Background
Epithelioid sarcoma (EpS) was first described in 1970 by Enzinger as a distinct soft-tissue tumor with mixed epithelial and mesenchymal phenotype [
1], but the origin and true nature of EpS remain controversial. In general, EpS is relatively rare and accounts for less than 1% of all soft-tissue sarcomas [
2]. The overall 5-year survival rates are 32%–78% [
2‐
5]. The clinical course of EpS is usually characterized by local recurrences and distant metastases to lymph nodes and lungs, but an effective chemotherapy has not yet been established [
2‐
5]. Therefore, novel therapeutic approaches against EpS are critically needed.
The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway, which drives cell proliferation, motility, and survival, is frequently hyperactivated in a variety of malignancies [
6,
7], and inhibition of this pathway has been considered an appropriate approach for cancer therapy. Integrase interactor 1 (INI-1) is the protein product of the tumor suppressor gene
hSNF5/INI1/SMARCB1/BAF47 located on 22q11.2. Loss of INI-1 serves as a diagnostic feature in malignant rhabdoid tumors (MRTs) and atypical teratoid/rhabdoid tumors (AT/RTs) [
8,
9]. Darr and colleagues reported that INI-1-deficient tumor cells exhibited persistent activation of AKT signaling [
10]. INI-1 expression is also lost in most EpS clinical samples [
11,
12], suggesting that AKT signaling may also be activated in EpS cells. In the present study, we detected loss of INI-1 expression and constitutive AKT activation in two human EpS cell lines, Asra-EPS [
13] and VAESBJ [
14].
AKT activation has been proposed as a predictor of response to rapamycin, which is an allosteric mTOR inhibitor [
15]; this concept raises the possibility that mTOR inhibitors may be effective on EpS. Administration of these drugs results in reduction of regulatory proteins involved in progression of cells from the G1 to S-phase of their growth cycle [
16]. The U.S. Food and Drug Administration has approved mTOR inhibitors for treatment of neuroendocrine tumors, renal cell carcinoma, and subependymal giant cell astrocytoma associated with tuberous sclerosis. However, the antitumor effects of mTOR inhibitors on patients with bone or soft-tissue sarcomas are limited, and responses are frequently short lived [
17,
18]. In addition, blocking mTOR activity inadvertently reactivates AKT signaling, which mitigates the antitumor effects of mTOR inhibitors, and this reactivation has been posited as a mechanism of intrinsic resistance to mTOR inhibitors [
19‐
22].
The AKT/mTOR signaling pathway is normally regulated by upstream receptor tyrosine kinases (RTKs) [
23‐
25]. The resistance to mTOR inhibitors has been reported to be caused by RTK-dependent AKT reactivation due to a release of negative feedback inhibition [
19‐
22]. Overexpression of hepatocyte growth factor (HGF) and its receptor, known as c-MET, is observed in most EpS clinical samples [
26]. We demonstrated that c-MET was highly activated via an autocrine HGF loop in both EpS cell lines. The HGF/c-MET signaling pathway is critical in cell proliferation, motility, and invasion of several human sarcomas [
27‐
29], but little is known about its biological functions in EpS.
In the present study, we first examined the therapeutic efficacy of an mTOR inhibitor, RAD001 (everolimus; Novartis Pharma AG, Basel, Switzerland), on two human EpS cell lines, Asra-EPS and VAESBJ. Next, we investigated whether RAD001-induced AKT reactivation was dependent on c-MET signaling. Finally, to seek a novel therapeutic modality for EpS, we evaluated the antitumor effects of combining RAD001 with a c-MET inhibitor, INC280 (Novartis Pharma AG), on the growth of EpS cell lines in vitro and in vivo.
Discussion
Activation of the AKT/mTOR signaling pathway through mutation of pathway components as well as through activation of upstream signaling molecules occurs in a majority of cancers and contributes to deregulation of proliferation and resistance to apoptosis [
6,
7]. Constitutive AKT activation was observed in MRTs characterized by loss of INI-1 expression, and a mechanism for AKT activation may be caused by aberrant activation of the insulin-like growth factor 1 receptor (IGF-1R) pathway [
33]. Moreover, AKT signaling was activated via autocrine signaling by insulin and the insulin receptor in INI-1-deficient AT/RTs [
34]. In addition, INI-1 loss is observed in the majority of EpS [
11,
12] and is responsible for the tumorigenic properties of EpS [
35], but it is unknown whether the AKT/mTOR pathway is activated in EpS. In the present study, we demonstrated loss of INI-1 expression and constitutive activation of the AKT/mTOR pathway in two human EpS cell lines, Asra-EPS and VAESBJ. Although the histological phenotypes of MRT, AT/RT, and EpS are different from each other, INI-1 expression is lost, and AKT signaling is activated among these distinct tumors. However, little is known about the relationship between INI-1 deficiency and AKT activation in EpS. Asra-EPS and VAESBJ cell lines can be useful tools to investigate this relationship in EpS.
mTOR inhibitors exert antitumor effects on several cancers in which the AKT/mTOR pathway is hyperactivated, but their effects are frequently modest in clinical trials [
17,
18]. Biopsy samples from patients treated with mTOR inhibitors confirmed that AKT reactivation occurred clinically and portended a poorer prognosis [
19,
36]. AKT reactivation induced by mTOR inhibition in tumor cells is likely to reduce its antitumor effects by activating pathways that attenuate its effects on proliferation and apoptosis; thus, it is an unexpected and potentially undesirable consequence of mTOR inhibition [
19]. Recently, it has been shown that mTOR inhibitors increased upstream RTK activity, which resulted in reactivation of not only AKT but also ERK [
32]. Here, we demonstrated that an mTOR inhibitor, RAD001, also inhibited EpS cell proliferation but induced reactivation of both AKT and ERK. These results suggested that blockade of this reactivation could enhance the antitumor effects of mTOR inhibitors on EpS.
It has been reported that mTOR inhibitors induced feedback reactivation of AKT signaling through an IGF-1R-dependent, a platelet-derived growth factor receptor A (PDGFRA)-dependent, or a PDGFRB-dependent mechanism [
19‐
22]. However, phospho-RTK array analyses did not show activation of IGF-1R, PDGFRA, or PDGFRB in EpS. Instead, we found that c-MET was the most highly activated RTK in both EpS cell lines and that reactivation of AKT and ERK by mTOR inhibition was c-MET-dependent in EpS. To the best of our knowledge, this is the first study to show that an mTOR inhibitor induces reactivation of AKT and ERK through a c-MET-dependent mechanism. These results provide a rationale for combining mTOR inhibitors with c-MET inhibitors to treat patients with EpS.
HGF stimulation induces c-MET phosphorylation, which in turn activates multiple downstream pathways, including PI3K/AKT and MAPK/ERK signaling [
25,
37]. Combined overexpression of HGF and c-MET have been observed in numerous sarcomas [
38‐
40], and HGF can activate c-MET in an autocrine manner in these tumors. We observed that both HGF and c-MET were also overexpressed in Asra-EPS and VAESBJ cells, indicating that c-MET was aberrantly activated by autocrine HGF stimulation in EpS. Cancer-associated c-MET activation triggers cell growth, survival, invasion, migration, and angiogenesis [
41‐
43]. c-MET inhibitors have shown antitumor efficacy in preclinical studies and are currently being evaluated in human cancer clinical trials [
44,
45]. In the present study, a selective c-MET inhibitor INC280 showed antitumor effects on EpS cell growth by blocking activation of AKT and ERK. These data indicated that one mechanism for activation of both AKT and ERK pathways was based on the HGF/c-MET autocrine signaling in EpS. However, the sensitivity of VAESBJ cells to INC280 was modest compared with that of Asra-EPS cells. AKT activation was completely blocked by treatment with INC280 in Asra-EPS cells but not in VAESBJ cells. These results suggested that the dependency of VAESBJ cells on HGF/c-MET signaling may differ from that of Asra-EPS cells.
PTEN counteracts the effects of PI3K on AKT, and loss of PTEN expression mediates AKT activation [
30]. PTEN status affected anti-c-MET therapies to glioblastomas in which PTEN protein expression was frequently low or absent, and combining anti-HGF/c-MET therapies with mTOR inhibitors additively inhibited growth of glioblastoma xenografts [
46]. Xie and colleagues demonstrated no or reduced PTEN expression in many EpS samples, indicating that PTEN deregulation was a common molecular aberration in human EpS [
47]. We found that PTEN expression was much lower in VAESBJ cells than in Asra-EPS or control HDF cells, suggesting that epithelioid sarcomas were heterogeneous malignancies in terms of PTEN expression. Our results indicated that reduction of PTEN expression in VAESBJ cells may contribute to sustained AKT activation after INC280 treatment and result in decreased sensitivity to c-MET inhibitors.
In fact, the growth of EpS was also significantly abrogated by treatment with the combination of RAD001 and INC280 in vitro and in vivo. Their combination notably inhibited mTOR and c-MET signaling pathways that were hyperactivated in human EpS; thus, we propose a combined therapeutic approach using mTOR and c-MET inhibitors for EpS lacking effective systemic treatment.
In the present study, the expression of proteins related to AKT/mTOR and HGF/c-MET pathways were detected in nearly all EpS clinical samples, as in EpS cell lines. However, expression levels of these proteins were different among the clinical samples of EpS patients. These results in clinical and experimental studies suggested that EpS cells exhibited heterogeneity in dependency on AKT and c-MET pathways within the tumor. The activation of these pathways contributes to the cell proliferation, survival, and resistance to chemotherapies in many cancers [
6,
7,
37,
41‐
43]. Therefore, dual targeting of AKT/mTOR and HGF/c-MET pathways may exert significant antitumor effects on EpS cells in which these pathways are activated and help us to overcome this devastating disease.
Materials and methods
Cell lines, reagents, and antibodies
We used two human EpS cell lines, Asra-EPS and VAESBJ. Asra-EPS was established from a primary tumor of the patient with angiomatoid type of EpS in our laboratory as previously described [
13]. VAESBJ, which was established from a bone marrow aspirate of the patient with EpS whose tumors metastasized to a bone marrow [
14], was purchased from the American Type Culture Collection. HDF cells were purchased from Kurabo. A human synovial sarcoma cell line, SYO-1, was kindly provided by Dr. Ozaki (Okayama University, Okayama, Japan). Cells were grown in Dulbecco’s Modified Eagle Medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured in a humidified atmosphere at 37°C in 5% CO
2.
An mTOR inhibitor, RAD001, and an ATP-competitive selective c-MET inhibitor, INC280, were provided by Novartis Pharma AG. According to the manufacturer’s instructions, RAD001 and INC280 were prepared in dimethyl sulfoxide (DMSO) before being added to cell cultures for in vitro studies. The oral RAD001 formulation provided by Novartis Pharma AG (everolimus microemulsion preconcentrate and corresponding placebo) for animal experiments was diluted with water to the optimal concentration just before administration via gavage. INC280 was diluted in 0.5% methylcellulose and 0.1% Tween 80 for in vivo experiments.
Antibodies against c-MET (#8198; WB, 1:1000; IHC, 1:300), p-MET (Tyr1234/1235; #3077; WB, 1:1000; IHC, 1:150), AKT (#4691; 1:1000), p-AKT (Ser473; #4060; WB, 1:1000; IHC, 1:50), ERK (#4695; 1:1000), p-ERK (Thr202/Tyr204; #4370; WB, 1:2000; IHC, 1:400), mTOR (#2983; 1:1000), p-mTOR (Ser2448; #5536; 1:1000), S6RP (#2217; 1:1000), p-S6RP (Ser235/236; #2211; 1:1000), PTEN (#9188; 1:1000), cleaved caspase-3 (#9661; 1:1000), and beta-actin (#4970; 1:1000) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). An antibody against Ki-67 (M7240; 1:50) was purchased from Dako (Glostrup, Denmark). An antibody against INI-1 (612110; 1:500) was purchased from Becton Dickinson Biosciences (BD Biosciences; San Jose, CA, USA). An antibody against HGF (AF-294-NA; 10 μg/ml) was purchased from R&D systems (Minneapolis, MN, USA). Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from GE Healthcare Life Sciences (Piscataway, NJ, USA).
Patients
Six patients with EpS (5 males and 1 female) were operated in Osaka University Hospital from 1998 to 2012. The mean age at the operation was 59.5 years (49 to 67). Tumor specimens were obtained with the patients’ informed consent and used for immunohistochemical studies.
Western blot analysis
For the lysate preparation, cells were first washed with PBS and lysed in RIPA buffer (Thermo Scientific, Waltham, MA, USA). Protein concentrations were determined according to the bicinchoninic acid method (Thermo Scientific). Then, the cell lysates were separated on 4%–12% Bis-Tris gels (Life Technologies) and transferred to polyvinylidene difluoride (PVDF) membranes (Nippon Genetics, Tokyo, Japan). The membranes were incubated in 5% skim milk in TBS with Tween 20 (TBS-T) at room temperature. Blocked membranes were incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1 hour. After washing in TBS-T, immunoreactive bands were visualized by enhanced chemiluminescence (ECL; GE Healthcare Life Sciences).
WST-1 cell proliferation assay
Cells were seeded at a density of 1 × 103 cells/well in 96-well plates for cell proliferation assays. The cells were incubated overnight and treated with various concentrations of drugs or vehicle (DMSO) for drug experiments. Cell viability was assessed using the Premix WST-1 cell proliferation assay system (Takara Bio, Inc., Otsu, Japan). Using a microplate reader, absorbance measurements read at 690 nm were subtracted from those read at 450 nm. Relative cell viability was expressed as (absorbance of treated cells minus absorbance of cell-free control)/(absorbance of untreated control minus absorbance of cell-free control).
Cell cycle analysis
EpS cells were seeded at a density of 5 × 105 cells/dish in 10-cm culture dishes and grown overnight, followed by treatment with RAD001, INC280, their combination, or vehicle. After 24-hour treatment, the cells were collected 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 minutes at room temperature. The cell cycle was analyzed using BD FACSCanto II flow cytometer (BD Biosciences).
In vivo animal xenograft models
Five-week-old athymic nude mice (BALB/c nu/nu; SLC, Shizuoka, Japan) were housed at the Institute of Experimental Animal Sciences Osaka University Medical School, in accordance with a guideline approved by the Institutional Animal Care and Use Committee of the Osaka University Graduate School of Medicine. For the xenograft tumor growth assay, 1 × 107 EpS cells were injected subcutaneously into the left side of the back. Therapy was initiated after tumor establishment (>5 mm in the longest diameter). RAD001 and INC280 were administered orally thrice a week and once a day, respectively. Xenograft tumor volume and mice body weight were measured twice a week. Tumor volume was measured with a caliper and calculated according to the formula (A × B2)/2, with A being the longest diameter and B the shortest diameter of the tumor. Mice were sacrificed when the total tumor burden reached 2 cm3, and the tumor weight was then measured. The tumors were resected for western blot analyses and immunohistochemical studies.
Immunohistochemistry
Specimens of tumors formed in nude mice and those of patients’ primary tumors were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned in 4-μm thicknesses. Paraffin-embedded sections were deparaffinized and dehydrated. Antigens were retrieved at 95°C for 10 minutes in a 10-mM citrate buffer. After blocking of endogenous peroxidase activity for 10 minutes with methanol containing 3% H2O2, the sections were reacted for 1 hour with TBS containing 2% bovine serum albumin at room temperature. The sections were incubated with primary antibodies at 4°C overnight. On the next day, sections were incubated for 1 hour with secondary antibodies and stained with 3,3’-diaminobenzidine tetrahydrochloride (DAB; Dako). The sections were finally counterstained with hematoxylin. Immunohistochemical protein expression levels were determined using NIS-Elements software (Nikon Corporation, Tokyo, Japan). Staining of p-AKT, HGF, c-MET, and p-MET in patients’ clinical samples was scored as follows: 0, undetectable (0% positive cells); 1+, focally positive (<10% positive cells); 2+, moderately positive (<50% positive cells), and 3+, intensely positive (more than 50% positive cells). Immunohistochemical results were interpreted as negative (0, 1+) or positive (2+, 3+).
Phospho-RTK array
To evaluate expression of phosphorylated RTKs, the phospho-RTK array was performed with the Proteome Profiler Array Kit (R&D Systems), according to the manufacturer’s protocol. In brief, the array membrane was blocked for 1 hour, incubated with cell lysates overnight, and then treated with HRP-conjugated anti-phospho-tyrosine antibody for 2 hours at room temperature. The membrane was developed with ECL detection reagent, and RTK spots were visualized.
ELISA
Cells were cultured at a density of 1 × 105 cells/well in 6-well plates. On the 4th day, cell culture supernatants were collected. When xenograft tumors reached 2 cm3, whole blood samples were collected by intracardiac puncture, and sera were obtained. HGF concentrations in cell-conditioned media or sera of xenografted mice were determined by ELISA using a Human HGF Quantikine ELISA kit (R&D Systems), according to the manufacturer’s instruction.
siRNA transfection
EpS cells were seeded at a density of 3 × 105 cells/well in 6-well plates and grown overnight. Cells were transfected with 20 nM siRNAs for 48 hours using Lipofectamine 2000 (Life Technologies). Two kinds of siRNAs targeting c-MET (constructs I and II; #6618 and #6622) and mTOR (construct I and II; #6381 and #6556), and a non-targeting siRNA (#6568) were purchased from Cell Signaling Technology, Inc.
Five thousand EpS cells were suspended in 1 ml of 0.5% SeaPlaque Agarose (Lonza, Basel, Switzerland) with normal growth medium and seeded over a basal layer of 0.6% agarose in 35-mm culture dishes. The number of colonies (>100 μm in diameter) per well was counted under a light microscope two weeks later.
Determination of cell number
EpS cells were plated at a density of 1 × 105 cells/well into 6-well plates and grown overnight before treatment with RAD001, INC280, their combination, or vehicle for 72 hours. Cells were trypsinized with 0.25% trypsin plus EDTA (Life Technologies), and a hemocytometer was used to count the cell number for each well every 24 hours.
Statistical analysis
Each experiment was performed in triplicate. All data are expressed as means ± SDs. Student’s t-test for biological assays and Mann–Whitney’s U test for animal experiments were used to evaluate the significance of differences. Values of p < 0.05 were considered statistically significant.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
YI and NN: conceived and designed the experiments; YI, HY, and HO: performed the experiments and analyzed the data; YI: wrote the manuscript; HY, HO, TW, KH, TN, and SY; helped to draft the manuscript; AM, NA, TU, KI, HY, and NN: revised the manuscript. All authors read and approved the final manuscript.