The human thyroid cancer cell lines SW579 (ATCC^® HTB-107™) and TT (ATCC^® CRL-1803™) were purchased from the American Type Culture Collection (ATCC). K1 cells and FTC133 cells were obtained from Guangzhou Cellcook Biotech Co., Ltd. The STR profiles obtained for the K1 cell in the present study match the K1 STR profile provided by the European Collection of Authenticated Cell Cultures for those loci tested in common (Table SI). The SW579 cell line is derived from thyroid squamous cell carcinoma, a different type of thyroid cancer, TT cells are derived from MTC, K1 cells from PTC and the FTC133 cell line is derived from FTC.

K1, SW579, FTC133 and TT cells were cultured in DMEM, L-15, RPMI-1640 and F-12K media (Gibco; Thermo Fisher Scientific, Inc.), respectively, supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.). The cells were maintained at 37°C in a humidified atmosphere (5% CO₂). The transfection reagent Simple-Fect was purchased from Signaling Dawn Biotech, and geneticin (G418) antibiotic was acquired from Sigma-Aldrich (Merck KGaA). The pcDNA3-HA-TOPK (8 µg) and empty pcDNA3 plasmid were transfected into K1 cells (80% confluency, 8×10⁶ cells/10 cm² dish) according to the manufacturer's protocol. Sulfasalazine was purchased from Shanghai Zhongxi Three-dimensional Pharmaceutical Co., Ltd. and phenformin hydrochloride (PFH) from Jiangsu Yabang Epson Pharmaceutical Co., Ltd.

Cells were harvested and lysed in radioimmunoprecipitation assay buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, 5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT) and 1% Triton X-100]. The samples were sonicated 3 times on ice for 15 sec and centrifuged at 9,550 × g at 4°C for a further 15 min. The proteins were quantified using Bradford assay reagent (Bio-Rad Laboratories, Inc.), 40–60 µg separated using SDS-PAGE with a 10% gel and transferred to a PVDF membrane (EMD Millipore). The membranes were blocked with 5% non-fat milk or 5% BSA (BioSharp, Inc.) for 30 min at room temperature and incubated with primary antibody (Mouse anti-TOPK (cat. no., sc-393313; Santa Cruz Technology, Inc.; dilution, 1:1,000); anti-β-actin (cat. no., sc-47778; Cruz Technology, Inc.; dilution, 1:1,000) Anti-total-protein kinase B (T-AKT; cat. no., 9272s; Cell Signaling Technology, Inc.; dilution, 1:1,000), anti-phospho-AKT (p-AKT; S473; cat. no., 9271s; Cell Signaling Technology, Inc.; dilution, 1:500), anti-histone H3 XP rabbit monoclonal (mAb; cat. no., 4499; Cell Signaling Technology, Inc.; dilution, 1:1,000) and anti-phospho-histone H3 XP rabbit mAb (p-H3; cat. no., 3377s; Cell Signaling Technology, Inc.; dilution, 1:500) at 4°C overnight. The membranes were subsequently incubated with HRP-conjugated secondary antibody (Rabbit IgG; cat. no., E030120-2; dilution, 1:3,000; Mouse IgG; cat. no., E030110-02; dilution, 1:3,000; EarthOx Life) for 1 h at room temperature, and the protein bands were visualized using chemiluminescence (Bio-Rad Laboratories, Inc.). All experiments were repeated in triplicate.

The thyroid carcinoma tissue arrays (cat. nos. BCC15014, TH961, TH641, TH242 and TH208) were purchased from Xi'an Elena Bio Co., Ltd. A total of 132 patients with thyroid cancer, including 44 males and 88 females, aged 20–86 years (median age, 48.43 years) provided samples for the tissue microarray; 56 tissues were from papillary carcinoma, 18 from follicular carcinoma, 42 from medullary carcinoma and 16 from undifferentiated carcinoma. There were also 11 tissues from cases of thyroid adenoma, 18 normal thyroid tissue samples and 1 normal testicular tissue samples. Detailed information on patients is shown in Table I. The microarray tissues were deparaffinized in xylene and rehydrated in ethanol; high pressure repair with citrate buffer (pH 6.0) for 1.5 min was conducted, followed by endogenous peroxidase inactivation using hydrogen peroxide (0.3%) for 10 min at room temperature. The sections were washed 3 times with PBS and incubated with an anti-TOPK antibody (dilution, 1:200) at room temperature for 1 h. Biotin-labeled secondary antibody (DAKO; Agilent Technologies, Inc.; cat. no., K5007; dilution, 1:1,000) was added for 30 min at room temperature, and the sections were stained with 3,3′-diaminobenzidine, counterstained with hematoxylin for 5 min at room temperature, dehydrated and mounted. Positive staining was distinguished as brownish-yellow particles located in the cytoplasm and nuclei. A total of 10 high-power fields were randomly selected from each section and observed under a light microscope (magnification, ×10). A total of 10 high-power fields were randomly selected from each section, and the scores were determined according to the staining intensity and percentage of positive cells. The hot-spot method was used for semi-quantification of immunohistochemical expression scores (35,36). Each case was evaluated by one observer and subsequently reviewed by another observer using the semi-quantitative classifications proposed by Yuan et al (35) and Montgomery et al (36). The semi-quantified positive expression index (PEI, 0–12) of TOPK was calculated based on the percentage of positivity of tumor cells (1, 0–25%; 2, 26–50%; 3, 51–75%; and 4, 76–100%), staining intensity score (0, none; 1, weak; 2, moderate; 3, intense), and an overall score (0–12) calculated for each case by multiplying percentage score with intensity score.

To estimate cell viability, 5×10³ cells/well were seeded in 96-well plates and cultured for 24, 48, 72 and 96 h. MTT was added to each well, and the cells were incubated for 4 h at 37°C. Following incubation, the formazan crystals were dissolved in 150 ml dimethyl sulfoxide, and absorbance was determined at a wavelength of 490 nm within 10 min. To assess sulfasalazine inhibition, 5×10³ cells/well were seeded in 96-well plates and cultured for 24 h; fresh medium containing different concentrations of sulfasalazine was applied, and cells were cultured for a further 72 h. Sulfasalazine cytotoxicity was measured using an MTT assay as described above. The experiments were performed in triplicate, and the mean absorbance values were calculated with non-treated cells as the negative control.

A total of 1×10⁴ cells/well were seeded in a 6-well plate; sample cells were treated with different concentrations of sulfasalazine, while control cells were left untreated. The cells were cultured in 1 ml Basal Medium Eagle agar (0.33%; Sigma-Aldrich; Merck KGaA) containing 10% FBS, 2 mM L-glutamine and 25 µg/ml gentamicin, maintained at 37°C (5% CO₂) for 5–10 days. Analysis of the resultant colonies under a white light, 4× microscope.

Stable cell lines were grown in 6-well plates and serum-starved overnight. To create a wound, the cell monolayer was scraped using a sterile pipette tip. The cells were washed with media and incubated in growth medium with or without inhibitors for 0, 24, 30 or 36 h. The cells were imaged under a light microscope (magnification, ×10), and each assay was repeated at least three times.

Active TOPK (EMD Millipore) phosphorylates and subsequently activates its downstream substrate histone H3. Active TOPK (0.2 mg) was incubated with sulfasalazine in 1X kinase buffer [25 mM Tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na₃VO₄, 10 mM MgCl₂ and 5 mM MnCl₂] at 32°C for 20 min. Inactive histone H3 (4 µg) and 100 µM ATP were added and the reaction was incubated at 32°C for 1.5 h. The reactions were treated with 5X SDS sample buffer and analyzed by western blotting, as aforementioned.

Recombinant TOPK was labeled with the Monolith NT™ Protein Labeling Kit RED (NanoTemper Technologies GmbH) according to the manufacturer's protocol. His tag labeled TOPK was used at 250 nM. The samples were diluted in 20 mM HEPES (pH 7.4) supplemented with 0.05% (v/v) Tween-20. Sulfasalazine was dissolved in 0.4% NaHCO₃ to a concentration of 50 mM and serially diluted from a starting concentration of 4 mM. Samples were incubated for 10 min at room temperature, and loaded into Monolith NT standard-treated capillaries; thermophoresis was measured at 25°C following a 30 min incubation on a Monolith NT.115 instrument (NanoTemper Technologies GmbH). The laser power was set to 20 or 40%, with 30 sec ‘on’-time, and an LED power of 100%. The dissociation constant (Kd) was distinguished using NTAnalysis software (NanoTemper Technologies GmbH) (37).

Sulfasalazine-conjugated and un-conjugated Sepharose^® 4B beads were prepared as previously reported (38). The Sepharose beads were incubated with His tag labeled recombinant TOPK protein in reaction buffer [50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP-40, 2 µg/ml BSA, 0.02 mM PMSF and 1 µg/ml protease inhibitor cocktail (Roche Applied Science)] with gentle rotation at 4°C overnight. The beads were subsequently rinsed 5 times in wash buffer [50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP-40 and 0.02 mM PMSF], and the bead-bound proteins were analyzed using western blotting with an anti-TOPK antibody, as aforementioned.

The crystal structure of IRAK-4 kinase (PDB code: 2NRU) was used as a template, and the homology model of human TOPK (Accession No.: NP_060962) was used with MODELLER, an automated homology modeling program (39,40). The subset zdd (ZINC Drug Database), which includes all commercially approved drugs and nutraceuticals worldwide, was downloaded as input from the ZINC as a docking input for the mol2 file (41). Docking was performed on the Intel i7 4960 using the ICM 3.8.1 modeling software processor (MolSoft L.L.C.) (42). Ligands that bind pocket residues were selected using graphical tools in ICM software, to create a boundary docking search. In docking calculations, the potential energy map of the receptor is calculated using default parameters. These compounds are imported into ICM and an index file is created. Conformation sampling is based on the Monte Carlo program, and finally the lowest energy and the most favorable orientation ligands were selected.

Clinical statistical analysis (Tables II and III) was conducted using SPSS version 13.0 software (SPSS Inc.), and the data were compared by χ² analysis. All other statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software, Inc.). Analysis of multiple groups was performed using one-way ANOVA and Tukey's post hoc test. Student's t-test was used to compared between two groups. All data are presented as the mean ± standard deviation, and P<0.05 was considered to indicate a statistically significant difference.

The expression level of TOPK was established in 132 thyroid cancer, 11 thyroid adenoma and 18 normal thyroid samples, and 1 testicular tissue sample using immunohistochemistry (Fig. 1). Expression levels of TOPK were scored from 0 to 12 according the definition described in the materials and methods section. PEI of 7–12 of TOPK for well-differentiated thyroid cancer (DTC) was 37.84%, and that for poorly differentiated thyroid cancer (MTC and ATC) was 68.97%, as presented in in Table II. There was a significant association between the pathological types of thyroid cancer and the intensity of TOPK (P<0.001). For thyroid cancer clinical stages I–II, PEI of 7–12 for TOPK is 43.59%, and that for clinical stages III–IV is 62.96% shown in Table III. The present results identified a significant association between TOPK expression and clinical stage of thyroid carcinoma (P=0.026). These results indicated that the high expression level of TOPK in patients with thyroid carcinoma is associated with pathological type and clinical stage of disease. This suggests that TOPK may be used as an index to evaluate malignant thyroid nodules.

The expression levels of TOPK were determined in different thyroid cancer cell lines (K1, SW579, TT and FTC133 cells), and the results revealed that TOPK expression level was lowest in K1 cells derived from a metastasis of PTC (Fig. 2A). PTC is the most common type of thyroid cancer, and therefore, the effects of TOPK-overexpression in K1 cells were investigated. A stable cell line overexpressing TOPK (K1-TOPK) was established to observe the effects on proliferation and metastasis of PTC-derived cells (Fig. 2B). TOPK overexpression significantly increased the proliferation of K1-TOPK cells compared with control cells (Fig. 2C; P<0.001). Anchorage-independent growth is a hallmark of in vitro transformed and cancer cells, thus anchorage-independent colony formation ability was assessed using a soft agar assay. The number and size of the colonies formed by K1-TOPK cells was greater compared with those of the control group (Fig. 2D) indicating that increased TOPK expression may promote thyroid cancer cell proliferation. TOPK promotion of metastasis in thyroid cancer cells was also assessed using a wound-healing assay, in which the overexpression of TOPK resulted in a marked increase in cell migration (P<0.001; Fig. 2E). A previous study confirmed that TOPK is involved in the MAPK pathway, and that this enhances cell migration by modulating PI3K/PTEN/AKT signaling (28). The MAPK and PI3K/AKT pathways are associated with molecular pathogenesis in thyroid cancer (43) and as such, the level of p-AKT was detected in K1-TOPK cells. The results demonstrated that the level of p-AKT in K1-TOPK cells was higher compared with that of the control cells (Fig. 2B), indicating that TOPK promotes the activation of AKT signaling.

The results of the present study suggest that TOPK may be an important target for the treatment of thyroid cancer; however, there are currently no compounds in clinical use that directly target TOPK. Using homologous modeling, potential TOPK inhibitors were screened in an FDA-supported database, and sulfasalazine and PFH were selected for further analysis. An in vitro kinase assay was performed using histone H3 as a substrate, and the results demonstrated that sulfasalazine inhibited TOPK activity (Fig. 3A). The data revealed that phosphorylation of histone H3 (Ser10) was substantially attenuated following treatment with sulfasalazine, but not PFH (Fig. 3A), suggesting that sulfasalazine is a potential inhibitor of TOPK. The molecular structure of sulfasalazine is presented in Fig. 3B. In addition, an in vitro pull-down assay was performed to detect the potential interaction between TOPK and sulfasalazine. TOPK was detected in the sample eluent from Sepharose 4B beads coupled with sulfasalazine, but not in the eluent from Sepharose beads alone, indicating that sulfasalazine directly binds TOPK (Fig. 3C). Furthermore, the affinity of TOPK for sulfasalazine was assessed using microscale thermophoresis, revealing a Kd of 228 µM (Fig. 3D). Using the stable cell line K1-TOPK, phosphorylation of histone H3 was significantly reduced in a dose-dependent manner following treatment with sulfasalazine, indicating that it may act as an inhibitor of TOPK in thyroid cancer cells (Fig. 3E). Thus, sulfasalazine can interact with TOPK and inhibit its activity in vitro.

As an inhibitor of TOPK, the antiproliferative and antimetastatic properties of sulfasalazine in thyroid cancer were further investigated. The cytotoxicity of sulfasalazine was assessed in K1 cells; cells were treated with increasing concentrations of sulfasalazine for 72 h, and cell viability was measured using an MTT assay. The results indicated K1 cell viability was 73% when treated with sulfasalazine at a concentration of 400 µM. (Fig. 4A). The expression levels of p-AKT in K1-TOPK cells and control cells were also determined. The level of p-AKT in K1-TOPK cells decreased with increasing concentrations of sulfasalazine (Fig. 4B), but in control group cells appears to be increased treated with sulfasalazine at 50 and 150 µM, and reduced at 450 µM. Additionally, the level of cleaved-PARP increased with increasing concentrations of sulfasalazine in K1-TOPK cells (Fig. 4B), suggesting that sulfasalazine induced apoptosis.

The effects of sulfasalazine on anchorage-independent colony formation ability were also investigated. The results revealed that K1-TOPK cells treated with sulfasalazine formed fewer colonies compared with untreated K1-TOPK cells (Fig. 4C; P<0.001), and that sulfasalazine may attenuate anchorage-independent cell growth. In addition, the effect of sulfasalazine on thyroid cancer cell metastasis was observed using a wound healing assay. The results presented in Fig. 4D demonstrate that at 36 h post treatment, sulfasalazine significantly inhibited the migration of K1-TOPK cells (P<0.001). Collectively, the results suggest that sulfasalazine-targeting of TOPK inhibits the proliferation and metastasis of thyroid cancer cells.