T‐LAK cell‐originated protein kinase (TOPK) is a Novel Prognostic and Therapeutic Target in Chordoma

Abstract Objectives To assess the expression, prognostic value, and functionality of T‐lymphokine‐activated killer (T‐LAK) cell‐originated protein kinase (TOPK) in chordoma pathogenesis. Materials and Methods TOPK expression in chordoma was assessed via immunohistochemical staining of a tissue microarray (TMA) and correlated with patient clinicopathology. TOPK expression in chordoma cell lines and fresh patient tissues was then evaluated by Western blot. TOPK small interfering RNA (siRNA) and the specific inhibitor OTS514 were applied to determine the roles of TOPK in chordoma pathogenicity. The effect of TOPK expression on chordoma cell clonogenicity was also investigated using clonogenic assays. A 3D cell culture model was utilized to mimic in vivo environment to validate the effect of TOPK inhibition on chordoma cells. Results TOPK was highly expressed in 78.2% of the chordoma specimens in the TMA and all chordoma cell lines. High TOPK expression significantly correlated with metastasis, recurrence, disease status and shorter overall survival. Knockdown of TOPK with specific siRNA resulted in significantly decrease chordoma cell viability. Inhibition of TOPK with OTS514 significantly inhibited chordoma cell growth and proliferation, colony‐forming capacity and ex vivo spheroid growth. Conclusions High expression of TOPK is an important predictor of poor prognosis in chordoma. Inhibition of TOPK resulted in significantly decrease chordoma cell proliferation and increase apoptosis. Our results indicate TOPK as a novel prognostic biomarker and therapeutic target for chordoma.

has remained the primary treatment modality for patients; however, its insidious course and proximity to vital neurovascular structures make complete resection challenging if not possible. 2 Additionally, some chordoma patients already have metastatic diseases upon initial diagnosis. 3,6,7 The overall survival for chordoma patients is 68.4% at five years and 39.2% at 10 years, with a median overall survival of 7.8 years. 1 The strong chemotherapeutic resistance and lack of validated prognostic biomarkers in chordoma has highlighted the need for new and robust therapeutic targets. 2,3,5,8 Recent studies suggest that T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK) has tumorigenic roles in various malignancies. [9][10][11][12] TOPK, also known as PDZ-binding kinase (PBK), is a 322-amino acid serine/threonine kinase encoded by the PBK gene on chromosome 8p21.1. Expression and activation of TOPK function as a mitogen-activated protein kinase kinase (MAPKK) which is essential for catalytic activity during mitosis. 9 Recent studies have shown that TOPK regulates mitosis through its governing of several DNA binding proteins. 13 While TOPK expression is low or undetectable in healthy tissues, 9 it is overexpressed in lung cancer, ovarian cancer, renal cancer, colorectal cancer, prostate cancer and haematologic malignancies and correlates with worse outcomes. 11,[14][15][16][17][18][19] Functionally, TOPK promotes cancer cell growth and proliferation, dissemination and apoptotic resistance via numerous mechanisms. 9 Moreover, TOPK is upregulated in and promotes the proliferation and self-renewal of cancer stem cells, thus prompting the aggression of multiple malignancies. 20,21 These findings have given TOPK recognition as an emerging prognostic biomarker and therapeutic target with specificity for cancer cells while sparing normal host tissue. Several TOPK-specific inhibitors have shown promising results in pre-clinical works and are thus anticipated to be used in clinical trials in the near future. 9,11,12,22 In this study, we systemically investigated: (

| Chordoma sample collection and tissue microarray
The tissue microarray (TMA) was constructed from 55 individual chordoma patient specimens within a formalin-fixed paraffin-embedded (FFPE) block as previously described. 23,24 The clinicopathological characteristics of the specimens were collected and are outlined in Table 1, including age, gender, tumour location, recurrence, metastasis and disease status. The samples included 39 (70.9%) males and 16 (29.1%) females with an average age of 58.9 years old (range: 25-88 years old).

| Immunohistochemistry
The expression of TOPK was evaluated using Immunohistochemistry (IHC) assays according to the manufacturer instructions for the TOPK antibody (Cell Signaling Technology, Danvers, MA, USA). In brief, the paraffin-embedded slide was baked for 1 hour at 60°C before xylene deparaffinization. The slide was subsequently rehydrated through graded ethanol (100% and 95%). After heated epitope retrieval, 3% hydrogen peroxide was used to quench endogenous peroxidase activity. The slide was then blocked for 1 hour with normal goat serum.
Afterwards, it was incubated with a polyclonal rabbit antibody to human TOPK (1:100 dilution, Cell Signaling Technology) in a humidified chamber at 4°C overnight. SignalStain ® Boost Detection Reagent (Cell Signaling Technology) and SignalStain ® DAB (Cell Signaling Technology) were then utilized to detect the bound antibody. Finally, all the sections were counterstained with Hematoxylin QS (Vector Laboratories), and the slide was mounted with VectaMount AQ (Vector Laboratories) for long-term preservation.
The low TOPK expression subset included groups 0 and 1+, while the groups 2+ and 3+ were defined as the high TOPK expression subset.

| Human chordoma cell lines and culture
The human chordoma cell line UCH1 and UCH2 were established and provided by Dr Silke Brüderlein (University Hospitals of Ulm). 25,26 The CH22 chordoma cell line was established in our laboratory as previously reported. 24,27 The cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, GIBCO, Grand Island, NY, USA). The media were supplemented with 10% foetal bovine serum (Sigma-Aldrich) and 2% penicillin/streptomycin (Life Technologies). All cell lines were incubated in a humidified 5% CO 2 atmosphere at 37°C.

| Protein preparation and western blotting
The protein was extracted from the cells and chordoma tissue specimens with 1x RIPA lysis buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail tablets (Roche Applied Science). The protein lysate concentrations were then determined by DC TM protein assay reagents (BIO-RAD, Hercules, CA, USA) and a spectrophotometer SPECTRA max 340PC (Molecular Devices, LLc.). Western blotting was performed using a similar method to those previously described.
In brief, equal amounts of protein were separated on 4%-12% Bis-Tris gels (NuPAGE ® , Thermo Fisher Scientific) before they were transferred to nitrocellulose membranes. The membranes were incubated with the following specific primary antibodies at 4°C overnight after they were blocked in 5% non-fat milk for 1 hour, TOPK (1:500 dilution, Cell dilution) (Li-COR Biosciences) for 1 hour at room temperature. After being washed with TBST another three times, the bands were detected using Odyssey ® CLx equipment (LI-COR Bioscience) and Odyssey software 3.0. The quantity of β-actin was measured to ensure equal loading.

| Immunofluorescence assay
The expression of TOPK in chordoma cells was visualized by immunofluorescence assays. The UCH2 and CH22 cells were grown for three days in 24-well plates and fixed with 4% paraformaldehyde for 15 minutes before being permeabilized with ice-cold 100% methanol and blocked with 1% bovine serum albumin. Immunostaining was performed with TOPK (1:200 dilution, Cell Signaling Technology) and β-actin (1:500 dilution, Sigma-Aldrich) antibody at 4°C overnight. The next day, the cells were incubated for an additional 1 hour with Alexa Fluor 488 (Green) conjugated goat anti-rabbit antibody or Alexa Fluor 594 (red) goat anti-mouse antibody (Invitrogen). Nuclei were counterstained with 1 μg/mL Hoechst 33 342 (Invitrogen). Cell images were obtained using a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc) equipped with a SPOT RT TM digital camera. Green colour highlights TOPK protein, blue highlights nucleus, and red highlights cytoplasm.

| Suppression of TOPK by OTS514 treatment and MTT assay
The highly selective and potent TOPK inhibitor OTS514, ((R)-9-(4- (1-aminopropan-2-yl)phenyl)-8-hydroxy-6-methylthieno(2,3-c)quinoline-4(5H)-one, (Selleckchem), has been shown to inhibit the effect of TOPK in lung cancer, ovarian cancer, kidney cancer and haematologic malignancies both in vitro and in vivo. 11,[16][17][18][19] OTS514 inhibited TOPK kinase activity at a half-maximal inhibitory concentration (IC50) value of 2.6 nmol/L. 20 More recently, fluorescently-labelled OTS514 has been used to generate intraoperative in vivo tumour imaging. 28 Here, UCH2 and CH22 cells were seeded into 96-well plates at a density of 4x10 3 cells/well or 4 × 10 4 cells/well in 12-well plates and incubated with increasing concentrations (0, 1, 2, 5 and 10 nmol/L) of OTS514 for 2, 3 or 5 days prior to the following experiments. Three days following treatment with OTS514, the proteins of UCH2 and CH22 cells were extracted for protein measurement via Western blot. After OTS514 treatment for 5 days, the cell proliferation of UCH2 and CH22 was investigated using the MTT assay (as previously mentioned in the experimental protocol above). A Nikon microscope (Nikon Instruments Inc) was used to evaluate the morphological changes of UCH2 and CH22 cells after 3 and 5 days of OTS514 treatment.

| Clonogenic assay
The clonogenic assay, as known as the colony formation assay, is a wellestablished in vitro cell survival experiment based on the ability of a single cancer cell to grow into a colony. 29 A clonogenic assay can be used to study the effect of a specific agent on cancer cell prolifera-

| Three-dimensional cell culture
Three-dimensional (3D) cell culture is an artificially created environment that allows cells in vitro to interact with their surroundings and to grow in all directions, similar to in vivo growth. 30 The hydrogel 3D culture system (VitroGel 3D-RGD, #TWG002, TheWell Bioscience) was prepared according to the manufacturer's protocol. In short, 250 μL of a 2 × 10 4 cells/mL suspension of UCH2 or CH22 was mixed with the prepared hydrogel 3D culture system and seeded into 24-well culture plates. An additional 250 μL of DMEM supplemented with 10% FBS and 2% penicillin/streptomycin was added to cover the hydrogel. Immediately following this, 5 nmol/L of OTS514 was added into the medium. Spheroids formed from untreated chordoma cells served as the negative control.
The culture plates were then incubated at 37°C in a humidified 5% CO 2 atmosphere. The medium was changed every five days to provide sufficient nutrients and to prevent an osmolarity shift in the culture sys-  and hazard ratio (HR) were reported along with a 95% confident interval (CI). A P < 0.05 was considered statistically significant.
We also found TOPK overexpression in the chordoma cell lines UCH1, UCH2 and CH22 via Western blot analysis (Figure 2A,B).
In order to exclude the possibility that TOPK expression is an artefact of in vitro propagation, we also investigated the expression of TOPK in six fresh human chordoma specimens and found varying degrees of positive expression in all tested specimens ( Figure 2C,D). Interestingly, the chordoma tissues from patients with either recurrent or metastatic disease showed higher TOPK expression ( Table 2). Among the high TOPK expressing chordoma tissues, two of the patients were alive with disease. In contrast, no evidence of disease was observed in the patients who had low TOPK expressing chordomas (Table 2). We also examined the localization of TOPK via immunofluorescence of UCH2 and CH22 cell lines and found that the TOPK protein was located primarily within the cytoplasm (Figure 3). This cell line work was consistent with the chordoma TMA, which also demonstrated high TOPK expression within chordoma tissues and a cytoplasmic localization.

| TOPK expression in chordoma correlates with patient clinical characteristics and prognosis
After confirming the high expression of TOPK in the chordoma TMA, we performed follow-up statistical analysis for clinical significance. We found higher TOPK expression significantly correlated with recurrent or metastatic chordoma compared to primary disease alone (P = .0004 and P = .019, respectively) ( Figure 4A). TOPK expression was also significantly higher in the chordoma tissues from patients who developed metastatic disease compared with those who did not (P = .0417) ( Figure 4B). In addition, TOPK was significantly overexpressed in the chordoma tissues from patients with recurrent disease compared to those without recurrence (P = .0083) ( Figure 4C). Moreover, the chordoma tissues from patients who died demonstrated significantly higher TOPK expression than those patients who survived (P = .0056) ( Figure 4D). Further analysis showed expression of TOPK had a statistically significant correlation with metastasis, recurrence and disease status in chordoma (P = .038, P = .003 and P = .011, respectively), while other clinical parameters such as patient age, gender and tumour location had no such significance (Table 1) (Table 3) (Figure 4E,F).
However, our multivariate analysis showed only metastasis was an independent risk factor for OS in patients with chordoma.

| TOPK knockdown by siRNA decreased proliferation of human chordoma cell lines
To evaluate the role of TOPK in chordoma cell proliferation and growth, we knocked down TOPK expression with TOPK siRNA and observed changes. We used an immunofluorescence assay and Western blots to assess the expression of TOPK in chordoma cell lines following TOPK siRNA transfection. Immunofluorescence studies revealed a marked reduction in TOPK in both UCH2 and CH22 following 60 nmol/L of TOPK siRNA transfection (Figure 3). Western blots further confirmed a significant reduction of TOPK expression with increasing concentrations of siRNA transfection in both UCH2 and CH22 compared to the cell only or non-specific siRNA transfected groups ( Figure 5A).
In MTT assays, a dose-dependent decrease in cell viability was observed in both UCH2 and CH22 cells with transfection of increasing concentrations of TOPK siRNA over five days. Similar findings were not observed in the control groups, including the non-specific siRNA transfected cells and the untreated cells ( Figure 5B-D).

F I G U R E 3 TOPK expression in
Similar results were also seen in the CH22 cell line, with the spheroid diameter of the CH22 cells being 52.8% of the untreated CH22 cells after the treatment period (P < .001, Figure 7D).

| D ISCUSS I ON
TOPK has become an attractive therapeutic target due to its oncogenic roles and comparatively high expression in malignancies to their normal tissue counterparts. 11,14-17 Therefore, we evaluated TOPK expression in chordoma tissues and cell lines to discern whether it was also significant in this rarer and untested neoplasm.
Our TMA revealed all chordoma tissues expressed TOPK, of which 78.2% had a high expression. Similarly, elevated TOPK protein expression occurred in half of our fresh chordoma tissues, with the tissues of patients with disease recurrence or metastasis having especially high TOPK expression. High TOPK expression was also confirmed in all of our tested human chordoma cell lines.
While the complete roles of TOPK in chordoma are unknown, several studies have shown that TOPK is a prognostic marker of poor outcomes in lung cancer, ovarian cancer, kidney cancer, colorectal cancer, leukaemia, melanoma and glioblastoma. 11,[14][15][16][17][18][19]31 Although brachyury is a vital biomarker for notochord-derived tissues such as chordoma, it is not a prognostic indicator in clinical use. 8,32,33 In this study, we found high    The curve of spheroid diameters of CH22 cells treated with OTS514 showed significant decrease compared with untreated cells. The data represent mean ± SD of the independent triple experiment. *P < .001 TOPK knockdown via siRNA or short hairpin RNA (shRNA) has been shown to decrease tumour cell growth and induce apoptosis in various cancers. 10,[34][35][36][37] To explore the roles of TOPK in chordoma, we performed a knockdown analysis using TOPK-specific siRNA.
We found a significant reduction in cell growth and viability in both UCH2 and CH22 chordoma cell lines. has been shown to positively regulate the TBX3 in TGF-β/Smad signalling pathway in breast cancer, thereby enhancing epithelial-mesenchymal transition (EMT) and tumour cell invasion. 36 Interestingly, the TGF-β signalling pathway acts upstream of brachyury and has vital roles in bone and cartilage development. 38,39 Overexpression and activation of either the TGF-β or Smad signalling pathways have been found in chordomas and can predict poor clinical outcomes as well. 40,41 However, correlations and detail underlying mechanisms of TOPK with these signalling pathways in chordoma need to be investigated.
Clonogenic assays are in vitro cell survival experiments that function based on the premise that single cancer cells can quickly proliferate and form colonies. 42,43 We found the number and size of UCH2 and CH22 chordoma cell colonies reduced in a dose-dependent manner following OTS514 treatment ( Figure 7A). Next, as 3D cell culture models are validated in vitro models mimicking the in vivo environment, 30,44 we investigated the effects of OTS514 on chordoma cells in this unique tumour culturing system. We found the spheroid diameters of chordoma cells treated with OTS514 were significantly decreased compared with untreated cells (P < .001, Figure 7B-D). Previous studies have also shown in vivo suppression of tumour growth and proliferation in murine xenografts following treatment with OTS514. 11 Taken together, TOPK is an emerging prognostic biomarker and therapeutic target for chordoma patient treatment.

| CON CLUS ION
Our study demonstrates TOPK is highly expressed in chordoma and significantly correlates with recurrence, metastasis and shorter overall survival. Inhibition of TOPK with siRNA or inhibitor significantly reduces chordoma cell growth and proliferation. Our findings support TOPK as a potential prognostic biomarker and therapeutic target in chordoma therapy, warranting future mechanistic and in vivo investigations.

ACK N OWLED G EM ENT
This work was supported, in part, by the Department of Orthopaedic Surgery at UCLA.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Pichaya Thanindratarn contributed to conception, design, experiment, data acquisition, analysis and interpretation, drafted and critically revised the manuscript. Dylan C. Dean, Scott D. Nelson, and Francis J. Hornicek contributed to analysis and interpretation and critically revised the manuscript. Zhenfeng Duan contributed to conception, design, analysis and interpretation and critically revised the manuscript. All authors gave final approval and agreed to be accountable for all aspects of the present work.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
All experiments were reviewed and approved by the Ethics Committee of David Geffen School of Medicine, University of California, Los Angeles.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.