Background

As the ninth most common cancer worldwide [1], bladder cancer (BCa) is having a gradually increasing incidence in China [2]. Most of the newly diagnosed cases are non-muscle invasive BCa. Even with transurethral resection of tumor, BCa still has a very high recurrence rate [3]. Chemotherapy based on cisplatin has improved the outcome modestly. For cisplatin-ineligible patients, T-cell checkpoint inhibitors have presented some benefits to those having high PD-L1 expression in some trials [4,5,6]. Except for the traditional chemotherapy drugs, there are no emerging new drugs for almost 30 years in BCa [7]. Therefore, to enhance the targeted and personalized therapy, molecular analysis to find more new specific markers and therapeutic targets is of great urgent.

BORA encoded protein activates kinase Aurora A, and is very important in spindle assembly, centrosome maturation and the process of mitosis. BORA was identified as a cell cycle co-factor protein of Aurora A in the first place [8]. Binding with pole-like kinase 1 (PLK1), BORA forms a PLK1/BORA complex and recruits Aurora A to the T-loop of PLK1 T210 phosphorylation site to activate PLK1, thus promote mitotic entry [9]. PLK1 and Aurora A are critical regulators of cell cycle, which has a fundamental role in cell proliferation, and related to the checkpoint recovery when DNA damage appears in cells where it leads to DNA repair or progress to apoptosis [10, 11]. A variety of cell cycle related regulators have been explored as therapeutic targets and biomarkers [35]. However, some studies also showed that PLK1 and Aurora A conversely had an influence on BORA activity through posttranslational modifications [8, 36]. Recent studies demonstrated that CDK1 regulated the activation of BORA by phosphorylating 3 conserved sites located at the N-terminal part of BORA, which are Cyclin docking sites [20, 37]. Once mutated the three phosphorylation sites of BORA, PLK1 could not be activated by Aurora A at T210 on the T-loop. During DNA damage recovery, it is important for cells to maintain the G2 checkpoint, which provides cells with time for DNA repair or progress to apoptosis. Cairns et al. reported that BORA was significantly related to radiosensitivity by regulating DNA repair and MDC1, and BORA affected irradiation response via a different pathway from PLK1 [19]. We detected the expression of cell cycle proteins and found that PLK1 and CDC25C were upregulated, while CDK1 and CDK2 were downregulated after knockdown of BORA. Moreover, the protein changes were reversed after BORA overexpression in BCa cells (Fig. 3). GSK3β activation was reported to be important for BORA [38]. We noticed a downregulation of GSK3β after BORA knockdown in UM-UC-3 cells (data not shown). However, the exact mechanism of how BORA regulates and be regulated in the feedback loops still needs to be further explored.

Interestingly, after knockdown of BORA, we noticed that BCa cell migration and invasion were inhibited, which has not been reported anywhere else (Fig. 4). We observed upregulated epithelial marker E-cadherin in BORA knockdown cells and downregulated N-cadherin, Vimentin and other proteins associated with EMT pathway, which has been verified to play a critical role in migration and invasion of cancer cells [39]. But how reduced BORA exactly regulates EMT still needs further detections. To confirm our in vitro results, we established stable cell lines of BORA knockdown through lentiviral packaging. Xenografts and pulmonary metastasis mice model were established by subcutaneously and intravenously injecting BCa cells, respectively. In vivo, we also found reduced BCa cell growth and migration after knockdown of BORA (Fig. 5).

Recently, a large number of Aurora A and PLK1 inhibitors were reported to be evaluated in clinical trials as anticancer drugs but showed modest effect against solid tumors [40]. BORA, as the key intermediate protein of Aurora A and PLK1, was reported to be a potential biomarker for prognosis in lung, breast, and gastric adenocarcinomas [18]. Our results also showed that BORA knockdown could suppress BCa cell growth and migration both in vitro and in vivo. Researches about the effect of BORA in cancers are still too less. And how PLK1 and Aurora A inhibitors affect solid tumors after blockading BORA needs to be further investigated. As a signaling node of the BORA–PLK1–CDC25–CDK1 feedback loop and Aurora A-BORA-PLK1 axis, BORA is likely to have exciting prospects as a potential target.

Conclusions

In conclusion, our study revealed that BORA positively associated with BCa cell growth and regulated cell cycle. For the first time, we found that BORA knockdown could suppress BCa cell migration and invasion possibly through EMT pathway. BORA has the potential to become a new biomarker and possible therapeutic target in BCa.