Supplementary Material for: BUB1B Overexpression Is an Independent Prognostic Marker and Associated with CD44, p53, and PD-L1 in Renal Cell Carcinoma

Introduction: BUB1 mitotic checkpoint serine/threonine kinase B encoded by BUB1B gene is a member of the spindle assembly checkpoint family. Several reports have demonstrated that overexpression of BUB1B is associated with cancer progression and prognosis. Objective: This study aims to clarify the expression and function of BUB1B in renal cell carcinoma (RCC). Methods: The expression of BUB1B was determined using immunohistochemistry and bioinformatics analysis in RCC. The effects of BUB1B knockdown on cell growth and invasion were evaluated. We analyzed the interaction between BUB1B, cancer stem cell markers, p53, and PD-L1 in RCC. Results: In 121 cases of RCC, immunohistochemistry showed that 30 (25%) of the RCC cases were positive for BUB1B. High BUB1B expression was significantly correlated with high nuclear grade, T stage, and M stage. A Kaplan-Meier analysis showed that the high expression of BUB1B was associated with poor overall survival after nephrectomy. High BUB1B expression was associated with CD44, p53, and PD-L1 in RCC. Knockdown of BUB1B suppressed cell growth and invasion in RCC cell lines. Knockdown of BUB1B also suppressed the expression of CD44 and increased the expression of phospho-p53 (Ser15). In silico analysis showed that BUB1B was associated with inflamed CD8+, exhausted T-cell signature, IFN-γ signature, and the response to nivolumab. Conclusion: These results suggest that BUB1B plays an oncogenic role and may be a promising predictive biomarker for survival in RCC.

引言：由BUB1B基因编码的BUB1有丝分裂检验点丝氨酸/苏氨酸激酶B（BUB1 mitotic checkpoint serine/threonine kinase B，以下简称BUB1B）属于纺锤体组装检验点家族成员。多项研究已证实，BUB1B过表达与癌症进展及预后密切相关。
研究目的：本研究旨在阐明BUB1B在肾细胞癌（renal cell carcinoma, RCC）中的表达模式与生物学功能。
研究方法：本研究通过免疫组织化学法与生物信息学分析，检测RCC组织中BUB1B的表达水平；评估BUB1B敲低对肾癌细胞生长与侵袭能力的影响；分析RCC中BUB1B与肿瘤干细胞标志物、p53及程序性死亡受体配体1（programmed death-ligand 1, PD-L1）之间的相互作用。
研究结果：在121例RCC组织样本中，免疫组织化学检测显示30例（25%）BUB1B表达呈阳性。BUB1B高表达与肿瘤高核分级、T分期及M分期显著相关。Kaplan-Meier分析法结果表明，BUB1B高表达与肾切除术后患者较差的总体生存率显著相关。RCC中BUB1B高表达与CD44、p53及PD-L1的表达水平密切相关。在RCC细胞系中，敲低BUB1B可抑制细胞生长与侵袭能力；同时可下调CD44的表达，并上调磷酸化p53（Ser15位点）的表达水平。生物信息学分析（in silico analysis）结果显示，BUB1B与炎性CD8+ T细胞特征基因集、耗竭性T细胞特征基因集、干扰素-γ（interferon-γ, IFN-γ）特征基因集以及纳武利尤单抗（nivolumab）的治疗响应显著相关。
研究结论：本研究结果表明，BUB1B在RCC中发挥致癌作用，或可成为预测RCC患者生存预后的潜在生物标志物。