Abstract 2053: Aurora-A kinase is required for rapid establishment of the mitotic checkpoint

Abstract

Abstract Aurora-A (AurA) is a conserved Serine/Threonine kinase and is mainly involved in multiple key steps in cell division. A high level of AurA is frequently detected in different human cancer types and is used as a poor prognostic biomarker. AurA inhibitors as potential anti-cancer drugs are widely being tested in clinical trials. To explore the mechanism(s) of possible efficacy, the roles of AurA in mitosis should be fully elucidated. AurA is known to be required for centrosome separation, spindle formation and kinetochore-microtubule attachment. However, the mechanism by which AurA is involved in regulation of the mitotic checkpoint is unclear. Most studies have used RNA interference, which does not abolish AurA activity, or inhibitors, which often target both AurA and AurB. In this study, we sought to dissect the role of AurA in checkpoint regulation by using cells derived from genetically engineered mice conditionally null for AurA, as well as a new selective AurA inhibitor. Upon deletion of AurA in mouse embryonic fibroblast (MEFs), 65% of cells formed monopolar spindles and exited mitosis without dividing. However, the average duration of mitosis in these cells was 88 minutes, significantly shorter than the 192 minutes in cells with monopolar spindles induced by Eg5 kinesin inhibition, in which checkpoint is fully functional. Previous work in our lab showed AurA is dispensable for checkpoint signaling in APC/C inhibition. Hence, we tested whether the truncated arrest in AurA-/- cells was due to defective checkpoint engagement and/or because of attenuated AurB mediated error correction, which reactivates checkpoint. Using live cell imaging followed by immunofluorescence, we found the recruitment of Mad2 to kinetochores at the start of mitosis was markedly delayed in AurA-/- MEFs. Mad2 levels at kinetochores were reduced to 28% of that in wild-type cells in the absence of microtubules. In addition, without AurA, AurB was not able to accumulate at the centromere at early prometa-phase. This observation may indicate that error correction is compromised, resulting in the abnormal acceleration of checkpoint satisfaction. Similar results were obtained using human immortalized RPE cells treated with a selective AurA inhibitor-MLN8237. Thus, preliminary data suggest AurA disruption causes delays in rapid establishment of a robust checkpoint and an effective error correction pathway at early mitosis. These defects accelerate mitotic exit in the presence of an aberrant spindle, leading to aneuploid progeny. Currently, we are assessing the dynamics of the checkpoint signaling on kinetochores throughout mitosis. We are also investigating whether AurA function in checkpoint regulation is directly through activation of AurB at centromere. This study is expected to further elucidate basic mechanisms of mitotic regulation and will provide important implications for the application of AurA kinase inhibitors in clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2053. doi:1538-7445.AM2012-2053