Inhibition of BUB1 suppresses tumorigenesis of osteosarcoma via blocking of PI3K/Akt and ERK pathways

Abstract Osteosarcoma (OS) is a primary malignant bone tumour that mainly affects teenagers, with patients displaying poor prognosis. Budding uninhibited by benzimidazoles 1 (BUB1), a type of serine/threonine kinase that is linked to pro‐tumorigenic phenomena, has not been well studied in OS. Hence, this study aimed to explore the role of BUB1 in OS. The expression of BUB1 in OS specimens and cell lines was assessed using immunohistochemistry and Western blot analysis. Univariate and multivariate analyses were applied to evaluate the impact of BUB1 on patient survival. Cell counting kit‐8, wound‐healing and Transwell assays, as well as flow cytometry, were used to investigate the influence of BUB1 inhibition on OS in vitro. Moreover, a tumour xenograft model was established to investigate the in vivo effect of BUB1 inhibition on OS tumour growth. Results showed that BUB1 was overexpressed in OS specimens and cell lines. Furthermore, BUB1 overexpression was closely associated with the poor clinical outcomes of patients with OS. Inhibition of BUB1 markedly suppressed cell proliferation and tumour growth, cell migration, invasion and induced cell apoptosis of OS by blocking the PI3K/Akt and ERK signalling pathways. Thus, our study suggested that overexpression of BUB1 protein contributed to poor survival of OS patients and that inhibition of BUB1 resulted in considerable anti‐tumour activity associated with proliferation, migration, invasion and apoptosis of OS.


| INTRODUC TI ON
Osteosarcoma (OS), the most common primary invasive bone tumour, often occurs to young adults. 1 The five-year overall survival rate of patients with OS has improved from 20% to 70% because of the development of active therapies. However, approximately 30% of patients are prone to relapse or metastasis, and their prognosis remains poor because of the limited efficacy of the present treatment strategies. 2 Thus, evaluation of efficient prognostic and therapeutic targets is essential to improve the poor clinical outcome of patients with OS.
Budding uninhibited by benzimidazoles 1 (BUB1) is a part of BUB family and mitotic arrest-deficient (MAD) families of proteins. 3 As a member of mitotic checkpoint serine/threonine kinases, BUB1 plays an important role in chromosome segregation. 4 BUB1 contains three primary regions: a conserved N-terminal region containing a kinetochore localization domain; an intermediate, non-conserved region that acts as a scaffold for the recruitment of proteins; and a C-terminal region that contains a catalytic serine/threonine kinase domain. 5 BUB1 has been identified as an oncogene in diverse types of tumours. 6 Overexpression of BUB1 is associated with tumour proliferation activity in human gastric carcinoma, 7 papillary renal cell carcinoma 8 and pancreatic ductal adenocarcinoma. 9 Bioinformatic analyses by Sun et al. 10 and Peng et al 11 have shown that BUB1 likely plays a crucial role in OS; however, the results have not been confirmed in in vitro and in vivo experiments.
Therefore, this study aimed to further explore the role of BUB1 in OS by investigating the effects of BUB1 inhibition using either an inhibitor of BUB1, BAY 1816032 or lentivirus-induced knockdown on the proliferation, migration, invasion, apoptosis and tumour growth of OS.

| Cell lines and culture
Human foetal osteoblastic cell line hFOB 1. 19

| Protein extraction and Western blot analysis
Proteins were extracted using the Membrane and Cytosol Protein Extraction Kit (Beyotime, Shanghai, China), and their concentrations were quantified using the Bicinchoninic Acid Protein Assay Kit (Beyotime, Shanghai, China). Next, the proteins were subjected to 8% SDS-polyacrylamide gel, blotted onto PVDF membranes (Millipore, Bedford, MA, USA) and then incubated in QuickBlock™ Blocking Buffer (Beyotime, Shanghai, China) for 15 min. Next, the membranes were incubated overnight at 4°C with primary antibodies against tubulin, BUB1, caspase 3, Bcl-2, Akt, ERK, P-Akt and P-ERK, and all were purchased from Affinity Biosciences, Cincinnati, USA. The membranes were then incubated with secondary antibodies (Affinity) at room temperature for 1 h. The blots were finally developed using the Affinity ECL Kit (Affinity, Biosciences, Cincinnati, USA) and the FluorChem R detection system (ProteinSimple, USA).

| Immunohistochemistry
The 61 samples collected from participants in this study were stained immunohistochemically with primary anti-BUB1 antibody (Affinity, Biosciences, Cincinnati, USA) using the PV9000 immunohistochemical kit (Origene Technologies, Inc., Beijing, China). Prior to that, the site of tumour in the specimens was identified using haematoxylin-eosin (HE) staining. Two independent pathologists evaluated the staining results. The labelling index of BUB1 expression was scored as 0 to 3 based on staining intensities: negative, 0; weakly positive, 1; moderately positive, 2; and strongly positive, 3.
The mean percentage based on staining from 10 random high-power fields of positive tumour cells was also scored as 1 to 3 as follows: 1, <25%; 2, 25%-75%; and 3, >75%. Total scores were determined based on the intensity and percentage of positive staining in cancer cells. Overexpression was defined as a score >2, and low expression was defined as a score ≤2. 12

| BUB1 knockdown cell line
Cell lines were infected with a knockdown lentivirus (sh-BUB1, TCCTACACTTCCTGATATT) and the corresponding negative control lentivirus (sh-NC, TTCTCCGAACGTGTCACGT), purchased from GeneChem (Shanghai, China). The cells were plated into 6-well plates at 1 × 10 5 cells/well and incubated overnight. Next, the medium was replaced with 1 ml fresh medium containing an appropriate amount of virus suspension. After 24 h, the virus-containing medium was replaced with fresh medium and the plates were incubated for another 48 h. Stably transfected clones were screened using puromycin. Transfection efficiency was confirmed by Western blot analysis.

| Cell viability assay
Cells were maintained at a density of 2 × 10 3

| Transwell assay
Transwell assay was performed in an 8μm pore size chamber    SPSS 19.0 software (SPSS Inc., Chicago, USA) was used for statistical analysis. The chi-square test or Fisher's exact test was performed to investigate the difference in BUB1 protein expression between OS and normal bone tissues, as well as the relationship between BUB1 protein expression and clinicopathological parameters. Differences in survival status were measured by log-rank test and Kaplan-Meier survival plots. Cox proportional hazards model was performed on the parameters that displayed significantly different in the univariate analysis. Difference between groups was analysed using Student's t test; data are presented as mean ± SD. A p < 0.05 was considered to be statistically significant.

| BUB1 expression is up-regulated in OS tissues and cell lines
Immunohistochemistry results demonstrated that the BUB1 protein was overexpressed in 39.02% (16/41) of OS tissues but not in the normal bone tissues ( Figure 1A

| BUB1 expression is associated with clinicopathological characteristics and clinical outcome
Among the clinicopathological parameters, including sex, age, tumour size, tumour location, histologic subtype, Enneking staging, response to chemotherapy and distant metastasis. Response to chemotherapy (p < 0.001) and distant metastasis (p = 0.036) were found to be closely related to BUB1 protein overexpression (Table 1). Independent prognostic value of BUB1 protein in OS was evaluated by performing univariate and multivariate analyses to investigate the relationships of BUB1 protein overexpression with progression-free survival (PFS) time and overall survival (OS) time in patients with OS. As shown in Figure 1D and Table 2

| BUB1 inhibition promotes cell apoptosis of OS cells in vitro
To explore cell death caused by inhibition of BUB1, we used the

| BUB1 inhibition suppresses tumour growth of OS in vivo
To evaluate the effect of BUB1 suppression and BUB1 gene knockdown on tumour growth of OS in vivo, an OS xenograft model was established in nude mice. We found that both BAY 1816032 treatment and BUB1 knockdown resulted in the regression tumour growth, as demonstrated by decreased tumour volumes and weights of mice in the experiment group ( Figure 6A,B).

| BUB1 affects the biological behaviours of OS via phosphatidylinositol 3 kinase (P13K)/Akt and ERK pathways
To investigate the effects of BUB1 on OS, we assessed the activity of PI3K/Akt and ERK pathways in Saos2 and U2OS cells by

TA B L E 1 Association between BUB1 expression and clinicopathological characteristics in osteosarcoma
Western blot analysis following inhibition of BUB1. As shown in Figure 6C,D, the expression of P-Akt and P-ERK proteins was reduced following treatment with BAY 1816032, similarly, BUB1

| DISCUSS ION
OS, which originates from mesenchymal tissue, is one of the most aggressive primary bone tumours among children and adolescents. 13 Moreover, the survival status of patients with metastatic or relapsed disease has not improved in the past two decades. 14

| CON CLUS IONS
This study reports up-regulation of BUB1 in OS, which is closely related to the adverse clinical outcomes of patients with OS.
Suppression of BUB1 protein could significantly reduce cell proliferation, invasion and migration, promote apoptosis of OS in vitro and inhibit tumour growth in vivo. Therefore, our study may provide a new therapeutic target for the treatment of OS, which may improve the current stagnant survival of patients. However, further F I G U R E 5 Effect of BUB1 inhibition on the cell apoptosis and apoptosis-related proteins. A, B, Increased total apoptosis rate in Saos2 and U2OS cells after treatment with different concentrations of BAY 1816032. C, D, Increased total apoptosis rate in Saos2 and U2OS cells with BUB1 gene knockdown. E, F, Expression of P53, caspase 3 and Bcl-2 proteins in Saos2 and U2OS cells following treatment with BAY 1816032 and BUB1 gene knockdown. **p < 0.01, ***p < 0.001.
investigations are required to confirm the therapeutic effect of BUB1 on OS.

ACK N OWLED G EM ENTS
Our research has been supported by the Natural Science Foundation of China (grant no. 31571292).