Jiao et al. Cell Death and Disease (2021) 12:63 https://doi.org/10.1038/s41419-020-03234-x Cell Death & Disease ARTICLE Open Access BUB1B promotes extrahepatic cholangiocarcinoma progression via JNK/c-Jun pathways Chen Yu Jiao1, Qin Chao Feng1,2, Chang Xian Li1,DongWang1,ShengHan1, Yao Dong Zhang1,WangJieJiang1, Jiang Chang1,XuehaoWang1 and Xiang Cheng Li1 Abstract Currently, the controversy regarding the expression profile and function of BUB1B in different malignancies still exist. In this project, we aimed to explore the role and molecular mechanism of BUB1B in the progression of extrahepatic cholangiocarcinoma (ECC). The expression levels of BUB1B in human ECC were evaluated by immunohistochemistry, western blot, and real-time PCR. The role and mechanism of BUB1B in CCA cell proliferation and invasion were investigated in both in vitro and in vivo functional studies. To indicate the clinical significance, a tissue microarray was performed on 113 ECC patients, followed by univariate and multivariate analyses. The expression of BUB1B was increased in both human CCA tissues and CCA cells. Results from loss-of-function and gain-of-function experiments suggested that the inhibition of BUB1B decreased the proliferation and invasiveness of CCA cells in vitro and in vivo, while overexpression of BUB1B achieved the opposite effect. Furthermore, the activation of c-Jun N-terminal kinase-c- Jun (JNK)-c-Jun pathway was regulated by BUB1B. BUB1B regulated the proliferation and invasiveness of CAA cells in a JNK-c-Jun-dependent manner. Clinically, ECC patients with BUB1B high expression had worse overall survival and recurrence-free survival than those with BUB1B low expression. Multivariate analysis identified that BUB1B was an independent predictor for postoperative recurrence and overall survival of ECC patients. In conclusion, BUB1B promoted ECC progression via JNK/c-Jun pathways. These findings suggested that BUB1B could be a potential 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; therapeutic target and a biomarker for predicting prognosis for ECC patients. Introduction Complete surgical resection remains the first choice for Cholangiocarcinoma (CCA), described as a malignancy the treatment of CCA patients. However, the rates of that arises from the epithelial cells of the bile duct, is the resectability and the long-term outcome after these second most common primary hepatobiliary malignancy1. therapies are less than satisfactory because of the high – CCA can be divided into intrahepatic cholangiocarcinoma post-surgical recurrence3 5. Therefore, a clearer under- (ICC) or extrahepatic cholangiocarcinoma (ECC) standing of CCA growth and metastasis mechanisms is according to the tumor location in the biliary tree. ECC is urgently necessary to find the potential therapeutic targets the most common CCA accounting for ~75% of all CCA2. for CCA. BUB1B (BUB1 mitotic checkpoint serine/threonine kinase B) is a member of the spindle assembly checkpoint 6 Correspondence: Chang Xian Li ([email protected])or (SAC) protein family . SAC prevents premature sister Xiang Cheng Li ([email protected]) chromatid separation until all kinetochores are properly 1 Key Laboratory of Liver Transplantation, Chinese Academy of Medical attached to the mitotic spindle during mitosis7. BUB1B Sciences, Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu Province, China plays a central role in SAC signaling and stable attach- 2Department of surgery, JiangYuan Hospital Affiliated to Jiangsu Institute of ment of kinetochores to spindle microtubules8,9. Nuclear Medicine, Wuxi, Jiangsu Province, China Although Huang et al.10 reported that the structure of the These authors contributed equally: Chen Yu Jiao, Qin Chao Feng, Chang Xian Li kinase domain of BUB1B in Drosophila melanogaster Edited by S. Tait © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Jiao et al. Cell Death and Disease (2021) 12:63 Page 2 of 13 folded into a conformation predicted to be catalytically routinely every 3–6 months during the first 5 years of active, more evidence showed that BUB1B, an inactive follow-up and once a year thereafter. Overall survival (OS) pseudokinase, interacts directly with Cdc20, BUB3, and time was defined as the period of time in months from MAD2, constituting the mitotic checkpoint complex, operation to death. Recurrence-free survival time was which inhibit the activity of the anaphase-promoting defined as the period of time in months from operation to complex or cyclosome (APC/C)7,11,12. Accordingly, the recurrence. To validate the clinical significance of BUB1B function of BUB1B is to ensure proper chromosome in CCA, a tissue microarray, as well as the staining of segregation by suppressing the onset of anaphase by BUB1B, was performed on 113 ECC patients. A total of 29 inhibiting APC/C activation13. Given the critical role of ECC samples with matched para-tumor tissues were BUB1B in mitotic checkpoint signaling and chromosome collected for detecting messenger RNA (mRNA) expres- congression, impairment in BUB1B and SAC often results sion. Furthermore, we also detected the mRNA expres- in aneuploidy and chromosomal instability, which can sion of BUB1B in 30 pairs of ICC samples. All CCA tissues – contribute to an increase in cancer incidence14 16. were collected using protocols approved by the Ethics The relationship between BUB1B expression and specific Committee of The First Affiliated Hospital of Nanjing human malignancies remains to be clarified. It has been Medical University, and written informed consent was reported that low expression of BUB1B contributed to the obtained from every patient. initiation and progression of colon adenocarcinoma17,18. However, a large number of reports have demonstrated that Tissue microarray overexpression of BUB1B was associated with progression To validate the clinical significance of BUB1B in ECC, a and recurrence of pancreatic ductal adenocarcinoma, tissue microarray was performed. The details of tissue prostate cancer, hepatocellular carcinoma, and some other microarray have been described in our previous article24. – cancers19 21. BUB1B can mediate anchorage-independent The quantitation of immunostaining for BUB1B was com- survival and growth, thereby facilitating lung adenocarci- pleted by Quick-score (Q-score) based on the intensity and noma dissemination during metastasis22.Moreover, heterogeneity by two independent researchers who were reduction of BUB1B level or inhibition of BUB1B kinase blinded regarding patient details. The positive rates were activity in human cancer cells resulted in massive chro- scored as 0 point (0–5%), 1 point (6–35%), 2 points mosome loss and apoptotic cell death23.TheCancerGen- (36–70%), and 3 points (71–100%). The score of the ome Atlas (TCGA) database data showed that the staining intensity was presented as 0 point (none), 1 point expression of BUB1B in CCA tissues was upregulated. (low), 2 points (medium), and 3 points (high). The Q-score However, the biological function and molecular regulatory was the product of heterogeneity and intensity. The mechanism of BUB1B in CCA remain unclear. Therefore, it expression was defined as high when the Q-score were >4. was worthwhile to further explore the roles and precise mechanisms of BUB1B in CCA. Cell culture and transfection In the current study, we aimed to investigate the roles CCA cell lines (RBE, QBC939, HCCC9810, and and mechanisms of BUB1B in CCA and then explore its HUCCT) and normal bile duct epithelial cell line (HiBEC) prognostic prediction value. We first detected the were obtained from the Cell Bank of the Chinese Acad- expression profile of BUB1B in CCA tissues and cell lines. emy of Science (Shanghai, China). These cell lines were Then, we investigated the functional role of BUB1B in cell cultured in Dulbecco’s modified Eagle’s medium (Gibco, proliferation and invasiveness of CCA and further USA) with 10% fetal bovine serum (Biological Industries, explored the underlying mechanism. After that, the clin- Israel), penicillin/streptomycin 100 U/ml at 37 °C in a 5% ical significance of BUB1B in ECC patients was explored. CO2 humidified incubator. The BUB1B-knockdown len- Together, our findings suggested that BUB1B promoted tivirus was designed and produced by Polybrene (Obio CCA proliferation and invasiveness, as well as a candidate Technology) using pLKD-CMV-G and PR-U6-shRNA biomarker of prognostic prediction and a potential ther- vector. BUB1B overexpression plasmid was designed by apeutic target for ECC patients. Shanghai Genechem Co., Ltd (GV144 vector, XhoI/ BamHI enzyme cleavage, seq: Materials and methods 5′-GTCCGGACTCAGATCTCGAGCTATGGCGG