferation rates were measured using a WST-1 assay. Bars represent the SD. * p < 0.05. c Growth of Yamato-SS (3 × 103), SYO-1 (5 × 103) and HS-SY-II (1 × 104) cells transfected with siRNAs against PDGFRα. Relative cell proliferation rates were determined using a WST-1 assay after 96 h. Bars represent the SD. * p < 0.05

Next, we used RNA interference technology to determine which RTK was crucial for the viability of the Yamato-SS, SYO-1 and HS-SY-II cell lines. Two kinds of small interfering RNAs (siRNAs) against c-MET or those against PDGFRα were transfected into Yamato-SS, SYO-1 and HS-SY-II cells. Silencing of c-MET expression significantly inhibited the growth of Yamato-SS cells but had little effect on the viability of the SYO-1 or HS-SY-II cells (Fig. 1b and Additional file 1: Figure S1A). By contrast, knockdown of PDGFRα expression markedly abrogated the proliferation of SYO-1 and HS-SY-II cells, but not that of Yamato-SS cells (Fig. 1c and Additional file 1: Figure S1B). These results suggested that Yamato-SS cell proliferation was highly addicted to the c-MET signalling pathway, whereas the proliferation of SYO-1 or HS-SY-II cells was dependent upon the PDGFRα signalling pathway.

TAS-115 suppresses the growth of both c-MET-dependent and PDGFRα-dependent SS cells in vitro

We performed WST-1 cell proliferation assays to examine the antitumour activity of TAS-115, known as a c-MET/VEGFR dual tyrosine kinase inhibitor, against SS cell lines in vitro. TAS-115 inhibited the growth of all three SS cell lines in a dose-dependent manner (Fig. 2a). In addition, the 50% inhibitory concentrations (IC50s) of TAS-115 in the Yamato-SS, SYO-1 and HS-SY-II cell lines were 0.52, 7.32 and 2.43 μM, respectively (Fig. 2b). These results suggested that the Yamato-SS cells, which were c-MET-dependent SS cells, were more sensitive to TAS-115 than the SYO-1 and HS-SY-II cells, which were PDGFRα-dependent SS cells.
Fig. 2

TAS-115 inhibits the growth of SS cells by inducing G0/G1 cell cycle arrest and apoptosis. a Yamato-SS, SYO-1 and HS-SY-II cells (3 × 103) were treated with TAS-115 in a concentration range of 0 to 10 μM for 72 h. Relative cell proliferation rates were determined using a WST-1 assay. Bars represent the SD. b Calculated IC50 values of each cell line. c The effect of TAS-115 on the cell cycle. Yamato-SS, SYO-1 and HS-SY-II cells were treated with control (0.1% DMSO) or 0.1–10-μM TAS-115 for 24 h. After treatment, the cells were stained with PI solution for flow cytometric analysis. d The effect of TAS-115 on PARP cleavage in Yamato-SS, SYO-1 and HS-SY-II cells. Cells were treated control (0.1% DMSO) or 0.001–10 μM of TAS-115 for 24 h

Flow cytometric analyses were conducted to elucidate the mechanisms by which TAS-115 inhibited SS cell proliferation. TAS-115 increased the percentage of cells in the G0/G1-phase and decreased the percentage of cells in the S-phase in the Yamato-SS, SYO-1 and HS-SY-II cells in a dose-dependent manner (Fig. 2c). Immunoblot analyses revealed that levels of cleaved poly ADP ribose polymerase (PARP) mildly increased in the Yamato-SS cells, whereas PARP cleavage did not occur in either the SYO-1 or HS-SY-II cells after treatment with TAS-115 for 24 h (Fig. 2d). These observations indicated that TAS-115 suppressed cell proliferation by inducing G0/G1 cell cycle arrest in all SS cell lines. TAS-115 also caused slight apoptosis in the Yamato-SS cells, but not in the SYO-1 or HS-SY-II cells.

TAS-115 blocks phosphorylation of PDGFRα and c-MET, as well as their downstream effectors in vitro

We investigated the effects of TAS-115 on the c-MET and PDGFRα signalling pathways by immunoblot analyses in vitro. c-MET phosphorylation was markedly suppressed in Yamato-SS cells after a 3-h incubation with TAS-115 at concentrations as low as 0.1 to 10 μM. Moreover, treatment with TAS-115 at these concentrations also inhibited the phosphorylation of downstream effectors, such as AKT and extracellular signal-regulated kinase (ERK) 1/2 (Fig. 3a).
Fig. 3

TAS-115 suppresses phosphorylation of c-MET and PDGFRα, as well as their downstream effectors. a Yamato-SS cells (c-MET-dependent SS cells) were treated with 0.001–10 μM of TAS-115 or control (0.1% DMSO) for 3 h. b SYO-1 and HS-SY-II cells (PDGFRα-dependent SS cells) were treated with 0.001–10 μM of TAS-115 or control (0.1% DMSO) for 3 h

PDGFRα has been reported to have more than 10 autophosphorylation sites. Among them, the PDGFRα residue Tyr849, which is located inside tyrosine kinase domain II, is critical for activation of the kinase [24]. Thus, we first focused on this tyrosine residue. TAS-115 at concentrations as low as 1–10 μM remarkably inhibited the phosphorylation of PDGFRα on Tyr849, as well as its downstream effectors AKT and ERK 1/2, in the SYO-1 and HS-SY-II cells (Fig. 3b). In rhPDGF-BB-stimulated SYO-1 and HS-SY-II cells, TAS-115 at concentrations of 0.01 μM or higher remarkably suppressed the phosphorylation of PDGFRα at Tyr849, as well as its downstream effectors (Additional file 2: Figure S2). These results indicated that TAS-115 inhibited the activity of PDGFRα and strongly suppressed c-MET phosphorylation.

Comparing the inhibitory effects of TAS-115 and pazopanib on c-MET or PDGFRα phosphorylation

We compared the abilities of TAS-115 and pazopanib to inhibit the c-MET and PDGFRα pathways using immunoblot analyses. TAS-115 at a concentration of 0.1 μM suppressed c-MET, AKT and ERK 1/2 phosphorylation, whereas pazopanib at the same concentration inhibited neither c-MET phosphorylation nor the phosphorylation of downstream effectors in the Yamato-SS cells (Fig. 4a). When increasing the drug concentration from 0.001 to 20 μM, pazopanib had no demonstrable effect on the phosphorylation of c-MET in the Yamato-SS cells (Fig. 4b).
Fig. 4

Inhibitory activities of TAS-115 and pazopanib on c-MET, PDGFRα and their downstream effectors in vitro. a Yamato-SS cells (c-MET-dependent SS cells) were treated with 0.1 μM TAS-115 or pazopanib or control (0.1% DMSO) for 3 h. b Yamato-SS cells were treated with 0.001–20 μM of pazopanib or control (0.1% DMSO) for 3 h. c SYO-1 and HS-SY-II (PDGFRα-dependent) SS cells were treated with 10-μM TAS-115 or pazopanib or control (0.1% DMSO) for 3 h, followed by an additional treatment with rhPDGF-BB at a concentration of 10 ng/ml for the last 15 min

In SYO-1 and HS-SY-II cells, rhPDGF-BB treatment enhanced the phosphorylation of PDGFRα at Tyr754, Tyr762, Tyr849, Tyr1018, as well as the phosphorylation of downstream effectors, AKT and ERK 1/2. Treatment with 10-μM TAS-115 inhibited phosphorylation at these sites of PDGFRα, which resulted in the subsequent suppression of AKT and ERK 1/2 activity. Pazopanib (10 μM) also attenuated the phosphorylation of these tyrosine residues of PDGFRα and the activity of downstream effectors (Fig. 4c).

To verify the inhibitory effects of TAS-115 and pazopanib on c-MET and PDGFRα in vivo, we administered TAS-115 (200 mg/kg) and pazopanib (100 mg/kg) orally to mice bearing Yamato-SS or SYO-1 xenograft tumours. TAS-115 at a dose of 200 mg/kg inhibited the phosphorylation of c-MET, AKT and ERK 1/2, while pazopanib did not affect c-MET signalling in Yamato-SS xenograft tumours (Fig. 5a). Both TAS-115 and pazopanib blocked the phosphorylation of PDGFRα at Tyr754, Tyr762, Tyr849, Tyr1018, as well as the phosphorylation of downstream effectors in SYO-1 xenograft tumours (Fig. 5b). These observations suggested that pazopanib attenuated the phosphorylation of PDGFRα, but not of c-MET. On the other hand, TAS-115 inactivated both c-MET and PDGFRα; moreover, the inhibitory activity of TAS-115 against PDGFRα phosphorylation was probably equivalent to that of pazopanib with respect to at least four representative autophosphorylation sites of the receptor in vitro and in vivo.
Fig. 5

Inhibitory effect of TAS-115 and pazopanib on c-MET, PDGFRα and their downstream effectors in vivo. a, b Mice bearing Yamato-SS (c-MET-dependent) cells and mice bearing SYO-1 (PDGFRα-dependent) SS cells were treated with orally administered TAS-115 (200 mg/kg) or pazopanib (100 mg/kg) or control for consecutive 3 days and euthanized 3 h after the final administration

TAS-115 abrogates the growth of Yamato-SS and SYO-1 xenograft tumours

We tested the antitumour effect of TAS-115 against Yamato-SS (c-MET-dependent SS cells) and SYO-1 (PDGFRα-dependent SS cells) xenograft tumours. Mice bearing tumours were treated daily with an oral dose of TAS-115 at 50 or 200 mg/kg, or a control. TAS-115 at a dose of 50 mg/kg showed moderate inhibitory activity for Yamato-SS xenograft tumours. Notably, treatment with 200 mg/kg of TAS-115 completely prevented the tumour growth during the treatment period (Fig. 6a, b). A remarkable decrease in tumour wet weight was observed in tumours treated with TAS-115 at a dose of 200 mg/kg (Additional file 3: Figure S3). No marked body weight loss was observed in TAS-115-treated mice (Additional file 4: Figure S4). Immunoblot analyses of resected tumours demonstrated that TAS-115 treatment inhibited the phosphorylation of c-MET, AKT and ERK 1/2 (Additional file 5: Figure S5). Histological analyses showed a decrease in the density of the tumour cells, as well as slight myxoid degeneration, with no signs of an inflammatory reaction or necrosis in TAS-115 treated groups (Additional file 6: Figure S6A). Additionally, Yamato-SS xenograft tumours sustained both spindle and epithelial cells after 28-day treatment in all treatment groups (Additional file 6: Figure S6A), indicating that TAS-115 might have similar effect on both spindle and epithelial components. Immunohistochemical analyses revealed that the number of proliferating cell nuclear antigen (PCNA)-positive tumour cells was significantly reduced in Yamato-SS xenograft tumours treated with TAS-115 (Fig. 6c, d). Besides, TAS-115 dose-dependently inhibited microvascular density (MVD) (Additional file 7: Figure S7A, B and Additional file 8: Supplementary methods). Unlike our in vitro experiments, cleaved caspase-3 was not detected in the Yamato-SS xenografts (data not shown). Apoptosis did not seem to be involved in the antitumour mechanisms of TAS-115 in vivo.
Fig. 6

TAS-115 strongly abrogates the growth of Yamato-SS and SYO-1 xenograft tumours. a The appearance of resected Yamato-SS tumours at the end of the experiments. b Mice bearing Yamato-SS xenografts were treated with 50 or 200 mg/kg of TAS-115, or control. Bars represent the SE. * p < 0.05. c Immunohistological analysis of PCNA (× 200). Scale bars, 100 μm. d PCNA-positive rate of each treatment group. Bars represent the SD. * p < 0.05. e The appearance of resected SYO-1 tumours at the end of the experiments. f Mice bearing SYO-1 xenografts were treated with 50 or 200 mg/kg of TAS-115, or control. Bars represent the SE. * p < 0.05. g Immunohistological analysis of PCNA (× 200). Scale bars, 100 μm. h PCNA-positive rate of each treatment group. Bars represent the SD. * p < 0.05

Similarly to experiments in the Yamato-SS xenografts, TAS-115 administration showed mild inhibition (50 mg/kg) and entire suppression (200 mg/kg) of the tumour growth and wet weight in SYO-1-transplanted mice (Fig. 6e, f and Additional file 3: Figure S3). Body weight loss was not observed in these TAS-115-treated mice (Additional file 4: Figure S4), either. Tumours treated with TAS-115 also showed a decrease in the cell density along with slight myxoid changes (Additional file 6: Figure S6B). As seen in Yamato-SS xenograft models, not only spindle cells but also epithelial tumour cells were observed in SYO-1 xenograft tumours after TAS-115 administration (Additional file 6: Figure S6B), which was confirmed by immunohistochemical staining of anti-vimentin and anti-cytokeratin (AE1/AE3) antibodies (Additional file 9: Figure S8 and Additional file 8: Supplementary methods). Treatment with TAS-115 also resulted in a significant reduction in PCNA-positive tumour cells and MVD in SYO-1 xenografts (Fig. 6g, h and Additional file 7: Figure S7C, D and Additional file 8: Supplementary methods).

These results suggested that TAS-115 exhibited strong antitumour effects for both c-MET-dependent and PDGFRα-dependent SS cells in vivo by inhibiting the proliferation of tumour cells, as well as tumour vascular development, without any demonstrable adverse events.

Discussion

Aberrant activation of HGF/c-MET signalling by mutation, autocrine or paracrine HGF stimulation, or overexpression has been implicated in the oncogenesis of large number of cancers [10, 2528]. Additionally, upregulation or mutational activation of PDGF ligand or PDGFR expression has also been reported in many types of malignancies [12, 2931]. Co-expression of HGF and c-MET has been seen in 14% of SS clinical samples, correlating with poor prognosis [32]. Likewise, we noted that 36% of SS specimens expressed both HGF and c-MET, which resulted in a significantly worse clinical course in SS patients [11]. A recent study revealed that PDGF-AA expression and phosphorylation of PDGFRα without any gene alteration led to AKT activation through an autocrine/paracrine-mediated loop in a subset of clinical SS tumour samples [33]. In the present study, we found c-MET activation in the Yamato-SS cells and PDGFRα phosphorylation in all three SS cell lines. Silencing of c-MET or PDGFRα expression revealed that the proliferation of the Yamato-SS cells sustained the high dependency upon c-MET signalling and that the viability of the SYO-1 or HS-SY-II cells was primarily driven by PDGFRα pathway. Based upon these observations, we could divide the SS cell lines tested in this study into two groups: c-MET-dependent SS cells (Yamato-SS) and PDGFRα-dependent SS cells (SYO-1 and HS-SY-II).

RTK cascades appear to be the principal activators of intracellular signalling through the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways. These downstream pathways have been reported as critical to SS viability [34, 35]. TAS-115, a novel c-MET/VEGFR-targeting dual tyrosine kinase inhibitor, has been reported to inhibit multiple RTKs besides c-MET and VEGFR under cell-free conditions [15]. TAS-115 significantly suppressed the proliferation of both c-MET-dependent and PDGFRα-dependent SS cells, mainly by inducing G0/G1 cell cycle arrest. Treatment with TAS-115 attenuated c-MET signalling in Yamato-SS cells, leading to subsequent suppression of AKT and ERK 1/2 phosphorylation. Similarly, blocking PDGFRα function with TAS-115 decreased intracellular signalling in SYO-1 and HS-SY-II cells. Interestingly, the TAS-115 concentration at which c-MET or PDGFRα signalling was inhibited in each cell line, using immunoblot analyses, corresponded to the IC50 value for TAS-115 against c-MET-dependent cells (Yamato-SS) or PDGFRα-dependent cells (SYO-1 or HS-SY-II) in WST-1 cell proliferation assays. In agreement with our in vitro findings, TAS-115 exerted anti-c-MET or anti-PDGFRα pathway activity and inhibited their downstream effectors in the Yamato-SS (c-MET-dependent) and SYO-1 (PDGFRα-dependent) cells in in vivo xenograft models. These insights indicate that the c-MET and PDGFRα cascades are the primary regulators of both the PI3K/AKT and MAPK pathways in c-MET-dependent and PDGFRα-dependent SS cells, respectively. This difference of signal dependency in SS cell lines may be ascribed to the diversity among individual tumours. Another possible explanation is an artificial selection of cells with activated c-MET or PDGFRα from heterogeneous cell populations within a tumour during cell-line establishment.

Pazopanib, a PDGFRα and PDGFRβ/ VEGFR/ c-kit-targeting tyrosine kinase inhibitor [13], has been approved by Food and Drug Administration (FDA) for soft tissue sarcoma and renal cell carcinoma [36, 37]. In Japan, this drug is the only tyrosine kinase inhibitor available for treatment of advanced soft tissue sarcomas. Accumulating data suggested that high plasma concentrations of HGF were significantly correlated with shorter progression-free survival in patients treated with pazopanib for metastatic renal cell carcinoma [38, 39]. These clinical studies give rise to an idea that the effect of pazopanib on cancers with active HGF/c-MET signalling might be limited. In the SS cell lines tested, pazopanib attenuated only PDGFRα signalling, while c-MET phosphorylation was not inhibited even at high concentration of pazopanib. By contrast, TAS-115 successfully suppressed both the c-MET and PDGFRα pathways. Moreover, TAS-115 treatment inhibited the phosphorylation of PDGFRα on at least four representative autophosphorylation sites of the receptor, as well as its downstream effectors, equivalently to pazopanib. These data suggest that TAS-115 will have therapeutic capability in SS with active c-MET or PDGFRα pathways, via inhibition of c-MET and PDGFRα signalling, indicating that it might be clinically applicable.

Recently, several investigators argued the possibility of personalized therapy by evaluating the specific signalling activation state of tyrosine kinases in different types of malignancies, including lung carcinoma [40], colorectal carcinoma [41] and ovarian carcinoma [42]. Most recently, we have reported that c-MET phosphorylation in clinical samples is a potential biomarker predicting response to a selective c-MET inhibitor for SS patients [11]. Treatment with a specific tyrosine kinase inhibitor selected by predicting therapeutic efficiency may prevent or delay non-specific deleterious side effects [43]. On the other hand, the possible advantages of a multi-targeting tyrosine kinase inhibitor include simplicity and capability of its use in daily practice for tumours with multiple signalling pathways. The tumours found in patients may have inter-individual differences in the activation states of RTKs that are involved in tumour growth or progression, even if they belong to a pathologically identical group. Moreover, the individual tumour may be a heterogeneous mixture of cell populations driven by multiple RTK signals. Indeed, our results indicated that monotherapy with TAS-115 suppressed both c-MET and PDGFRα signalling, resulting in strong therapeutic effects against two types of SS cells both in vitro and in vivo without outstanding side effects. These observations support the view that inhibition of both c-MET and PDGFRα with a single agent is a practical consideration for SS treatment.

The cellular origins of SS are still unknown. While several potential cellular origins of SS have been reported, such as cells of a myogenic or neurogenic lineage [4446], we have claimed that a multipotent mesenchymal stem cell may be a cell of origin for SS [19]. In this study, we noted the activation of c-MET or PDGFRα and the importance of these signals for viability in SS cell lines. In some translocation-related sarcomas, fusion genes are assumed to contribute to the upregulation of RTK cascades. For instance, c-MET was identified as a direct target of PAX3-FOXO1 in alveolar rhabdomyosarcoma and EWS-ATF1 in clear cell sarcoma [27, 47]. Additionally, EWS-FLI1 directly upregulated PDGF-C expression, which in turn activated the PDGFRα pathway in Ewing sarcoma [48]. Correspondingly, EWS-WT1, which underlay desmoplastic small round cell tumours, induced PDGF-A expression [49]. By sharp contrast, our parallel study showed that SS18-SSX silencing did not elicit the downregulation or inactivation of either c-MET or PDGFRα [11]. These data suggest that the activation of c-MET or PDGFRα might not be evoked by SS18-SSX. Rather, it reflects a prerequisite cellular background to be permissive for SS18-SSX. It has been reported that the window of permissive cells might be relatively narrow for SS18-SSX [44, 50]. Thus we speculated that SS might arise from a multipotent mesenchymal stem cell natively driven by HGF/c-MET or PDGF/PDGFR signalling [8]. These findings raise the possibility that SS inherits phosphorylated RTKs from a multipotent mesenchymal stem cell at a certain phase of differentiation and that these RTKs are suitable therapeutic targets for SS. Taken together, RTKs innately present in cells of origin, in addition to those epigenetically altered by fusion genes, might be plausible target molecules for the treatment of translocation-related sarcomas.

Although the findings detailed above do seem clear, there are some limitations in this study. First, since an environment in which either cultured cells or mouse xenografts grow is significantly different from that of their originating tumour, those model systems cannot always provide useful information to generate clinically relevant treatments. Second, in the clinical setting, distant metastases have been reported to be strongly associated with worse outcome of patients with SS [3, 5, 7]. However, experimental models of human SS with spontaneous metastatic potential have not been successfully developed. Thereby, whether TAS-115 monotherapy is sufficient to treat metastatic SS remains unknown. In October 2016, olaratumab, a monoclonal antibody of PDGFRα, received FDA approval for clinical use in combination with doxorubicin for unresectable and metastatic soft tissue sarcomas based upon the clinical data that olaratumab plus doxorubicin showed gains in median progression free and overall survival as compared to doxorubicin alone [51]. Those results lead us to hypothesize that combined therapy of conventional chemotherapeutic agents and TAS-115 targeting PDGFRα and c-MET may be effective for metastatic SS and warrants further investigations. Third, though treatment of TAS-115, introduced as an angiogenesis inhibitor via VEGFR signalling [15], decreased MVD in our in vivo experiments, the extent to which VEGFR inhibition by TAS-115 in the tumour microenvironment contributed to suppression of tumour growth was unclear in this study. Since HGF/c-MET as well as PDGF/PDGFR systems have been recognised as important mediators of angiogenesis [10, 12, 52], it could not be excluded that the antiangiogenic mechanisms of TAS-115 in SS at least in partly encompassed targeting c-MET or PDGFRα. Further clarification would be required in order to make definitive answers to these challenging issues in the future.

Conclusions

In conclusion, c-MET and PDGFRα signalling are essential for the growth and/or survival of SS tumours. By inhibiting c-MET or PDGFRα signalling, TAS-115 achieves significant therapeutic effects in both c-MET-dependent and PDGFRα-dependent SS cells in vitro and in vivo. Our observations provide strong evidence that TAS-115 can serve as a multiple tyrosine kinase inhibitor which can impede c-MET and PDGFRα signalling and may offer the distinct clinical advantages even when used as a monotherapy for patients with SS tumours driven by either c-MET or PDGFRα pathways.

Abbreviations

c-kit: 

Stem cell factor receptor

DMSO: 

Dimethyl sulfoxide

ERK: 

Extracellular signal-regulated kinase

FDA: 

Food and Drug Administration

HGF: 

Hepatocyte growth factor

IC50

50% inhibitory concentration

MAPK: 

Mitogen-activated protein kinase

MVD: 

Microvascular density

PARP: 

Poly ADP-ribose polymerase

PCNA: 

Proliferating cell nuclear antigen

PDGF: 

Platelet-derived growth factor

PDGFR: 

Platelet-derived growth factor receptor

PI3K: 

Phosphatidylinositol 3-kinase

RTK: 

Receptor tyrosine kinase

siRNA: 

small interference RNA

SS: 

Synovial sarcoma

VEGFR: 

Vascular endothelial growth factor receptor

Declarations

Acknowledgements

We thank Prof. Toshifumi Ozaki for the gift of the SS cell line SYO-1. We also thank Mari Shinkawa, Asa Tada and Yukiko Eguchi for excellent technical assistance and Hidenori Fujita for insightful comments and suggestions.

Funding

This study was supported by grants from the Japan Society for the Promotion of Science, JSPS KAKENHI (Grant Numbers: 26462264 and 16H05448), the Osaka Medical Research Foundation for Intractable Diseases, MSD K.K. and the Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development, AMED. None of the funding bodies were involved in in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

Datasets used and/or analyzed during this study are available from the corresponding author on reasonable request.

Authors’ contributions

SY and NN conceived and designed the study. YI, HO, KI and NN developed the methodology. SY, YI, TN, SN, NY, KK and HO collected the data. SY, YI, ST, KH, NA, TU, KI and NN analysed and interpreted the data. AM and HY provided study materials. SY, YI and ST drafted the final version of the manuscript and figure legends. KI and NN revised the figures, added critical content to the discussion and were responsible for revising all portions of the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

The animal studies were performed in accordance with a guideline approved by the Institutional Animal Care and Use Committee of the Osaka University Graduate School of Medicine.

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Authors’ Affiliations

(1)
Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine
(2)
Musculoskeletal Oncology Service, Osaka International Cancer Institute
(3)
Department of Orthopaedic Surgery, Osaka National Hospital
(4)
Research Institute, Nozaki Tokushukai

References

  1. Clark J, Rocques PJ, Crew AJ, Gill S, Shipley J, Chan AM, Gusterson BA, Cooper CS. Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11.2;q11.2) translocation found in human synovial sarcoma. Nat Genet. 1994;7:502–8.View ArticlePubMedGoogle Scholar
  2. Goldblum JR, Folpe AL, Weiss SW. Synovial sarcoma. In: Enzinger and Weiss’s soft tissue tumors. 6th ed. Philadelphia: Elsevier; 2013. p. 1052–70.Google Scholar
  3. Krieg A, Hefti F, Speth B, Jundt G, Guillou L, Exner U, von Hochstetter A, Cserhati M, Fuchs B, Mouhsine E, Kaelin A, Klenke F, Siebenrock K. Synovial sarcomas usually metastasize after >5 years: a multicenter retrospective analysis with minimum follow-up of 10 years for survivors. Ann Oncol. 2011;22:458–67.View ArticlePubMedGoogle Scholar
  4. Lewis JJ, Antonescu CR, Leung DH, Blumberg D, Healey JH, Woodruff JM, Brennan MF. Synovial sarcoma: a multivariate analysis of prognostic factors in 112 patients with primary localized tumors of the extremity. J Clin Oncol. 2000;18:2087–94.View ArticlePubMedGoogle Scholar
  5. Spillane AJ, A’Hern R, Judson IR, Fisher C, Thomas JM. Synovial sarcoma: a clinicopathologic, staging, and prognostic assessment. J Clin Oncol. 2000;18:3794–803.View ArticlePubMedGoogle Scholar
  6. Naing KW, Monjazeb AM, Li CS, Lee LY, Yang A, Borys D, Canter RJ. Perioperative radiotherapy is associated with improved survival among patients with synovial sarcoma: a SEER analysis. J Surg Oncol. 2015;111:158–64.View ArticlePubMedGoogle Scholar
  7. Takenaka S, Ueda T, Naka N, Araki N, Hashimoto N, Myoui A, Ozaki T, Nakayama T, Toguchida J, Tanaka K, Iwamoto Y, Matsumine A, Uchida A, Ieguchi M, Kaya M, Wada T, Baba I, Kudawara I, Aoki Y, Yoshikawa H. Prognostic implication of SYT-SSX fusion type in synovial sarcoma: a multi-institutional retrospective analysis in Japan. Oncol Rep. 2008;19:467–76.PubMedGoogle Scholar
  8. Yasui H, Naka N, Imura Y, Outani H, Kaneko K, Hamada K, Sasagawa S, Araki N, Ueda T, Itoh K, Myoui A, Yoshikawa H. Tailored therapeutic strategies for synovial sarcoma: receptor tyrosine kinase pathway analyses predict sensitivity to the mTOR inhibitor RAD001. Cancer Lett. 2014;347:114–22.View ArticlePubMedGoogle Scholar
  9. Bottaro DP, Rubin JS, Faletto DL, Chan AM, Kmiecik TE, Vande Woude GF, Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science. 1991;251:802–4.View ArticlePubMedGoogle Scholar
  10. Birchmeier C, Birchmeier W, Gherardi E, Vande Woude GF. Met, metastasis, motility and more. Nat Rev Mol Cell Biol. 2003;4:915–25.View ArticlePubMedGoogle Scholar
  11. Imura Y, Nakai T, Yamada S, Outani H, Takenaka S, Hamada K, Myoui A, Araki N, Itoh K, Yoshikawa H, Naka N. Functional and therapeutic relevance of hepatocyte growth factor/c-MET signaling in synovial sarcoma. Cancer Sci. 2016;107:1867–76.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Paulsson J, Ehnman M, Östman A. PDGF receptors in tumor biology: prognostic and predictive potential. Future Oncol. 2014;10:1695–708.View ArticlePubMedGoogle Scholar
  13. Kumar R, Knick VB, Rudolph SK, Johnson JH, Crosby RM, Crouthamel MC, Hopper TM, Miller CG, Harrington LE, Onori JA, Mullin RJ, Gilmer TM, Truesdale AT, Epperly AH, Boloor A, Stafford JA, Luttrell DK, Cheung M. Pharmacokinetic-pharmacodynamic correlation from mouse to human with pazopanib, a multikinase angiogenesis inhibitor with potent antitumor and antiangiogenic activity. Mol Cancer Ther. 2007;6:2012–21.View ArticlePubMedGoogle Scholar
  14. Hosaka S, Horiuchi K, Yoda M, Nakayama R, Tohmonda T, Susa M, Nakamura M, Chiba K, Toyama Y, Morioka H. A novel multi-kinase inhibitor pazopanib suppresses growth of synovial sarcoma cells through inhibition of the PI3K-AKT pathway. J Orthop Res. 2012;30:1493–8.View ArticlePubMedGoogle Scholar
  15. Fujita H, Miyadera K, Kato M, Fujioka Y, Ochiiwa H, Huang J, Ito K, Aoyagi Y, Takenaka T, Suzuki T, Ito S, Hashimoto A, Suefuji T, Egami K, Kazuno H, Suda Y, Nishio K, Yonekura K. The novel VEGF receptor/MET-targeted kinase inhibitor TAS-115 has marked in vivo antitumor properties and a favorable tolerability profile. Mol Cancer Ther. 2013;12:2685–96.View ArticlePubMedGoogle Scholar
  16. Zhao L, Yasumoto K, Kawashima A, Nakagawa T, Takeuchi S, Yamada T, Matsumoto K, Yonekura K, Yoshie O, Yano S. Paracrine activation of MET promotes peritoneal carcinomatosis in scirrhous gastric cancer. Cancer Sci. 2013;104:1640–6.View ArticlePubMedGoogle Scholar
  17. Nakade J, Takeuchi S, Nakagawa T, Ishikawa D, Sano T, Nanjo S, Yamada T, Ebi H, Zhao L, Yasumoto K, Matsumoto K, Yonekura K, Yano S. Triple inhibition of EGFR, met, and VEGF suppresses regrowth of HGF-triggered, erlotinib-resistant lung cancer harboring an EGFR mutation. J Thorac Oncol. 2014;9:775–83.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Kunii E, Ozasa H, Oguri T, Maeno K, Fukuda S, Uemura T, Takakuwa O, Ohkubo H, Takemura M, Niimi A. Reversal of c-MET-mediated resistance to cytotoxic anticancer drugs by a novel c-MET inhibitor TAS-115. Anticancer Res. 2015;35:5241–7.PubMedGoogle Scholar
  19. Naka N, Takenaka S, Araki N, Miwa T, Hashimoto N, Yoshioka K, Joyama S, Hamada K, Tsukamoto Y, Tomita Y, Ueda T, Yoshikawa H, Itoh K. Synovial sarcoma is a stem cell malignancy. Stem Cells. 2010;28:1119–31.PubMedGoogle Scholar
  20. Kawai A, Naito N, Yoshida A, Morimoto Y, Ouchida M, Shimizu K, Beppu Y. Establishment and characterization of a biphasic synovial sarcoma cell line, SYO-1. Cancer Lett. 2004;204:105–13.View ArticlePubMedGoogle Scholar
  21. Sonobe H, Manabe Y, Furihata M, Iwata J, Oka T, Ohtsuki Y, Mizobuchi H, Yamamoto H, Kumano O, Abe S. Establishment and characterization of a new human synovial sarcoma cell line, HS-SY-II. LabInvest. 1992;67:498–505.Google Scholar
  22. Podar K, Tonon G, Sattler M, Tai YT, Legouill S, Yasui H, Ishitsuka K, Kumar S, Kumar R, Pandite LN, Hideshima T, Chauhan D, Anderson KC. The small-molecule VEGF receptor inhibitor pazopanib (GW786034B) targets both tumor and endothelial cells in multiple myeloma. Proc Natl Acad Sci U S A. 2006;103:19478–83.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Paesler J, Gehrke I, Gandhirajan RK, Filipovich A, Hertweck M, Erdfelder F, Uhrmacher S, Poll-Wolbeck SJ, Hallek M, Kreuzer KA. The vascular endothelial growth factor receptor tyrosine kinase inhibitors vatalanib and pazopanib potently induce apoptosis in chronic lymphocytic leukemia cells in vitro and in vivo. Clin Cancer Res. 2010;16:3390–8.View ArticlePubMedGoogle Scholar
  24. Heldin CH, Ostman A, Rönnstrand L. Signal transduction via platelet-derived growth factor receptors. Biochim Biophys Acta. 1998;1378:F79–F113.PubMedGoogle Scholar
  25. Imura Y, Yasui H, Outani H, Wakamatsu T, Hamada K, Nakai T, Yamada S, Myoui A, Araki N, Ueda T, Itoh K, Yoshikawa H, Naka N. Combined targeting of mTOR and c-MET signaling pathways for effective management of epithelioid sarcoma. Mol Cancer. 2014;13:185.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Torres KE, Zhu QS, Bill K, Lopez G, Ghadimi MP, Xie X, Young ED, Liu J, Nguyen T, Bolshakov S, Belousov R, Wang S, Lahat G, Liu J, Hernandez B, Lazar AJ, Lev D. Activated MET is a molecular prognosticator and potential therapeutic target for malignant peripheral nerve sheath tumors. Clin Cancer Res. 2011;17:3943–55.View ArticlePubMedPubMed CentralGoogle Scholar
  27. Davis IJ, Mcfadden AW, Zhang Y, Coxon A, Teresa L, Wagner AJ, Fisher DE. Identification of the receptor tyrosine kinase c-met and its ligand, hepatoocyte growth factor, as therapeutic targets in clear cell sarcoma. Cancer Res. 2010;70:639–45.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Outani H, Tanaka T, Wakamatsu T, Imura Y, Hamada K, Araki N, Itoh K, Yoshikawa H, Naka N. Establishment of a novel clear cell sarcoma cell line (Hewga-CCS), and investigation of the antitumor effects of pazopanib on Hewga-CCS. BMC Cancer. 2014;14:455.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Shimizu A, O’Brien KP, Sjöblom T, Pietras K, Buchdunger E, Collins VP, Heldin CH, Dumanski JP, Ostman A. The dermatofibrosarcoma protuberans-associated collagen type Iα1/platelet-derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB. Cancer Res. 1999;59:3719–23.PubMedGoogle Scholar
  30. Corless CL, Schroeder A, Griffith D, Town A, McGreevey L, Harrell P, Shiraga S, Bainbridge T, Morich J, Heinrich MC. PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib. J Clin Oncol. 2005;23:5357–64.View ArticlePubMedGoogle Scholar
  31. McDermott U, Ames RY, Iafrate AJ, Maheswaran S, Stubbs H, Greninger P, McCutcheon K, Milano R, Tam A, Lee DY, Lucien L, Brannigan BW, Ulkus LE, Ma XJ, Erlander MG, Haber DA, Sharma SV, Settleman J. Ligand-dependent platelet-derived growth factor receptor (PDGFR)-α activation sensitizes rare lung cancer and sarcoma cells to PDGFR kinase inhibitors. Cancer Res. 2009;69:3937–46.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Oda Y, Sakamoto A, Saito T, Kinukawa N, Iwamoto Y, Tsuneyoshi M. Expression of hepatocyte growth factor (HGF)/scatter factor and its receptor c-MET correlates with poor prognosis in synovial sarcoma. Hum Pathol. 2000;31:185–92.View ArticlePubMedGoogle Scholar
  33. Bozzi F, Ferrari A, Negri T, Conca E, Luca DR, Losa M, Casieri P, Orsenigo M, Lampis A, Meazza C, Casanova M, Pierotti MA, Tamborini E, Pilotti S. Molecular characterization of synovial sarcoma in children and adolescents: evidence of akt activation. Transl Oncol. 2008;1:95–101.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Friedrichs N, Trautmann M, Endl E, Sievers E, Kindler D, Wurst P, Czerwitzki J, Steiner S, Renner M, Penzel R, Koch A, Larsson O, Tanaka S, Kawai A, Schirmacher P, Mechtersheimer G, Wardelmann E, Buettner R, Hartmann W. Phosphatidylinositol-3′-kinase/AKT signaling is essential in synovial sarcoma. Int J Cancer. 2011;129:1564–75.View ArticlePubMedGoogle Scholar
  35. Ishibe T, Nakayama T, Okamoto T, Aoyama T, Nishijo K, Shibata KR, Shima Y, Nagayama S, Katagiri T, Nakamura Y, Nakamura T, Toguchida J. Disruption of fibroblast growth factor signal pathway inhibits the growth of synovial sarcomas : potential application of signal inhibitors to molecular target therapy. Clin Cancer Res. 2005;11:2702–12.View ArticlePubMedGoogle Scholar
  36. van der Graaf WT, Blay JY, Chawla SP, Kim DW, Bui-Nguyen B, Casali PG, Schöffski P, Aglietta M, Staddon AP, Beppu Y, Le Cesne A, Gelderblom H, Judson IR, Araki N, Ouali M, Marreaud S, Hodge R, Dewji MR, Coens C, Demetri GD, Fletcher CD, Dei Tos AP, Hohenberger P. Pazopanib for metastatic soft-tissue sarcoma (PALETTE): a randomised, double-blind, placebo-controlled phase 3 trial. Lancet. 2012;379:1879–86.View ArticlePubMedGoogle Scholar
  37. Sternberg CN, Davis ID, Mardiak J, Szczylik C, Lee E, Wagstaff J, Barrios CH, Salman P, Gladkov OA, Kavina A, Zarba JJ, Chen M, McCann L, Pandite L, Roychowdhury DF, Hawkins RE. Pazopanib in locally advanced or metastatic renal cell carcinoma: results of a randomized phase III trial. J Clin Oncol. 2010;28:1061–8.View ArticlePubMedGoogle Scholar
  38. Tran HT, Liu Y, Zurita AJ, Lin Y, Baker-Neblett KL, Martin AM, Figlin RA, Hutson TE, Sternberg CN, Amado RG, Pandite LN, Heymach JV. Prognostic or predictive plasma cytokines and angiogenic factors for patients treated with pazopanib for metastatic renal-cell cancer: a retrospective analysis of phase 2 and phase 3 trials. Lancet Oncol. 2012;13:827–37.View ArticlePubMedGoogle Scholar
  39. Pal SK, Hossain DM, Zhang Q, Frankel PH, Jones JO, Carmichael C, Ruel C, Lau C, Kortylewski M. Pazopanib as third line therapy for metastatic renal cell carcinoma: clinical efficacy and temporal analysis of cytokine profile. J Urol. 2015;193:1114–21.View ArticlePubMedGoogle Scholar
  40. Zupa A, Improta G, Silvestri A, Pin E, Deng J, Aieta M, Musto P, Nitti D, Mammano E, Liotta L, Belluco C, Wulfkuhle J, Petricoin E. A pilot characterization of human lung NSCLC by protein pathway activation mapping. J Thorac Oncol. 2012;7:1755–66.View ArticlePubMedGoogle Scholar
  41. Harlé A, Salleron J, Perkins G, Pilati C, Blons H, Laurent-Puig P, Merlin JL. Expression of pEGFR and pAKT as response-predictive biomarkers for RAS wild-type patients to anti-EGFR monoclonal antibodies in metastatic colorectal cancers. Br J Cancer. 2015;113:680–5.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Sereni MI, Baldelli E, Gambara G, Deng J, Zanotti L, Bandiera E, Bignotti E, Ragnoli M, Tognon G, Ravaggi A, Meani F, Memo M, Angioli R, Liotta LA, Pecorelli SL, Petricoin E, Pierobon M. Functional characterization of epithelial ovarian cancer histotypes by drug target based protein signaling activation mapping: implications for personalized cancer therapy. Proteomics. 2015;15:365–73.View ArticlePubMedGoogle Scholar
  43. Hojjat-Farsangi M. Small-molecule inhibitors of the receptor tyrosine kinases: promising tools for targeted cancer therapies. Int J Mol Sci. 2014;15:13768–801.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Haldar M, Hancock JD, Coffin CM, Lessnick SL, Capecchi MR. A conditional mouse model of synovial sarcoma: insights into a myogenic origin. Cancer Cell. 2007;11:375–88.View ArticlePubMedGoogle Scholar
  45. Ishibe T, Nakayama T, Aoyama T, Nakamura T, Toguchida J. Neuronal differentiation of synovial sarcoma and its therapeutic application. Clin Orthop Relat Res. 2008;466:2147–55.View ArticlePubMedPubMed CentralGoogle Scholar
  46. Tamaki S, Fukuta M, Sekiguchi K, Jin Y, Nagata S, Hayakawa K, Hineno S, Okamoto T, Watanabe M, Woltjen K, Ikeya M, Kato T Jr, Toguchida J: SS18-SSX , the oncogenic fusion protein in synovial sarcoma , is a cellular context-dependent epigenetic modifier. PLoS One 2015, 10:e0142991.Google Scholar
  47. Cao L, Yu Y, Bilke S, Walker RL, Mayeenuddin LH, Azorsa DO, Yang F, Pineda M, Helman LJ, Meltzer PS. Genome-wide identification of PAX3-FKHR binding sites in rhabdomyosarcoma reveals candidate target genes important for development and cancer. Cancer Res. 2010;70:6497–508.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Zwerner J, May W. PDGF-C is an EWS/FLI induced transforming growth factor in Ewing family tumors. Oncogene. 2001;20:626–33.View ArticlePubMedGoogle Scholar
  49. Lee SB, Kolquist KA, Nichols K, Englert C, Maheswaran S, Ladanyi M, Gerald WL, Haber DA. The EWS-WT1 translocation product induces PDGFA in desmoplastic small round-cell tumour. Nat Genet. 1997;17:309–13.View ArticlePubMedGoogle Scholar
  50. Toguchida J, Nakayama T. Molecular genetics of sarcomas: applications to diagnoses and therapy. Cancer Sci. 2009;100:1573–80.View ArticlePubMedGoogle Scholar
  51. Tap WD, Jones RL, Van Tine BA, Chmielowski B, Elias AD, Adkins D, Agulnik M, Cooney MM, Livingston MB, Pennock G, Hameed MR, Shah GD, Qin A, Shahir A, Cronier DM, Ilaria R, Conti I, Cosaert J, Schwartz GK. Olaratumab and doxorubicin versus doxorubicin alone for treatment of soft-tissue sarcoma: an open-label phase 1b and randomised phase 2 trial. Lancet. 2016;388:488–97.View ArticlePubMedGoogle Scholar
  52. Folkman J. Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov. 2007;6:273–86.View ArticlePubMedGoogle Scholar

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© The Author(s). 2017