T‐LAK cell‐originated protein kinase (TOPK): an emerging prognostic biomarker and therapeutic target in osteosarcoma

T‐lymphokine‐activated killer (T‐LAK) cell‐originated protein kinase (TOPK) is an emerging target with critical roles in various cancers; however, its expression and function in osteosarcoma remain unexplored. We evaluated TOPK expression using RNA sequencing and gene expression data from public databases (TARGET‐OS, CCLE, GTEx, and GENT2) and immunohistochemistry in an osteosarcoma tissue microarray (TMA). TOPK gene expression was significantly higher in osteosarcoma than normal tissues and directly correlated with shorter overall survival. TOPK was overexpressed in 83.3% of the osteosarcoma specimens within our TMA and all osteosarcoma cell lines, whereas normal osteoblast cells had no aberrant expression. High expression of TOPK associated with metastasis, disease status, and shorter overall survival. Silencing of TOPK with small interfering RNA (siRNA) decreased cell viability, and inhibition with the selective inhibitor OTS514 suppressed osteosarcoma cell proliferation, migration, colony‐forming ability, and spheroid growth. Enhanced chemotherapeutic sensitivity and a synergistic effect were also observed with the combination of OTS514 and either doxorubicin or cisplatin in osteosarcoma cell lines. Taken together, our study demonstrated that TOPK is a potential prognostic biomarker and therapeutic target for osteosarcoma treatment.

T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK) is an emerging target with critical roles in various cancers; however, its expression and function in osteosarcoma remain unexplored. We evaluated TOPK expression using RNA sequencing and gene expression data from public databases (TARGET-OS, CCLE, GTEx, and GENT2) and immunohistochemistry in an osteosarcoma tissue microarray (TMA). TOPK gene expression was significantly higher in osteosarcoma than normal tissues and directly correlated with shorter overall survival. TOPK was overexpressed in 83.3% of the osteosarcoma specimens within our TMA and all osteosarcoma cell lines, whereas normal osteoblast cells had no aberrant expression. High expression of TOPK associated with metastasis, disease status, and shorter overall survival. Silencing of TOPK with small interfering RNA (siRNA) decreased cell viability, and inhibition with the selective inhibitor OTS514 suppressed osteosarcoma cell proliferation, migration, colonyforming ability, and spheroid growth. Enhanced chemotherapeutic sensitivity and a synergistic effect were also observed with the combination of OTS514 and either doxorubicin or cisplatin in osteosarcoma cell lines. Taken together, our study demonstrated that TOPK is a potential prognostic biomarker and therapeutic target for osteosarcoma treatment.

Introduction
Osteosarcoma is the common primary malignancy of the bone, disproportionately affects children and adolescents, yet accounts for < 1% of all cancer diagnoses within the United States [1]. While most arise within the metaphysis of long bones, axial lesions do occur and tend to be more aggressive [2]. Despite an aggressive approach and often burdensome treatment, the 5-year overall survival rate has plateaued at approximately 67% for nonmetastatic osteosarcoma, with no significant progress in the past four decades especially for those with recurrence, metastasis, or cytotoxic drug resistance [2,3]. Despite increasing efforts of targeted therapy for osteosarcoma, including some tyrosine kinase inhibitors (pazopanib, sorafenib, and regorafenib), these agents have failed to improve patient outcomes [4][5][6]. Several cancer immunotherapeutics including immune checkpoint inhibitors have been investigated as well; however, their efficacy has been dampened by the prominent heterogeneity of receptors and tumor microenvironment within osteosarcoma [7,8]. The limitations of these approaches highlight the urgent need for novel therapeutic strategies.
Recent works have shown T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK) is instrumental in the pathogenesis of various cancers [9][10][11][12]. Also called PBK (PDZ-binding kinase), this is a 322-amino-acid serine/threonine kinase encoded by the PBK gene located on chromosome 8p21.1. When activated, it functions as a mitogen-activated protein kinase (MAPK) kinase (MAPKK) essential for cell division [9]. TOPK has extensive mitotic roles and is a regulator of numerous DNA-binding proteins [13]. It is expressed mainly in the cytoplasm [9]. While TOPK expression is low to undetectable in normal tissues [9], higher expression exists in various human cancers including lung cancer, colorectal cancer, ovarian cancer, kidney cancer, prostate cancer, and hematologic malignancies [11,[14][15][16][17][18][19]. TOPK has garnered clinical interest as heightened expression correlates with poor clinical outcomes [11,[14][15][16][17][18][19]. Functionally, TOPK sustains cancer cell growth and proliferation, and tumor dissemination, and enhances apoptotic resistance [9]. In addition, it is upregulated in cancer stem cells where it promotes their proliferation and self-renewal in various malignancies [20,21]. Accumulating research supports TOPK as an emerging prognostic biomarker and therapeutic target that is highly specific for cancer cells, which has prompted the development of several specific and potent inhibitors of TOPK. These inhibitors have shown encouraging results in preclinical cancer models and will likely move to the clinical trial phase within the near future.
Given the encouraging results of TOPK targeting in other cancers and the limitations of current osteosarcoma regimens, we investigated the following: (a) expression of TOPK in public databases, with additional validation of this expression in human osteosarcoma tissues and cell lines; (b) correlation between TOPK expression and clinicopathology and outcomes of osteosarcoma patients; (c) functions of TOPK in osteosarcoma cell growth and proliferation; and (d) effects of a specific TOPK inhibitor on osteosarcoma cell proliferation, migration, and chemosensitivity.

Materials and methods
2.1. TOPK gene expression and RNA sequencing data from public databases >Publicly available genomic databases with their immense DNA and RNA sequencing data have streamlined the identification of aberrantly expressed genes and their molecular mechanisms driving tumor progression. Expression of the TOPK gene across various tumor tissues was contrasted to their healthy controls within the Gene Expression database of Normal and Tumor tissue 2 (GENT2) database [22] at https:// gent2.appex.kr/gent2/ (Fig. 1A). The Therapeutically Applicable Research to Generate Effective Treatment on Osteosarcoma (TARGET-OS) is a comprehensive genomic database which serves to delineate the molecular changes driving osteosarcoma. The TARGET-OS database contains comprehensive genomic profiles of clinically annotated patient cases within the discovery dataset. Each fully characterized TARGET-OS case includes data from nucleic acid samples extracted from osteosarcoma tissue. In our study, RNA sequencing data of TOPK in osteosarcoma tissues were obtained from TARGET-OS (https://portal.gdc.cancer.gov/ projects/TARGET-OS) and downloaded from the UCSC Xena browser at https://xenabrowser.net. As the control, normal bone and muscle tissue expressions of TOPK from RNA sequencing were obtained from the Genotype-Tissue Expression (GTEx) project [23]. The expressions of TOPK in osteosarcoma cell lines were collected from the Cancer Cell Line Encyclopedia (CCLE) [24] (Fig. 1B). Transcripts per million unit (TPM) was used to compare TOPK gene expression from RNA sequencing [25]. The immunostained slides were than underwent microscopic evaluation (Nikon Instruments Inc., Melville, NY, USA) and TOPK expression was categorized into four groups based on cytoplasmic staining intensity: 0, no staining, < 10% positive cells; 1+, weak staining, 10-25% positive cells; 2+, moderate staining, 26-50% positive cells; and 3+, strong staining, > 50% positive cells ( Fig. 2A). The low TOPK expression subset comprised groups 0 and 1+, while groups 2+ and 3+ were defined as the high TOPK expression subset.

Human osteosarcoma cell lines
The human osteoblast cell line NHOST was acquired from Lonza Walkersville Inc. (Walkersville, MD, USA) and cultured in osteoblast growth medium (PromoCell, Heidelberg, Germany). The human osteosarcoma cell line KHOS was generously provided by Dr. Efstathios Gonos (Institute of Biological Research & Biotechnology, Athens, Greece) [27]. Other human osteosarcoma cell lines MG63, MNNGHOS, U2OS, and 143B were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). The recurrent human osteosarcoma cell line OSA1777 was established in our lab and previously authenticated by the ATCC database [28]. These cell lines were cultured in RPMI 1640 (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 2% penicillin/ streptomycin (Life Technologies, Carlsbad, CA, USA). All cell lines were cultured in a humidified 5% CO 2 atmosphere at 37°C.

Protein extraction and western blotting
The protein was extracted from cells and osteosarcoma tissue specimens by 19 RIPA lysis buffer (Sigma-Aldrich) supplemented with protease inhibitor cocktail tablets (Roche Applied Science, Indianapolis, IN, USA). The concentrations of protein lysate were then evaluated by DC TM protein assay reagents (Bio-Rad, Hercules, CA, USA) and a spectrophotometer SPEC-TRA max 340PC (Molecular Devices, LLC, San Jose, CA, USA).
Western blotting was carried out as previously described [29]. In brief, equal amounts of protein were first separated on 4-12% Bis-Tris gels (NuPAGE Ò , Life Technologies) before transferred to nitrocellulose membranes. The membranes were then incubated at 4°C overnight after they were blocked in 5% nonfat milk for 1 h with the following specific primary antibodies, TOPK   Mann-Whitney U-test was used to analyze statistical significance. ***P < 0.001. (J) Comparison of TOPK immunohistochemistry staining scores between osteosarcoma tissues from survival and nonsurvival patients at the end of follow-up time. The error bars indicate SEM with 95% CI of each group. Mann-Whitney U-test was used to analyze statistical significance. ***P < 0.001. (K) Kaplan-Meier OS curve of osteosarcoma patients in our study were subgrouped into either the metastatic or nonmetastatic groups. Significantly shorter OS was observed in patients with metastasis. Cox regression analysis was used to determine statistical significance. *P < 0.05, **P < 0.01, ns; no statistical significance. (L) Kaplan-Meier OS curve of osteosarcoma patients in our study subgrouped as having either low (staining score ≤ 1+) or high (staining score ≥ 2+) TOPK expression. Significantly shorter OS was observed in patients with high TOK expression. Cox regression analysis was used to determine statistical significance. *P < 0.05, **P < 0.01, ns; no statistical significance.
for an additional 1 h with Alexa Fluor 488 (Green)conjugated goat anti-rabbit antibody or Alexa Fluor 594 (red) goat anti-mouse antibody (Li-COR Biosciences, Carlsbad, CA, USA). Nucleus counterstaining was performed with 1 lgÁmL À1 Hoechst 33342 (Life Technologies). Cell images were obtained using a Nikon Eclipse Ti-U fluorescence microscope (Diagnostic Instruments Inc., Sterling Heights, MI, USA) equipped with a SPOT RT TM digital camera. Green color reflects TOPK expression, blue represents nucleus, and red represents cytoplasm.

Silence TOPK expression by siRNA transfection and MTT assay
Silence TOPK expression in osteosarcoma cells was performed via small interfering RNA (siRNA) transfection. KHOS and U2OS cells were grown at a density of 4 9 10 3 cells per well in 96-well plates or 2 9 10 5 cells per well in 12-well plates and transfected with increasing concentrations (0, 10, 30, or 60 nM) of TOPK siRNA (5 0 -GACCAUAGUUUCUUGUUAA-3 0 ) (Sigma-Aldrich) using the Lipofectamine RNAiMax reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer instructions. Nonspecific siRNA (SIC001, Sigma-Aldrich) was used as a negative control. Three days following transfection with TOPK siRNA, the proteins of KHOS and U2OS cells were extracted for protein measurement by western blotting. Cellular proliferation was evaluated by conventional 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays. At the end of the 5-day treatment, 20 µL of MTT (5 mgÁmL À1 , Sigma-Aldrich) was added to each well of the 96-well culture plates. After incubating at 37°C in a humidified 5% CO 2 atmosphere for 4 h, the resulting formazan product was solubilized with 100 µL of acid isopropanol and the absorbance was measured at a 490 nm wavelength on the SpectraMax Microplate Ò Spectrophotometer (Molecular Devices LLC, Sunnyvale, CA, USA).

TOPK suppression by OTS514 inhibitor and MTT assay
The selective and potent TOPK inhibitor OTS514 ((R)-9-(4-(1-aminopropan-2-yl)phenyl)-8-hydroxy-6methylthieno(2,3-c)quinoline-4(5H)-one, Selleckchem, Houston, TX, USA) has been proven to inhibit the effects of TOPK in cancers such as lung cancer, kidney cancer, ovarian cancer, and hematologic malignancies in vitro and in vivo [11,[16][17][18][19]. OTS514 inhibited TOPK activity with a half-maximal inhibitory concentration (IC50) value of 2.6 nM [20]. Here, KHOS and U2OS cells were seeded into 96-well plates at a concentration of 4 9 10 3 cells per well and incubated with increasing concentrations (0, 6.25, 12.5, 25, and 50 nM) of OTS514 for 2, 3, or 5 days prior to the following experiments. After OTS514 treatment for 5 days, KHOS and U2OS proliferation was investigated using MTT assays as previously mentioned. A Nikon microscope (Nikon Instruments Inc.) was used to examine the morphological changes of KHOS and U2OS cells after 3 and 5 days of OTS514 treatment. The effect of OTS514 on the chemosensitivity of osteosarcoma cell lines was also investigated. KHOS and U2OS cells were cultured in 96-well plates as previously described and incubated with different concentrations of doxorubicin and cisplatin. Then, 10, 20, and 30 nM of OTS514 were added to each well of the treatment groups. At the end of the 5-day treatment, MTT assays were used to determine the cytotoxic effects in both cell lines as previously described. The synergistic effects of OTS514 were further evaluated by SYNERGYFINDER 2.0, a well-established web application for multidrug combination synergy analysis [30] (https://synergyfinder.fimm.fi). The degree of combination synergy was evaluated using a zero interaction potency (ZIP) model [31], which defines drug interactions as either synergistic (synergy score > 10), additive (synergy score À10 to 10), or antagonistic (synergy score < À10) [30].

Clonogenic assay
Also called a colony formation assay, the clonogenic assay is a well-established in vitro cell survival model that quantifies a single cell's ability to grow into a colony [32]. A clonogenic assay can be used to study the effectiveness of specific agents on cell proliferation and survival. Osteosarcoma KHOS and U2OS cells were seeded at 400 cells per well in 12-well plates and treated with OTS514 at different concentrations (0, 6.25, 12.5, 25 nM) and then incubated for 10 days at 37°C. Afterward, colonies were fixed with methanol for 10 min and then washed three times with PBS before staining with 10% Giemsa stain (Sigma-Aldrich) for 20 min. Finally, cells were washed with flowing water and allowed to dry. Images of the stained colonies were captured using a digital camera (Olympus, Tokyo, Japan).

Wound healing-cell migration assay
Wound healing assays were utilized to test cell migration activities. KHOS and U2OS cells were seeded into 6-well plates at a density of 4 9 10 5 cells per well and 2.11. Three-dimensional (3D) cell culture 3D cell culture is an artificial environment that allows in vitro tumor cells to interact with their surroundings and grow in all the directions they would in vivo [33]. In our experiment, we prepared the hydrogel 3D culture system according to manufacturer protocol (VitroGel 3D-RGD, #TWG002, TheWell Bioscience, Township, NJ, USA). We began with 250 µL of a 2 9 10 4 cellsÁmL À1 suspension of KHOS and U2OS mixed with the prepared hydrogel 3D culture suspension in 24-well culture plates. The other 250 µL of RPMI 1640 supplemented with 10% FBS and 2% penicillin/streptomycin was added to cover the hydrogel. Following, 10 nM of OTS514 was immediately put into the mixture. Spheroid formation of the osteosarcoma cells without treatment was considered as the negative control. The culture plates were then incubated at 37°C in a humidified 5% CO 2 atmosphere. Medium was changed every 48 h to provide sufficient nutrients and to prevent an osmolarity shift in the system. Images of spheroids were taken under the microscope every other day with NIS-Elements platform (Nikon Instruments Inc.). At 10 days, the spheroid pictures were also taken on a Nikon Eclipse Ti-U inverted fluorescence microscope (Nikon Instruments Inc.) after 15 min of incubation with 0.25 µM Calcein AM (Life Technologies).

Statistical analysis
GRAPHPAD PRISM 8 software (GraphPad Software, San Diego, CA, USA) and SPSS 23.0 (IBM Corp., Armonk, NY, USA) were utilized for statistical analyses. Nonparametric testing (Mann-Whitney U-test) was utilized to compare and determine statistical significance of two independent groups. A one-way analysis of variance (ANOVA) was utilized for multiple comparisons. The survival analyses were performed by Kaplan-Meier models. The correlations between different clinical characteristics and overall survival (OS) or recurrence-free survival (RFS) was determined by Cox regression analysis. Only those parameters that were statistically significant (P < 0.05) in the univariate analysis were included in the multivariate analysis. The median OS, RFS, and hazard ratio (HR) were reported with a 95% confident interval (CI). A P value < 0.05 was considered statistically significant.

TOPK gene is overexpressed in osteosarcoma
We first assessed TOPK gene expression from public databases, which included more than 68 000 samples and 72 different paired tissues from the GPL96 platform (HG-U133A) of Affymetrix mRNA gene array GENT2 database [22,34]. TOPK expression was significantly higher in cancerous samples compared with their normal tissue counterparts (P < 0.001). These included cancers of the breast, ovary, endometrium, cervix, colon, esophagus, stomach, kidney, liver, pharynx, thyroid, bone, and soft tissue sarcoma (Fig. 1A). TOPK was significantly overexpressed in osteosarcoma within the RNA sequencing database. The osteosarcoma expression profile of TOPK mRNA was available from 88 samples from TARGET-OS, six osteosarcoma cell lines from CCLE, and 396 normal bone or muscle tissues from GTEx. TOPK mRNA was significantly elevated in osteosarcoma tissue samples (40.8 AE 27.0 TPM, P < 0.001) and cell lines (101.0 AE 68.7 TPM, P < 0.001) compared with normal tissues (0.13 AE 0.22 TPM) (Fig. 1B). In addition, we found that TOPK gene expression significantly correlated with disease recurrence (P = 0.003) but not clinical characteristics such as age, gender, chemosensitivity, metastasis, or death (Fig. S1).

TOPK was highly expressed in our human osteosarcoma TMA, cell lines, and fresh tissues
To further support our findings, we evaluated TOPK expression in an osteosarcoma TMA. Of these patient tissues, 65 of 66 (98.5%) showed positive TOPK  (Fig. 2A,B). Then, these stained specimens were divided into two categories: 0 and 1+ were defined as the low TOPK expression group (16.67%), whereas the 2+ and 3+ staining groups as being the high TOPK expression group (83.33%) (Fig. 2C, Table 1).
We also revealed elevated TOPK expression in osteosarcoma cell lines via western blot, including KHOS, MG63, MNNGHOS, OSA1777, U2OS, and 143B, with the human osteoblast cell line NHOST serving as negative control (Fig. 2D,E). We also investigated TOPK expression in fresh human osteosarcoma tissue specimens and found high expression in seven of the eight samples (87.5%) (Fig. 2F,G). We also localized TOPK expression via immunofluorescence of KHOS and U2OS and detected that TOPK protein was located primarily within the cytoplasm (Fig. S2). This result was consistent with our osteosarcoma TMA findings, which showed high TOPK expression in cytoplasmic localization within osteosarcoma tissues.

TOPK expression correlates with osteosarcoma clinical characteristics and prognosis
Based on the overexpression of TOPK in our osteosarcoma TMA, we analyzed whether TOPK expression correlates with patient clinical characteristics and prognosis. Higher TOPK expression was significantly associated with metastatic or recurrent osteosarcoma compared with primary disease alone (P = 0.009 and P = 0.03, respectively) (Fig. 2H). Additionally, the TOPK staining score was significantly higher in the osteosarcoma tissues from patients who later developed metastatic disease than those who did not (P = 0.008) (Fig. 2I). Moreover, the osteosarcoma tissues from nonsurvival patients showed significantly higher TOPK staining score than those patients who survived (P = 0.008) (Fig. 2J). Although a higher immunostaining score was observed among osteosarcoma tissues from those with recurrent, high-grade, or chemoresistant (< 90% tumor necrosis) groups, statistical significance was not met. Expression of TOPK significantly correlated with metastasis and disease status (P = 0.039 and P = 0.046, respectively) but not other clinical parameters such as patient age, gender, tumor location, or histologic grade (Table 1).
Next, we performed an OS analysis to determine the prognostic value of TOPK expression in osteosarcoma. In our TMA analysis, the OS was 60.34% at three years, 55.60% at 5 years, and 45.51% at ten years. The median OS was 91.0 months (22.7-159.3 months). The OS of the high TOPK expression group was 54.55% at 3 years, 48.96% at 5 years, and 39.20% at ten years, with median OS at 65.40 months. In contrast, the OS of the low TOPK expression group was 90.48% at 3 and 5 years, and 79.17% at 10 years, with a median OS of 252.00 months ( Table 2). A univariate analysis demonstrated metastasis (HR = 11.12 (2.68-46.19), P = 0.001) and TOPK expression (HR = 3.33 (1.03-10.82), P = 0.045) were poor prognostic predictors for OS ( Table 2) (Fig. 2K,L). However, our multivariate analysis revealed only metastasis was an independent risk factor of OS for osteosarcoma patients.
As a tertiary hospital and referral center, our samples included more advanced osteosarcoma patients, and therefore, more metastatic osteosarcomas were present in our TMA. To increase external validity to the general osteosarcoma population, our TMA was further analyzed in cases of osteosarcoma tissues from patients with only localized disease. After the exclusion of patients with metastasis at presentation, 53 osteosarcoma samples including 31 (58.5%) males and 22 (41.5%) females were analyzed. Among these patients, TOPK overexpression was significantly correlated with disease status, similar to the previous analysis, while no statistical significance with metastasis was observed (Table S1). The OS analysis revealed a 5-year survival rate at 59.74% with a median OS of 137.07 months (45.9-230.1 months). The OS of the high TOPK expression group was 58.14% at 3 years, 50.97% at 5 years, and 43.69% at 10 years, with a median OS of 76.78 months. In contrast, the OS of the low TOPK expression group was 100% at 3 and 5 years and 87.5% at 10 years, with a median OS of 252.00 months (Table S2). In our univariate analysis, TOPK overexpression, metastasis, and recurrence were poor prognostic factors for OS, similar to the previous analysis. However, multivariate analysis revealed only metastasis was an independent risk factor (Table S2). In cases of localized osteosarcoma, presenting variables upon diagnosis are those which inform initial therapeutic decisions and therefore do not include subsequent variables such as recurrence or metastasis. Interestingly, TOPK expression has independent prognostic value for OS when only baseline variables are taken into consideration (except for subsequent recurrence and metastasis), with a hazard ratio of 4.48 (1.07-18.85, 95% CI) (Table S2). However, since there was no sufficient information about time to progression such as recurrence or metastasis, we cannot confirm TOPK as prognostic predictor for progression-

TOPK knockdown by siRNA decreases osteosarcoma cell proliferation
To investigate the function of TOPK in osteosarcoma growth and proliferation, we knocked down its expression via TOPK siRNA and quantified the subsequent changes within cell lines. We first used immunofluorescent assays and western blots to assess TOPK expression in osteosarcoma cell lines after TOPK siRNA transfection. Immunofluorescent study unveiled a marked decrease in TOPK fluorescence in both KHOS and U2OS following 60 nM of TOPK siRNA transfection (Fig. S2). Western blots further confirmed a notable decrease in TOPK expression with increasing concentrations of siRNA in KHOS and U2OS. This effect was absent in cells transfected with nonspecific siRNA (Fig. 3A).
In MTT assays, osteosarcoma cell viability was decreased in a dose-dependent manner in KHOS and U2OS following transfection with increasing concentrations of TOPK siRNA over 5 days. Similar finding was not observed in the control groups, including untreated cells and the nonspecific siRNA-transfected cells (Fig. 3B-D).

Pharmacological TOPK inhibition with OTS514 in osteosarcoma cells
We assessed whether these findings occurred with TOPK inhibition within KHOS and U2OS via the TOPK inhibitor OTS514. A dose-and time-dependent decrease in osteosarcoma cell viability was observed in KHOS and U2OS, with IC50 values for 5 days of OTS514 treatment at 4.77-21.17 nM and 6.34-42.10 nM, respectively (Fig. 4A,B). Similarly, we observed a reduction in viable cells and morphologic changes with increasing concentrations of OTS514 in KHOS and U2OS after 3 days of treatment (Fig. 4C). Western blots demonstrated TOPK and antiapoptotic proteins Mcl-1 and Survivin significantly decreased in a dosedependent manner, while apoptotic cleavage of PARP increased after incubation of KHOS and U2OS with  (Fig. 4D). To exclude the confounding effect of DMSO, we also treated the osteosarcoma cell lines with different concentrations of DMSO and found no significant biological effect of DMSO to osteosarcoma cell growth and proliferation. We next investigated the effect of OTS514 on osteosarcoma cell colony formation within a clonogenic assay. KHOS and U2OS showed a dosedependent reduction in colony formation with OTS514 treatment compared with the untreated cells (Fig. 5A). Furthermore, as cell migration and invasion are hallmark features of metastasis and the primary cause of osteosarcoma patient death, we began a second set of experiments. After confirming in our TMA that TOPK expression significantly correlated with osteosarcoma metastasis, we exposed our cell lines with 10 nM of OTS514 to reveal the effect of TOPK inhibition on in vitro osteosarcoma cell migration. This resulted in a significant time-dependent decrease in cell migration in both KHOS and U2OS (Fig. 5B-D).
Additionally, we examined whether suppression via OTS514 would affect spheroid formation in a 3D cell culture. The spheroid diameters in OTS514-treated KHOS and U2OS were significantly smaller than the untreated cells (Fig. 5E-G). After 10 days of 10 nM OTS514 treatment, the spheroid diameters of KHOS were 52.1% of the untreated KHOS group (P < 0.001, Fig. 5F). A similar result was also observed in U2OS, where the spheroid diameters were 61.5% of the untreated U2OS group (P < 0.001, Fig. 5G).

TOPK inhibition promotes chemosensitivity in osteosarcoma cells
TOPK inhibitors have known anticancer effects when combined with additional chemotherapy or radiation therapy [9]. As doxorubicin and cisplatin are among the most popular chemotherapeutics in osteosarcoma [3,35], we sought to investigate whether additional TOPK inhibition would enhance osteosarcoma chemosensitivity. MTT assays were used to compare viabilities of KHOS and U2OS treated with combinations of increasing concentrations of doxorubicin or cisplatin alongside the TOPK inhibitor OTS514. We found increasing concentrations of OTS514 decreased   (Fig. 6). A synergistic analysis revealed OTS514 did result in a synergistic anticancer effect on KHOS (ZIP synergy score = 10.379) (Fig. 6A-D); however, the same combination had an additive effect in U2OS (ZIP synergy score = 7.917) (Fig. 6E-H). Conversely, OTS514 therapy produced an additive anticancer effect alongside cisplatin in KHOS (ZIP synergy score = 9.945) and a synergistic effect in U2OS (ZIP synergy score = 11.336) (Fig. S3).

Discussion
TOPK has recently become a potential therapeutic target in cancer as it has shown overexpression within various tumors compared with their normal counterparts [11,[14][15][16][17]. This observation has been supported by the recent expansion of cancer genome databases and has made the selection of cancer targets such as TOPK a higher-yield process for preclinical research [22,[36][37][38][39]. In our present study, analysis of the GENT2 database revealed a notably higher TOPK expression within bone tumors and the TARGET-OS RNA sequencing database showed a significantly higher TOPK mRNA expression in osteosarcoma tissues compared with normal bone and muscle tissues in the GTEx database. Furthermore, TOPK mRNA was highly expressed in osteosarcoma cell lines from the CCLE repository compared with healthy tissues. These finding were clinically relevant, as a combined database analysis supported TOPK gene overexpression as a poor prognostic indicator for osteosarcoma patient survival. Drawing from our promising bioinformatic analysis, we evaluated and validated TOPK expression within our own osteosarcoma tissues and cell lines. The TMA data showed 98.5% of osteosarcoma tissue samples expressed TOPK, of which a majority 83.3% had high expression (staining score ≥ 2+). Similarly, a high quantity of TOPK protein was detected in fresh osteosarcoma tissues, and expression was elevated in all tested osteosarcoma cell lines compared with normal osteoblast cells.
Recent studies have indicated TOPK expression is a poor prognostic factor in cancers of the lung, ovary, kidney, colon, as well as leukemia, glioblastoma, and melanoma [11,[14][15][16][17][18][19]39]. In our TMA analysis, higher TOPK expression associated with metastasis and shorter OS. Particularly, 42 of 47 (89.4%) of the tissue samples from the patients with metastatic disease had high TOPK expression. Additionally, osteosarcoma patients with elevated TOPK expression had shorter OS compared with those with low expression, with a HR of 3.33 by univariate analysis. These results were significant and consistent with our TOPK gene expression analysis from the TARGET-OS and GENT2 public databases, and endorse the prognostic significance of TOPK expression in osteosarcoma.
Knockdown of TOPK using either siRNA or short hairpin RNA (shRNA) has been proven to decrease tumor cell proliferation and induce apoptosis in multiple cancers [10,[40][41][42][43]. To verify the functional roles of TOPK in osteosarcoma growth and proliferation, we conducted a knockdown experiment using a TOPKspecific siRNA. Accordingly, there was a significant reduction in cell viability and growth in KHOS and U2OS upon TOPK suppression.
Of the various TOPK inhibitors available such as OTS514, HI-TOPK-032, and ADA-07, we elected to use OTS514 as it is the most potent and target specific [9,11,18]. Recently, preclinical studies including xenograft models have shown OTS514 effectively inhibits tumor growth and dissemination in a dose-dependent manner for cancers such as lung cancer, kidney cancer, ovarian cancer, myeloma, and leukemia [11,[16][17][18][19]. In our work, we performed in vitro TOPK loss-offunction studies to determine its significance in osteosarcoma cell proliferation and growth. Similarly, TOPK inhibition with OTS514 decreased KHOS and U2OS growth and proliferation in a dose-and timedependent manner. While the exact molecular mechanism of TOPK inhibition in osteosarcoma is unclear, we report a marked decrease in the antiapoptotic proteins Mcl-1 and Survivin alongside increased apoptotic cleavage of PARP. Therefore, TOPK likely promotes proliferation through an inhibition of apoptosis. Previous studies have also found OTS514 to associate with FOXM1 and MELK in TOPK-expressing tumors [11,[16][17][18]. Most recently, TOPK was shown to positively regulate TBX3 in the TGF-b/Smad signaling pathway in breast cancer, hence enhancing epithelialmesenchymal transition (EMT) and tumor cell invasion [42]. These studies in other cancers warrant additional work to assess whether these TOPK pathways exist in osteosarcoma, as they may highlight potent and combined targeted therapy selection. Clonogenic assays quantify the ability of a single cancer cell to form colonies in vitro [44,45]. We show the number and size of colonies from KHOS and U2OS were reduced in a dose-dependent manner with increasing OTS514 (Fig. 5A). As TOPK was highly expressed in 89.4% of the tissues of osteosarcoma patients with metastatic disease, we further examined the role of TOPK within in vitro osteosarcoma cell migration. Cell migration significantly decreased in both KHOS and U2OS following treatment with OTS514 in a timedependent manner (P < 0.001, Fig. 5B-D). It is therefore likely that TOPK contributes to osteosarcoma cell migration and eventual distant metastasis. This finding is consistent with results in colon cancer, where TOPK regulates p53-and Akt-mediated migration and metastasis to mouse liver tissue [46]. TOPK also promotes cancer stem cell self-renewal, migration, and metastasis in neuroblastoma [21]. Taken together, our results demonstrate the importance of TOPK in metastasis and the ability of OTS514 to mitigate this effect in osteosarcoma. This is especially important clinically because pulmonary metastasis remains the primary mode of osteosarcoma patient mortality, and currently used chemotherapeutics have limited benefit in cases of tumor dissemination.
Given 3D cell culture is an approved in vitro model of the in vivo environment [33,47], we sought to validate the effects of OTS514 on osteosarcoma cell proliferation within this medium. We show the diameters of osteosarcoma spheroid treated with OTS514 are notably decreased compared with untreated cells (P < 0.001, Fig. 5E-G). Previous studies have also demonstrated the reduction in in vivo tumor growth and dissemination in mouse models following OTS514 treatment [11]. Finally, we demonstrate OTS514 inhibits osteosarcoma growth synergistically when used alongside doxorubicin and cisplatin in both KHOS and U2OS. Our work show TOPK is a promising biomarker and therapeutic target for osteosarcoma treatment, particularly when administered in combination with standard osteosarcoma therapeutics.
Previous studies suggest that TOPK plays a role in cell cycle regulation and mitotic progression. Moreover, TOPK expression is minimal in differentiated cells, whereas its overexpression is a pathophysiological feature of various malignancies. Therefore, a specific TOPK inhibitor may have anticancer activity while minimizing offtarget toxicity. Our findings similarly suggest that a specific TOPK inhibitor may have therapeutic roles in osteosarcoma treatment; however, its clinical application for osteosarcoma is complex. Because of its genomic heterogenicity, no single specific genomic and molecular target in osteosarcoma tumorigenesis has been identified. This study provides promising new data into the molecular biology of osteosarcoma, but further investigation into the molecular mechanisms behind TOPK in osteosarcoma is needed.

Conclusions
In summary, our study shows that TOPK is aberrantly expressed in osteosarcoma and significantly associates with shorter OS. Therapeutically, TOPK inhibition decreases osteosarcoma cell growth, proliferation, migration, and dissemination. Of note, application of a potent and specific TOPK inhibitor has synergistic effects alongside the commonly used osteosarcoma therapeutics cisplatin and doxorubicin. These results support TOPK as a prognostic predictor for OS and potential target in osteosarcoma treatment that warrants further mechanistic and in vivo investigation.

Supporting information
Additional supporting information may be found online in the Supporting Information section at the end of the article.