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

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 [19‐21]. 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.

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 (Tyr^1234/1235; #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).

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).

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.

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.

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).

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].

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.

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.

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.

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).

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.

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.

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.

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).

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.

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).

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.

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.

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.