Pb2101 functional significance of overexpression of mitotic checkpoint protein bub1 in multiple myeloma

Background: Multiple myeloma (MM) is a cytogenetically and molecularly heterogeneous hematological malignancy that remains mostly incurable, despite recent therapeutic progress. Clonal evolution due to chromosomal instability (CIN) in MM is considered to be one of the major driving forces of disease progression and acquisition of treatment resistance; however, the mechanisms underlying CIN are unclear. Faithful chromosomal segregation is tightly regulated by the coordinated functions of a series of mitotic checkpoint proteins, while the failure of this process potentially causes acquisition of additional chromosomal abnormalities that lead to karyotypic evolution in cancer. High expression of BUB1, one of the mitotic checkpoint proteins, has been associated with a poor prognosis of several types of cancers. However, the functional involvement of BUB1 in the development and progression of MM is unknown. Aims: The goal of this study was to define the relationship between BUB1 mRNA expression and disease progression in MM. The functional significance of BUB1 in myeloma pathophysiology, including cell survival and proliferation, clonogenic growth and cell division, was also examined. Methods BUB1 mRNA levels were evaluated in myeloma cells from 39 patients with MM and 15 with monoclonal gammopathy of undetermined significance (MGUS), in normal plasma cells from 3 healthy controls, and in 10 human MM cell lines (HMCLs). Studies using patient samples were approved by the institutional review board of Kyoto Prefectural University of Medicine (RBMR-G-124-7), and these samples were collected with informed consent in accordance with the Declaration of Helsinki. BUB1 mRNA levels were determined using quantitative RT-PCR. To investigate the functional role of BUB1, mRNA expression was knocked down using lentivirus-mediated shRNA in two HMCLs: KMS-18 cells (a kind gift from Dr. T. Ohtsuki) and AMO-1 cells (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH). The functional impacts of BUB1 knockdown were examined in cell viability, colony forming (MethoCult semi-solid medium), and cell division assays. Chromosome segregation abnormalities were evaluated by immunofluorescence staining of chromosomes in mitotic cells. Results: BUB1 mRNA expression was significantly higher in patient-derived myeloma cells compared to normal plasma cells, and also in myeloma cells from patients in an advanced stage (relapsed and refractory MM) compared to those in an early stage (MGUS and newly diagnosed MM), suggesting involvement of increased BUB1 expression in disease progression. In HMCLs, BUB1 knockdown resulted in reduction of chromosome segregation abnormalities, such as chromosomal breaks, bridging or lagging. These abnormalities were found in 33% and 39% of anaphase cells of parental KMS-18 and AMO-1 cells, respectively, but in only 12% and 22% after BUB1 knockdown in these respective cells. BUB1 knockdown did not cause a significant change in cell proliferation and viability, but did reduce the colony-forming ability of KMS-18 cells by more than 70%. In addition, BUB1 knockdown had no impact on the sensitivities of the cells to DNA-damaging agents. Summary/Conclusion: This study suggests that enhanced BUB1 expression is associated with an increase of clones that are prone to acquire additional chromosomal abnormalities due to CIN, resulting in increased mitotic segregation errors in MM. Further studies of the function and regulatory mechanism of BUB1 may contribute to development of a new treatment strategy for MM by prevention of clonal evolution with acquisition of additional chromosomal abnormalities.