BAP1 loss induces BRCA1 dependent and independent defective spindle organization, checkpoint dysfunction and genomic instability


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Anita Singh1
1university of leicester

Abstract

Background

BRCA1 associated protein 1 (BAP1) is a tumour suppressor that is commonly inactivated in the majority of mesotheliomas. We have previously reported that loss of BRCA1 expression in mesothelioma is a common event, and is associated with resistance to spindle checkpoint activator vinorelbine, a drug with relevance to treatment of mesothelioma. However 1.The mechanism of BRCA1 loss is unknown, and 2) the potential functional interaction between BRCA1 and BAP1 linked to spindle checkpoint is unknown. The aim of this study was to assess the functional relationship between BAP1 and BRCA1 and examine their role in genome stability in mesothelioma cells. 

 

Method

We conducted functional genetic analysis of BAP1 and BRCA1 in two MPM cell lines, MSTO and H2452, the latter carrying an inactivating A95D mutation in the UCH domain of BAP1. BAP1 knockdown was achieved by siRNA transfection, while BRCA1 knockdown was achieved by doxycycline induction of an integrated shRNA. 

Results

Loss of BAP1 expression led to reduced expression of BRCA1. Treatment with the proteasome inhibitor, MG132, restored BRCA1 expression in the absence of BAP1 indicating that BAP1contributes to post-translational stabilization of BRCA1 protein. Consistent with previous data, knockdown of BAP1 induced SAC deficiency and vinorelbine resistance concurrent with reduced expression of BRCA1. Loss of BAP1 and BRCA1 also led to an increased frequency of amplified centrosomes. Unexpectedly though, additional defects were observed in mitotic spindle architecture in response to BAP1 loss that were not seen upon loss of BRCA1. 

Conclusion

Our data demonstrate that BAP1 controls BRCA1 expression through regulating its protein stability. They also demonstrate both BRCA1-dependent and independent roles for BAP1 in mitotic progression. These findings suggest that BAP1 loss may disrupt spindle checkpoint function and predict resistance to agents such as vinorelbine, a hypothesis that we will test in our randomised trial VIM.