FTY720 (Fingolimod) attenuates basal and sphingosine-1-phosphate-evoked thyroid cancer cell invasion

in Endocrine-Related Cancer

The bioactive lipid sphingosine-1-phosphate (S1P) is a potent inducer of ML-1 thyroid cancer cell migration and invasion. It evokes migration and invasion by activating S1P receptor 1 and 3 (S1P1,3) and downstream signaling intermediates as well as through cross-communication with vascular endothelial growth factor receptor 2 (VEGFR2). However, very little is known about the role of S1P receptors in thyroid cancer. Furthermore, the currently used treatments for thyroid cancer have proven to be rather unsuccessful. Thus, due to the insufficiency of the available treatments for thyroid cancer, novel and targeted therapies are needed. The S1P receptor functional antagonist FTY720, an immunosuppressive drug currently used for treatment of multiple sclerosis, has shown promising effects as an inhibitor of cancer cell proliferation and invasion. In this study, we investigated the effect of FTY720 on invasion and proliferation of several thyroid cancer cell lines. We present evidence that FTY720 attenuated basal as well as S1P-evoked invasion of these cell lines. Furthermore, FTY720 potently downregulated S1P1, protein kinase Cα(PKCα), PKCβI, and VEGFR2. It also attenuated S1P-evoked phosphorylation of ERK1/2. Our results also showed that FTY720 attenuated S1P-induced MMP2 intracellular expression, S1P-induced secretion of MMP2 and MMP9, and decreased basal MMP2 and MMP9 activity. Moreover, in FTY720-treated cells, proliferation was attenuated, p21 and p27 were upregulated, and the cells were arrested in the G1 phase of the cell cycle. FTY720 attenuated cancer cell proliferation in the chick embryo chorioallantoic membrane assay. Thus, we suggest that FTY720 could be beneficial in the treatment of thyroid cancer.

Abstract

The bioactive lipid sphingosine-1-phosphate (S1P) is a potent inducer of ML-1 thyroid cancer cell migration and invasion. It evokes migration and invasion by activating S1P receptor 1 and 3 (S1P1,3) and downstream signaling intermediates as well as through cross-communication with vascular endothelial growth factor receptor 2 (VEGFR2). However, very little is known about the role of S1P receptors in thyroid cancer. Furthermore, the currently used treatments for thyroid cancer have proven to be rather unsuccessful. Thus, due to the insufficiency of the available treatments for thyroid cancer, novel and targeted therapies are needed. The S1P receptor functional antagonist FTY720, an immunosuppressive drug currently used for treatment of multiple sclerosis, has shown promising effects as an inhibitor of cancer cell proliferation and invasion. In this study, we investigated the effect of FTY720 on invasion and proliferation of several thyroid cancer cell lines. We present evidence that FTY720 attenuated basal as well as S1P-evoked invasion of these cell lines. Furthermore, FTY720 potently downregulated S1P1, protein kinase Cα(PKCα), PKCβI, and VEGFR2. It also attenuated S1P-evoked phosphorylation of ERK1/2. Our results also showed that FTY720 attenuated S1P-induced MMP2 intracellular expression, S1P-induced secretion of MMP2 and MMP9, and decreased basal MMP2 and MMP9 activity. Moreover, in FTY720-treated cells, proliferation was attenuated, p21 and p27 were upregulated, and the cells were arrested in the G1 phase of the cell cycle. FTY720 attenuated cancer cell proliferation in the chick embryo chorioallantoic membrane assay. Thus, we suggest that FTY720 could be beneficial in the treatment of thyroid cancer.

Introduction

Sphingosine-1-phosphate (S1P) is a member of the sphingomyelin family of bioactive lipids regulating several central cellular processes. It is generated in cells from sphingosine by the action of sphingosine kinase 1 (SK1) and is transported out from the cells by the aid of several transport mechanisms. However, a role for S1P as an intracellular messenger has become evident during the past few years (Alvarez et al. 2007, Strub et al. 2010, Alshaker et al. 2013, Pyne & Pyne 2013, Takabe & Spiegel 2014).

Extracellular S1P elicits its effects through binding to one of its five highly specific G-protein-coupled receptors, termed S1P1–5, and by activating different G-proteins. The final outcome of S1P treatment, however, is dependent on which receptor and G-protein it activates. As most cell types express receptors for S1P, this metabolite is unambiguously an important regulator of cell function during normal development and normal physiology. In addition, a substantial amount of evidence has implicated S1P in the progression of several types of cancers by enhancing many central events such as proliferation, angiogenesis, migration, and invasion (Alvarez et al. 2007, Strub et al. 2010, Alshaker et al. 2013, Pyne & Pyne 2013, Takabe & Spiegel 2014).

The importance of sphingolipids in thyroid cancer has not been studied in great detail. We have shown that S1P is without an effect on the proliferation and invasion of normal thyroid cells but induces a marked migration and invasion of ML-1 follicular thyroid cancer cells. This effect is mediated by S1P1/3, Gi/0, PI3-kinase, protein kinase C (PKC), and ERK1/2 kinase (Balthasar et al. 2006, Bergelin et al. 2010). Furthermore, we have identified hypoxia-induced factor-1α (HIF-1α) (Kalhori et al. 2013) and matrix metalloproteinases 2 and 9 (MMP2 and MMP9) (Kalhori & Törnquist 2015) as mediators of the S1P-evoked migration and invasion of ML-1 cells. Moreover, SK1 enhances migration and invasion through an autocrine mechanism in these cells (Bergelin et al. 2009, 2010). Thus, in thyroid cancer cells, S1P-evoked signaling induces an aggressive and invasive phenotype. This is strengthened by observations that SK1 is overexpressed in thyroid cancer and correlates with a high degree of thyroid malignancy and poor prognosis (Guan et al. 2011).

In almost all forms of thyroid cancer, vascular endothelial growth factor receptor 2 (VEGFR2) and the secretion of VEGF are upregulated, enhancing the development of metastasis through an autocrine mechanism (Turner et al. 2003, Vieira et al. 2005, Kim et al. 2006). Our investigations have shown a complex cross talk between VEGFR2 and S1P receptors in ML-1 cells. This communication is of crucial importance for migration (Balthasar et al. 2008, Bergelin et al. 2010).

Fingolimod (FTY720) is an immunosuppressive drug approved by both the FDA and the EMA for treatment of multiple sclerosis (MS) and is presently the only available S1P receptor-modulating drug in clinical use. It has affinity for all S1P receptors, except for S1P2 (but see Sobel et al. 2015). Exogeneous FTY720 is taken up by cells, phosphorylated by SK2 (Paugh et al. 2003), and finally secreted into the extracellular space, where it binds to S1P receptors. This results in internalization and proteasomal degradation of the S1P receptors, events highlighting the inhibitory effects of FTY720 (Oo et al. 2007). In addition to its immunosuppressive effects in the treatment of MS, FTY720 has shown promising effects as an inhibitor of cancer cell migration and proliferation. This effect is due to, for example, inhibition of several key players in these cellular events, as well as by acting as an inhibitor of SK1 (Alshaker 2013, Kunkel et al. 2013, Pyne & Pyne 2013, Selvam & Ogretmen 2013).

The incidence of thyroid cancer has increased significantly worldwide. The prognosis for many forms of thyroid cancer is very good. However, there are certain aggressive forms with extremely poor and unfavorable prognosis. The available treatments for these forms have shown poor overall effects. Thus, to overcome the difficulties in treating thyroid cancer, new approaches are needed (Haugen & Sherman 2013, Perri et al. 2014).

In this study, we have investigated the effect of FTY720 on the invasion and proliferation of thyroid cancer cells in vitro. Our results show that FTY720 efficiently inhibits both basal and S1P-evoked invasion by downregulating signaling intermediates of crucial importance for ML-1 cell migration. In addition, FTY720 attenuates the proliferation of thyroid cancer cells. Given the fact that FTY720 is already in clinical use for treatment of MS, we believe that it could be evaluated and considered for new medical indications. Thus, we suggest that FTY720 could be beneficial in the treatment of thyroid cancer.

Materials

Reagents

Dulbecco’s modified Eagle’s medium (DMEM), fatty acid-free bovine serum albumin (BSA), sodium pyruvate solution (1mM), propidium iodide (PI), Bafylomycin, MG132, 3H-thymidine, and FTY720 were purchased from Sigma-Aldrich. RMPI 1640 medium (without l-glutamine) was from Lonza (Basel, Switzerland). Fetal bovine serum (FBS), 2.5% trypsin, l-glutamine, penicillin/streptomycin, and F-12 (Ham’s nutrient medium) were from GIBCO. S1P was from Biomol (Plymouth, PA, USA). The CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit was from Promega. S1P1, S1P3, mouse VEGFR-2, PKCα and PKCβI, and Hsc70 primary antibodies were from Santa Cruz Biotechnology. Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Bio-Rad Laboratories. Antibodies against phospho- and total ERK1/2, HIF-1α (WB), β-actin, SK1, MMP2, p21, p27, and anti-rat and anti-mouse IgG were from Cell Signalling Technology. MMP9 antibodies were from Abcam. Cell culture plastic ware and human collagen type IV were from BD Biosciences. The Trans-well inserts for migration assays were from Coring. The nitrocellulose membrane was from PerkinElmer. The BCA (bicinchoninic acid) Protein Assay Reagent kit was from Pierce Biotechnology.

Methods

Cell lines

The follicular ML-1 thyroid cancer cells, provided by Dr Johann Schönberger (University of Regensburg, Germany), were cultured in DMEM supplemented with 2mM l-glutamine, 10% FBS and 100IU/mL penicillin–streptomycin (Schonberger et al. 2000). The FTC-133 follicular thyroid cancer cells were obtained from Banca Biologica e Cell Factory, National Institute for Cancer Research (Genova, Italy). The cells were grown in DMEM and Ham’s F12 medium (1:1) supplemented with 10% FBS and 2mM l-glutamine. The C643 anaplastic thyroid cancer cells, provided by Dr Nils-Erik Heldin (Karolinska Institute, Stockholm, Sweden), were cultured in DMEM supplemented with 10% FBS, 1% l-glutamine, and 1% penicillin–streptomycin. Normal human thyroid N-Thyr-Ori 3-1 cells (Culture Collections, Public Health England, Porton Down, Salisbury, UK) were grown in RPMI 1640 medium supplemented with 2mM glutamine, 10% FBS, and 1% penicillin–streptomycin. All cell lines were maintained in a humidified incubator at 37°C and 5% CO2. The cell lines not obtained from cell banks (the ML-1 and C643 cells) have been analyzed by DNA profiling by us (the ML-1 cells) and in the paper by Schweppe and coworkers (Schweppe et al. 2008). The C643 cells were obtained from the original source (Heldin), the same as Schweppe and coworkers (Schweppe et al. 2008). The FTC-133 cells were also analyzed by Schweppe and coworkers (Schweppe et al. 2008). All cell lines are authentic thyroid cancer cell lines. The cell passage never exceeded passage 28.

Western blot

Cells were treated as indicated, washed 3 times with ice-cold PBS, and lysed in cell lysis buffer (10mM Tris (pH 7.7), 150mM NaCl, 7mM EDTA, 0.5% NP-40, 0.2mM phenylmethylsulfonyl fluoride (PMSF), and 0.5%g/mL leupeptin). Thereafter, the lysates were centrifuged at 15,000g for 15min at 4°C, after which the supernatants were collected, protein concentration was determined using the BCA Protein Assay Kit, and Laemmli sample buffer was added to the samples. The samples were subjected to SDS/PAGE (8–12% polyacrylamide); thereafter, proteins were transferred onto nitrocellulose membrane by Western blotting. Next, membranes were incubated with primary antibodies targeted against specific proteins overnight, followed by incubation with secondary antibodies for 1h at room temperature. Finally, the proteins were detected by enhanced chemiluminescence, and densitometric analysis was performed using ImageJ software (http://rsb.info.nih.gov/ij/). Results were normalized against β-actin expression in each sample. In experiments where collected medium was subjected to SDS/PAGE, the results were analyzed against total protein concentrations in respective samples.

Proliferation assay

CellTiter assay

A total of 10,000 cells/well were cultured in a 96-well plate for 24h. Next, complete medium was changed to serum-free medium (SFM) supplemented with 0.2% fatty acid-free BSA for another 24h, after which cells were treated with FTY720 as indicated. Proliferation was measured using the CellTiter 96 Aqueous One Solution cell proliferation assay kit, according to the manufacturer’s instructions.

3H-thymidine incorporation assay

For this assay, 75,000 cells were cultured for 24h on 35mM as described previously. After being serum starved for 24h, the cells were treated as indicated. Four hours before ending the experiments, cells were incubated with 3H-thymidine (0.4μCi/mL). Then, the cells were washed 3 times with ice-cold PBS. After that, the cells were incubated with 5% trichloroacetic acid (TCA) for 10min followed by incubation with 0.1 N NaOH for another 10min. The radioactivity was measured by using a Wallac 1410 liquid scintillation counter (Wallac OY, Turku, Finland).

Fluorescence-activated cell sorting

Cells were grown in 10cm plates until 70% confluency was reached. Next, complete medium was changed to SFM overnight, and cells were treated with 1μM FTY720 for 24h. After being washed with ice-cold PBS and detached, 500,000 cells were suspended in PI solution (0.05mg/mL PI, 3.8μM sodium citrate, and 0.1% Triton-X-100 in PBS) and incubated for 15min at room temperature. Finally, the samples were analyzed by flow cytometry using ModFit LT 4.1 software (Verity Software House, Inc., Topsham, ME, USA).

Invasion assay

Invasion experiments were performed on 6.5mm-diameter Trans-well chambers with 8m pore size. The membranes were coated with 5g/cm2 collagen IV overnight and reconstituted with SFM for 1h at 37°C prior to the experiment. After being serum starved overnight, cells were preincubated for 1h with 1 or 10μM FTY720. A cell suspension containing 100,000 cells was added to the upper wells, and SFM with or without S1P and 10% lipid-stripped FBS (LS-FBS) was added to the lower wells. In experiments with FTY720, FTY720 was present in both chambers during the entire experiment. The cells were allowed to migrate in the incubator as indicated. Next, unmigrated cells from the topside of the membrane were wiped off with a cotton swab. The migrated cells were fixed in 2% paraformaldehyde in PBS for 10min and stained with 0.1% crystal violet in 20% methanol for 5min. The membranes were rinsed and allowed to dry. The cells were counted with 40× magnification in eight microscopic fields in a straight line bisecting the membrane.

Zymography

Cells were stimulated as indicated, conditioned medium was collected, and cells were lysed as described previously. Next, equal volumes of sample medium were mixed with loading buffer (0.1M Tris-phosphate buffer (pH 6.8) containing 20% glycerol, 6% SDS, 0.04% bromphenol blue). The samples were electrophoresed on 10% SDS–polyacrylamide gels containing 1mg/mL gelatin. Gels were then incubated in zymo buffer I (50mM Tris-HCl containing 2.5% Tween 80 and 0.02 NaN3 (pH 7.5)) for 30min, followed by incubation for another 30min in the zymo buffer II (zymo buffer I in which 1μM ZnCl2 and 5mM CaCl2 were added). Finally, to allow gelatinolytic activity, gels were incubated in zymo buffer III containing 50mM Tris-HCl, 5mM CaCl2, 1µM ZnCl2, and 0.02% NaN3 (pH 7.5) overnight at 37°C. To detect bands representing gelatinolytic activity, gels were stained with Coomassie Brilliant Blue R250 for 1–2h and destained for 30–60 min. Gels were then packed in plastic bags and scanned. Clear bands showing gelatinolytic activity were quantified by ImageJ software. MMP activity was normalized with the respective total protein concentrations in the plates.

Chick embryo chorioallantoic membrane model

The chorioallantoic membrane (CAM) model has been described elsewhere (Ossowski & Reich 1980, Björk et al. 2015). In brief, fertilized chicken eggs were placed with sharp edge down in a humidified egg incubator (37°C) and incubated for 3 days in the rocking mode. On egg development day (EDD) 4, 2-mm holes were made on the sharp edge of the eggs. The holes were covered with tape and the eggs were incubated stationary for 3 days, sharp edge up. On EDD 7, the holes were enlarged to approximately 2cm in diameter. On EDD 8, ML-1 cells were harvested, suspended in DMEM medium without supplements (1×106 cells per 10µL medium), and mixed 1:1 with ice-cold Matrigel. Then, 20µL of the cell suspension was pipetted onto the CAM within the perimeter of a silicone ring. The holes were sealed with sealing film, whereafter the eggs were placed back into the incubator. The ML-1 tumors were treated on EDD 9–11 with the control solution (100µL PBS with 0.05% DMSO) or with FTY720 (100µL PBS with 1µg FTY720 and 0.05% DMSO), respectively. The eggs were photographed and the tumor areas from EDD 9 and EDD 12 were measured using ImageJ software. Relative tumor areas were calculated by dividing the tumor area by the silicone ring area of each sample. The obtained values were used to calculate the percentage change in relative tumor area from EDD 9 to EDD 12. The normalized percentage change values were used for statistical analysis.

Scratch-wound experiment

FTC133 cells were seeded onto 96-well plates at densities of 1×105 and 1.5×105 cells per well, respectively. The cells were grown for 24h, whereafter the wounds were created by employing a wound maker tool (WoundMaker, Essen Bioscience, Ann Arbor, MI, USA). Then, the cells were washed twice with medium without supplements. Thereafter, the medium wash changed to DMEM and Ham’s F12 medium (1:1) supplemented with 5% LS-FBS and 1% penicillin–streptomycin and mitomycin C (1µg/mL) containing either the control solution (DMSO) or FTY720. The imaging was conducted with an automated live-cell imaging apparatus (Cell-IQ, CM Technologies, Tampere, Finland). The wound area analysis was conducted with the Cell-IQ Analyzer software.

Statistics

All numerical data are presented as the mean±s.e.m. for at least three independent measurements. Statistical analysis of two means was performed with Student’s t-test. Three or more means were analyzed with one-way ANOVA and Bonferroni’s post hoc test. A probability value of less than 0.05 was considered statistically significant.

Results

The effect of FTY720 on invasion of thyroid cancer cells

Our previous investigations had shown that S1P is a potent enhancer of ML-1 follicular thyroid cancer cell migration via S1P1,3, but not of normal thyroid cells (Balthasar et al. 2006, Bergelin et al. 2009, 2010). Furthermore, FTY720 has been shown to inhibit migration of both noncancerous (Matloubian et al. 2004, Lo et al. 2005, Mousseau et al. 2012) and cancerous cells (Tonelli et al. 2010, Lim et al. 2011). Thus, the aim of this study was to investigate the effect of FTY720 on invasion of thyroid cancer cells, and for this purpose, we tested several thyroid cell lines. As can be seen in Fig. 1A, the invasion of normal thyroid cells was not affected by FTY720, but the basal invasion of all thyroid cancer cells tested was decreased after incubation with 10μM FTY720 (Fig. 1B–D). Moreover, invasion of both ML-1 cells (Fig. 1E and F) and FTC-133 cells (Fig. 1G) evoked by 100nM S1P was attenuated by FTY720. Next, we investigated if the phosphorylated form of FTY720 (p-FTY720) could show similar effects in ML-1 cells. Similar to FTY720, p-FTY720 (1μM and 1h preincubation) was able to attenuate the S1P-evoked invasion of ML-1 cells (Fig. 1H). Taken together, FTY720 inhibits both basal and S1P-induced invasion of thyroid cancer cells.

Figure 1
Figure 1

The effect of FTY720 on invasion of thyroid cancer cells. (A) Lack of an effect of 10μM FTY720 on invasion (17h) of N-thy-Ori cells. (B–D) Invasion of anaplastic C643 cells (6 h), follicular ML-1 cells (6h), and follicular FTC-133 (20h) thyroid cancer cells is blocked by 10μM FTY720. (E–F) FTY720 attenuates S1P-evoked invasion of ML-1 cells. Cells were preincubated for 1h with 1μM FTY720 (E) or 10μM FTY720 (F) and allowed to invade toward 100 nM S1P and 10% LS-FBS for 4–6h. The scale bar in the photos are 50µm. (G) FTY720 (10μM and 1h preincubation) attenuates S1P-evoked invasion of FTC-133 cells (20h). (H) Cells were preincubated for 1h with 1μM p-FTY720 and allowed to invade toward 100nM S1P and 10% LS-FBS for 4h. p-FTY720 was present in both the upper and lower chambers during the entire experiments. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control. °indicates a statistically significant difference between S1P treatment and FTY720+S1P. #indicates a statistically significant difference between FTY720 treatment and FTY720+S1P.

Citation: Endocrine-Related Cancer 23, 5; 10.1530/ERC-16-0050

FTY720 causes lysosomal degradation of S1P1

Previous studies have shown that FTY720 has the ability to downregulate and degrade S1P1,3 in the proteasome (Oo et al. 2007, Mousseau et al. 2012). As FTY720 was able to inhibit invasion in our experiments, we thus tested whether FTY720 could affect the expression of S1P1,3 in ML-1 cells. We treated the cells with FTY720 (0.1, 1, 3, 5, and 10μM) for different times (1, 3, 6, and 24h), and could show that FTY720 was able to downregulate S1P1 in a concentration- and time-dependent manner (Fig. 2A–B). However, to downregulate S1P3, the cells had to be incubated with 10μM FTY720 (the highest concentration tested) for 24h (results not shown). Next, we asked if FTY720 caused degradation of S1P1; therefore, we tested the effect of the lysosome inhibitor Bafilomycin and the proteasome inhibitor MG132. Bafilomycin (100nM, 1h preincubation) was able to abolish the downregulation of S1P1 evoked by 1μM FTY720 (Fig. 2C). Interestingly, MG132 increased the expression of S1P1. However, MG132 was not able to abolish the response caused by 1μM FTY70 (Fig. 2D). To further confirm our results on the role of proteasome in degradation of S1P1 caused by FTY720, we performed ubiquitination studies. In these studies, we were not able to detect any ubiquitination of the receptor (results not shown). Collectively, FTY720 downregulates S1P1 by inducing lysosomal degradation. In contrast to its rapid effects at low concentrations on S1P1, FTY720 requires longer incubations and higher concentrations to be able to downregulate S1P3.

Figure 2
Figure 2

FTY720 causes lysosomal degradation of S1P1. (A–B) The expression of S1P1 is downregulated by FTY720 after 5h in a concentration- (A) and time-dependent (B) manner. (C) The lysosomal inhibitor Bafilomycin (100nM, 1h preincubation) attenuates downregulation of S1P1 evoked by1μM FTY720. The cells were incubated with FTY720 for 24h. (D) Lack of an effect of the proteasome inhibitor MG132 (10μM, 1h preincubation) on the FTY720-evoked (1μM, 24h) downregulation of S1P1. The upper panel shows Western blot pictures and the lower panel shows densitometric analyses. The arrows in C and D indicate the S1P1 protein. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control. °indicates a statistically significant difference between FTY720 treatment and FTY720+inhibitor. #indicates a statistically significant difference between inhibitor treatment and FTY720+inhibitor.

Citation: Endocrine-Related Cancer 23, 5; 10.1530/ERC-16-0050

The effects of FTY720 on signaling intermediates in ML-1 cells

Previously, we showed that the S1P-induced migration of ML-1 cells is mediated by PKC isoforms, ERK1/2, SK1 (Balthasar et al. 2008, Bergelin et al. 2010), and HIF-1α (Kalhori et al. 2013). Moreover, a cross talk between S1P1 and VEGFR2 of crucial importance for migration exists in ML-1 cells. Hence, we investigated the effect of FTY720 (0.1, 1, and 10μM) for different times (1, 3, 6, and 24h) on these signaling intermediates. Our results showed that 1μM FTY720 had no effect on the expression of neither PKCα nor PKCβI (results not shown). However, after incubating the cells with 10µM FTY720, the expression of PKCα was downregulated in a time-dependent manner, whereas the expression of PKCβI was significantly downregulated after 24h only (Fig. 3A). Interestingly, FTY720 was without an effect on both the total amount of ERK1/2 and the basal phosphorylation of ERK1/2 (results not shown). However, FTY720 attenuated the S1P-evoked phosphorylation of ERK1/2 (Fig. 3B). We also investigated the expression of HIF-1α but were not able to show a statistically significant decrease of HIF-1α expression after incubation with 10μM FTY720 (Fig. 3C). An important observation was that FTY720 decreased the expression of VEGFR2 in a time- and concentration-dependent fashion (Fig. 3D–E).

Figure 3
Figure 3

The effect of FTY720 on signaling intermediates in ML-1 cells. (A) 10μM FTY720 attenuates the expression of PKCα in a time-dependent manner and PKCβI expression after 24h. (B) FTY720 (1h preincubation and 1μM) attenuates phosphorylation of ERK1/2 evoked by S1P (100nM and 30 min). (C) Lack of a significant effect ofFTY720 (10μM) on HIF-1α expression. (D–E) FTY720 inhibits the expression of VEGFR2 in a concentration- (FTY720, 5 h, and 0.1–10μM) and time- dependent (FTY720, 10μM and 1–24h) fashion. The upper panel shows Western blot pictures and the lower panel shows densitometric analyses. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control. °Indicates a statistically significant difference between S1P treatment and FTY720+S1P. #indicates a statistically significant difference between FTY720 treatment and FTY720+S1P.

Citation: Endocrine-Related Cancer 23, 5; 10.1530/ERC-16-0050

We have previously shown that SK1 is of importance in regulating ML-1 cell migration (Bergelin et al. 2009), and previously, Tonelli and coworkers showed that FTY720 downregulated SK1 (Tonelli et al. 2010). We were, however, not able to show any effects of FTY720 on the levels of SK1 in ML-1 cells (results not shown). Taken together, FTY720 inhibits signaling intermediates of crucial importance for ML-1 cell invasion.

FTY720 modulates MMP2 and MMP9

Previous studies have shown that FTY720 inhibits MMP2 and MMP9 in human glioblastoma cell lines (Zhang et al. 2014). In addition, we showed earlier that S1P via S1P1,3 induced the secretion of MMP2 and MMP9 in ML-1 cells, and that these MMPs are of importance for S1P-induced invasion (Kalhori & Törnquist 2015). In accordance with previous results, FTY720 attenuated the S1P-evoked upregulation of MMP2 after 48h (Fig. 4A), and the S1P-evoked secretion of both MMP2 and MMP9 (Fig. 4B). Interestingly, FTY720 per se did not decrease the secretion of MMP2 or MMP9 (Fig. 4B). However, an important observation was that when we exposed ML-1 cells to 10 μM FTY720 for 24h, FTY720 decreased the activity of the secreted MMP2 and MMP9 (Fig. 4C). In conclusion, FTY720 attenuates S1P-evoked secretion of MMP2 and MMP9 as well as MMP2 expression. In addition, FTY720 decreases MMP2 and MMP9 activity.

Figure 4
Figure 4

FTY720 attenuates MMP2 and MMP9 secretion. (A) Cells were stimulated with S1P (100nM for 48h) in the presence or absence of 10μM FTY720 (1h preincubation) and the expression of MMP2 was measured. (B) FTY720 (10μM, 1h preincubation) attenuates S1P-evoked (100nM, 4h) MMP2 and MMP9 secretion. In (A–B), FTY720 was present during the entire experiment. (C) Incubating the cells with 10µM FTY720 for 24h decreased basal MMP2 and MMP9 activity. The upper panel shows Western blot or zymography pictures and the lower panel shows densitometric analyses. The amount of secreted MMPs was normalized against total protein concentrations in respective samples. The results are the mean±s.e.m., n≥3. *P<0.05 and **P<0.01 indicate a statistically significant difference between FTY720 or S1P treatment and respective control.

Citation: Endocrine-Related Cancer 23, 5; 10.1530/ERC-16-0050

FTY720 inhibits thyroid cancer cell proliferation

As FTY720 decreased the invasion of the thyroid cancer cells, we investigated the effect of different concentrations of FTY720 on the proliferation of normal thyroid cells and several thyroid cancer cell lines. Results obtained using the CellTiter assay showed that FTY720 was able to attenuate proliferation of normal N-Thyr-Ori 3-1 and anaplastic C643 cells in a concentration-dependent manner (Fig. 5A–B). By performing both CellTiter and 3H-thymidine incorporation assays, we could show that FTY720 is able to inhibit proliferation of ML-1 and FTC-133 cells in a concentration-dependent fashion (Fig. 5C–D, CellTiter; Fig. 5E–F, 3H-thymidine incorporation). Next, by performing FACS analysis, we found that FTY720 inhibited proliferation of ML-1 and FTC-133 cells by accumulating cells in the G1 phase and decreasing the amount of cells in the S phase of the cell cycle (Fig. 5G–H). Next, we investigated the expression of proteins involved in cell cycle regulation and found that incubating the cells with 1 µM FTY720 for 24h induced an upregulation of p21 and p27 in ML-1 cells (Fig. 5I). To investigate the significance of p21 and p27 in proliferation, ML-1 cells were transfected with siRNA against p21 and p27. The results showed that the basal proliferation was significantly enhanced and the 3µM FTY720-induced inhibition of proliferation was attenuated (Fig. 5J–K).

Figure 5
Figure 5

FTY720 inhibits thyroid cell proliferation. FTY720 attenuated in a concentration-dependent manner the proliferation of (A) N-Thy-Ori thyroid cells, (B) anaplastic C643 cancer cells, (C) follicular ML-1 cancer cells, and (D) follicular FTC-133 cancer cells using the CellTiter analysis methods. The cells were incubated with FTY720 for 24h. (E–F) FTY720 blocked proliferation of follicular ML-1 and FTC-133 cells investigated with the 3H-thymidine incorporation assay. (G–H) Incubating with 1μM FTY720 for 24h accumulated ML-1 and FTC-133 cells in the G1 phase and decreased cells in the S phase of the cell cycle. (I) Incubating ML-1 cells with 1µM FTY720 for 24h upregulated the expression of p21 and p27. (J) ML-1 cells transfected with control siRNA or p21/p27 siRNA. The representative blots show the expression of p21 and p27 compared to the control. Hsc70 was used as loading control. (K) Treatment with p21+p27 siRNA attenuates the effect of FTY720 on the proliferation of ML-1 cells. Proliferation was measured using the CellTiter proliferation assay kit. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, ¤¤P<0.01, ¤¤¤P<0.001, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control.

Citation: Endocrine-Related Cancer 23, 5; 10.1530/ERC-16-0050

To increase the efficiency of therapeutic drugs, combined therapy is becoming an attractive tool in the treatment of different diseases. Therefore, we tested both FTY720 and p-FTY720 (1μM) in combination with doxorubicin (0.1, 0.5, 1, and 5μM) to investigate whether an additive or synergistic inhibitory effect on proliferation could be obtained. However, we were not able to see any additive or synergetic effects of these compounds (results not shown). Taken together, FTY720 inhibits proliferation of thyroid cancer cell lines. In ML-1 cells, this inhibition is due to an increase in p21 and p27, and an arrest in the G1 phase of the cell cycle.

FTY720 inhibits thyroid cancer cell growth in the chick CAM model and cell migration in the scratch-wound assay

To assess the antiproliferative effect of FTY720 in vivo, we employed the chick CAM model (Ribatti 2014). ML-1 cells were implanted in to the CAM and allowed to develop into a tumor. The growth of these tumors was slightly but significantly attenuated by FTY720 treatment (Fig. 6A–B). The effect of FTY720 on FTC133 cell migration in a scratch-wound model was investigated, and the results showed a significant attenuation in wound healing upon 3μM FTY720 treatment (Fig. 6C–D).

Figure 6
Figure 6

FTY720 inhibits the growth of ML-1 cells in a chick embryo CAM model and blocks the migration of FTC133 cells in a scratch-wound experiment. (A) Representative images of control and FTY720-treated ML-1 cell tumors in the CAM. (B) Quantification of the relative change in ML-1 cell tumor area. The results are mean±s.e.m., n=13 eggs (control), 14 eggs (FTY720 treated), *P<0.05. (C) Representative images of FTC133 cells in a scratch-wound experiment. (D) Quantification of the wound closure at the 48-h time point. The results are mean±s.e.m., n=4, *P<0.05.

Citation: Endocrine-Related Cancer 23, 5; 10.1530/ERC-16-0050

Discussion

Currently, not many studies address the importance of sphingolipid signaling in thyroid cancer progression, although we have shown that S1P and SK1 are important players in regulating many essential cellular events in different types of thyroid cancer cells (Balthasar et al. 2006, Bergelin et al. 2009, 2010, Kalhori et al. 2013, Törnquist 2013, Kalhori & Törnquist 2015). In addition, our in vitro results have been corroborated by investigations on human thyroid samples (Guan et al. 2011). In this study, we have investigated a novel approach to curtail the proliferation and invasion of thyroid cancer cells. We evaluated the effect of FTY720, a drug that is approved by both the FDA and the EMA to be clinically used for treatment of MS on thyroid cancer cell proliferation and invasion.

Our results showed that FTY720 did not affect the invasion of normal thyroid cells; however, the basal invasion of all thyroid cancer cell lines tested and the S1P-evoked invasion of ML-1 and FTC-133 cells were attenuated by FTY720. Furthermore, p-FTY720 was able to block the S1P-induced invasion of ML-1 cells. FTY720 also potently attenuated migration of FTC-133 cells in a scratch-wound assay. Our results are in line with previously published data, showing that FTY720 is able to inhibit migration of noncancerous cells (Mousseau et al. 2012) as well as several cancer cell types, including breast cancer cells (Tonelli et al. 2010, Lim et al. 2011), liver cancer cells (Li et al. 2012), and prostate cancer cells (Tonelli et al. 2010). Thus, we suggest that FTY720 acts as an anti-invasive drug in thyroid cancer cells.

Results published earlier by Mousseau and coworkers showed that FTY720, at low concentrations (100nM), caused both internalization and downregulation of S1PR1,3 in vascular smooth muscle cells (Mousseau et al. 2012). Our study showed that FTY720 clearly downregulated S1P1 in a concentration- and time-dependent manner. Surprisingly, low concentration of FTY720 could not downregulate S1P3 in ML-1 cells. However, we were able to see decreased expression of S1P3 after incubating the cells with 10µM FTY720 for 24h.

Oo and coworkers showed that FTY720 caused proteasomal degradation of S1PR1,3 (Oo et al. 2007). However, our findings suggested that FTY720 induced degradation of S1P1 in the lysosome, but the normal maintenance of S1P1 in ML-1 cells occurs through proteasomal degradation. The observation that FTY720 is not able to degrade S1P1 in the proteasome was strengthened by the fact that we could no detect any ubiquitination of S1P1 in our investigations. Thus, based on our results, we conclude that FTY720 is a potent inhibitor of S1P1 and that it causes lysosomal degradation of the receptor. High concentrations of FTY720 are required to downregulate S1P3, highlighting the importance of cell specificity, and concentrations used in different studies.

S1P induces migration and invasion of ML-1 cells through several mediators, such as ERK1/2, PKCα and PKCβI, and HIF-1α. Furthermore, S1P communicates with VEGFR2, which is essential for migration (Balthasar et al. 2006, Bergelin et al. 2009, Kalhori et al. 2013, Kalhori & Törnquist 2015). In this study, we were able to show that FTY720 downregulated both PKCα and PKCβI, but we were not able to see a statistically significant decrease in HIF-1α expression. Moreover, FTY720 was also able to attenuate S1P-evoked phosphorylation of ERK1/2. Similarly, Zhang and coworkers could show that FTY720 inhibited ERK1/2 in human glioblastoma cell lines (Zhang et al. 2014). Interestingly, Tonelli and coworkers showed that FTY720, albeit at lower concentrations, was able to downregulate SK1 expression (Tonelli et al. 2010). We were, however, not able to see this in our study. Furthermore, FTY720 inhibited the expression of VEGFR2 in a time- and concentration-dependent fashion.

We have previously shown that S1P enhances MMP2/9 secretion and the expression of intracellular MMP2 in ML-1 cells (Kalhori & Törnquist 2015). In this investigation, we show that these effects of S1P can be attenuated by FTY720. Furthermore, although FTY720 per se did not attenuate the secretion of MMP2/9, the activity of MMP2/9 in collected media was attenuated by FTY720. Our results are in line with the results of Zhang and coworkers, who demonstrated that FTY720 inhibited MMP2 and MMP9 expression and activity in human glioblastoma cell lines (Zhang et al. 2014). Taken together, FTY720 is able to downregulate signaling intermediates of importance for ML-1 cell invasion and migration.

FTY720 has been proven to inhibit proliferation of both normal and cancerous cells (Mousseau et al. 2012, Permpongkosol et al. 2002). In our study, FTY720 was able to block proliferation significantly in a concentration-dependent fashion with two methods tested. Of these, the 3H-thymidine incorporation assay proved to be a more sensitive method. Furthermore, using the CAM model, we could show that FTY720 slightly, but significantly, attenuated ML-1 tumor growth in ovo. In a previous report, Permpongkosol and coworkers showed that FTY720 inhibited proliferation of human prostate carcinoma DU145 cells by inducing apoptosis and arresting cells in the G1 phase of the cell cycle (Permpongkosol et al. (2002). In our study, we could also see an accumulation of both ML-1 cells and FTC-133 cells in the G1 phase. Furthermore, we observed an upregulation of p21 and p27, two important regulators of the G1 phase. Nevertheless, we were not able to strengthen the effect of FTY720 on proliferation by cotreatment with doxorubicin.

In conclusion, we suggest that FTY720, which is currently in clinical use for the treatment of MS, is an inhibitor of thyroid cancer cell proliferation and invasion. Perri and coworkers and Manfredi and coworkers described in recent reviews, and Dicitore and coworkers in a recent report, that there are, in fact, several small-molecule inhibitors targeted to thyroid cancer, albeit they have mostly been proven to have small over all effects (Perri et al. 2014, Manfredi et al. 2015, Dicitore et al. 2016). Thus, novel approaches to curtail and stop the progression of aggressive thyroid cancers to a metastatic and terminally fatal disease are urgently warranted. Therefore, we suggest that FTY720 could be beneficial in the treatment of thyroid cancer.

Declaration of statement

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

The study was supported in part by the Sigrid Juselius Foundation, the Liv och Hälsa Foundation, The Academy of Finland, the Magnus Ehrnrooth foundation, the Swedish cultural foundation, the Minerva Foundation for Medical Research (all in Helsinki, Finland), and the Centre of Excellence in Cell Stress and Molecular Ageing (Åbo Akademi University), the Doctoral Network in Molecular Biosciences (Åbo Akademi University), and the Receptor Research Program (Åbo Akademi University and the University of Turku), all in Turku (Finland), which are gratefully acknowledged.

Acknowledgments

The authors thank Drs Johann Schönberger, Nils-Erik Heldin, and John Copeland for generously providing cell lines, and Erik Niemelä and Dr John Eriksson for invaluable advice on the use of the CAM model.

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    The effect of FTY720 on invasion of thyroid cancer cells. (A) Lack of an effect of 10μM FTY720 on invasion (17h) of N-thy-Ori cells. (B–D) Invasion of anaplastic C643 cells (6 h), follicular ML-1 cells (6h), and follicular FTC-133 (20h) thyroid cancer cells is blocked by 10μM FTY720. (E–F) FTY720 attenuates S1P-evoked invasion of ML-1 cells. Cells were preincubated for 1h with 1μM FTY720 (E) or 10μM FTY720 (F) and allowed to invade toward 100 nM S1P and 10% LS-FBS for 4–6h. The scale bar in the photos are 50µm. (G) FTY720 (10μM and 1h preincubation) attenuates S1P-evoked invasion of FTC-133 cells (20h). (H) Cells were preincubated for 1h with 1μM p-FTY720 and allowed to invade toward 100nM S1P and 10% LS-FBS for 4h. p-FTY720 was present in both the upper and lower chambers during the entire experiments. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control. °indicates a statistically significant difference between S1P treatment and FTY720+S1P. #indicates a statistically significant difference between FTY720 treatment and FTY720+S1P.

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    FTY720 causes lysosomal degradation of S1P1. (A–B) The expression of S1P1 is downregulated by FTY720 after 5h in a concentration- (A) and time-dependent (B) manner. (C) The lysosomal inhibitor Bafilomycin (100nM, 1h preincubation) attenuates downregulation of S1P1 evoked by1μM FTY720. The cells were incubated with FTY720 for 24h. (D) Lack of an effect of the proteasome inhibitor MG132 (10μM, 1h preincubation) on the FTY720-evoked (1μM, 24h) downregulation of S1P1. The upper panel shows Western blot pictures and the lower panel shows densitometric analyses. The arrows in C and D indicate the S1P1 protein. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control. °indicates a statistically significant difference between FTY720 treatment and FTY720+inhibitor. #indicates a statistically significant difference between inhibitor treatment and FTY720+inhibitor.

  • View in gallery

    The effect of FTY720 on signaling intermediates in ML-1 cells. (A) 10μM FTY720 attenuates the expression of PKCα in a time-dependent manner and PKCβI expression after 24h. (B) FTY720 (1h preincubation and 1μM) attenuates phosphorylation of ERK1/2 evoked by S1P (100nM and 30 min). (C) Lack of a significant effect ofFTY720 (10μM) on HIF-1α expression. (D–E) FTY720 inhibits the expression of VEGFR2 in a concentration- (FTY720, 5 h, and 0.1–10μM) and time- dependent (FTY720, 10μM and 1–24h) fashion. The upper panel shows Western blot pictures and the lower panel shows densitometric analyses. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control. °Indicates a statistically significant difference between S1P treatment and FTY720+S1P. #indicates a statistically significant difference between FTY720 treatment and FTY720+S1P.

  • View in gallery

    FTY720 attenuates MMP2 and MMP9 secretion. (A) Cells were stimulated with S1P (100nM for 48h) in the presence or absence of 10μM FTY720 (1h preincubation) and the expression of MMP2 was measured. (B) FTY720 (10μM, 1h preincubation) attenuates S1P-evoked (100nM, 4h) MMP2 and MMP9 secretion. In (A–B), FTY720 was present during the entire experiment. (C) Incubating the cells with 10µM FTY720 for 24h decreased basal MMP2 and MMP9 activity. The upper panel shows Western blot or zymography pictures and the lower panel shows densitometric analyses. The amount of secreted MMPs was normalized against total protein concentrations in respective samples. The results are the mean±s.e.m., n≥3. *P<0.05 and **P<0.01 indicate a statistically significant difference between FTY720 or S1P treatment and respective control.

  • View in gallery

    FTY720 inhibits thyroid cell proliferation. FTY720 attenuated in a concentration-dependent manner the proliferation of (A) N-Thy-Ori thyroid cells, (B) anaplastic C643 cancer cells, (C) follicular ML-1 cancer cells, and (D) follicular FTC-133 cancer cells using the CellTiter analysis methods. The cells were incubated with FTY720 for 24h. (E–F) FTY720 blocked proliferation of follicular ML-1 and FTC-133 cells investigated with the 3H-thymidine incorporation assay. (G–H) Incubating with 1μM FTY720 for 24h accumulated ML-1 and FTC-133 cells in the G1 phase and decreased cells in the S phase of the cell cycle. (I) Incubating ML-1 cells with 1µM FTY720 for 24h upregulated the expression of p21 and p27. (J) ML-1 cells transfected with control siRNA or p21/p27 siRNA. The representative blots show the expression of p21 and p27 compared to the control. Hsc70 was used as loading control. (K) Treatment with p21+p27 siRNA attenuates the effect of FTY720 on the proliferation of ML-1 cells. Proliferation was measured using the CellTiter proliferation assay kit. The results are the mean±s.e.m., n≥3. *P<0.05, **P<0.01, ¤¤P<0.01, ¤¤¤P<0.001, and ***P<0.001 indicate a statistically significant difference between FTY720 treatment and respective control.

  • View in gallery

    FTY720 inhibits the growth of ML-1 cells in a chick embryo CAM model and blocks the migration of FTC133 cells in a scratch-wound experiment. (A) Representative images of control and FTY720-treated ML-1 cell tumors in the CAM. (B) Quantification of the relative change in ML-1 cell tumor area. The results are mean±s.e.m., n=13 eggs (control), 14 eggs (FTY720 treated), *P<0.05. (C) Representative images of FTC133 cells in a scratch-wound experiment. (D) Quantification of the wound closure at the 48-h time point. The results are mean±s.e.m., n=4, *P<0.05.

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    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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