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Increased expression of the mitotic kinase Aurora A occurs with high incidence in a variety of human tumors, and has been correlated with tumor initiation and progression. Aurora A localizes to centrosomes and proximal mitotic spindles and phosphorylates a variety of proteins, which collectively play a role in centrosome maturation/separation and mitotic spindle formation. We investigated the effect of Aurora A inhibition in cultured human tumor cells using MLN8054, a potent and selective Aurora A small molecule inhibitor. MLN8054 inhibits Aurora A autophosphorylation on Thr288 and causes cells to accumulate in mitosis, phenotypes consistent with suppression of Aurora A activity. The effect of MLN8054 on centrosome maturation and spindle formation was examined in mitotic tumor cells. MLN8054 treated cells presented with a high incidence of monopolar and tripolar spindles, indicating defects in spindle pole formation resulting from Aurora A inhibition. Moreover, the majority of spindle fibers in MLN8054 treated cells were abnormally formed and DNA was not tightly aligned to the metaphase plate, even in cells containing bipolar spindles. Time lapse video microscopy experiments were performed on tumor cells labeled with fluorescent α-tubulin and Histone H2B to monitor mitotic events during MLN8054 treatment. MLN8054 significantly delayed anaphase onset relative to untreated cells, indicating that the “wait-anaphase” spindle assembly checkpoint was prolonged by improper attachment of spindle fibers to kinetochores. However, despite the mitotic delay and formation of abnormal spindles, many MLN8054 treated cells completed mitosis and underwent cytokinesis, often with apparent unequal segregation of DNA. To examine the combined effect of Aurora A inhibition in tumor cells arrested in mitosis with microtubule disorganizers, cells were simultaneously treated with MLN8054 and paclitaxel or nocodazole. Combined treatment with these compounds caused cells to rapidly exit mitosis without undergoing cytokinesis, resulting in the formation of multinucleated cells demonstrating a role for Aurora A in maintaining the spindle assembly checkpoint in the presence of spindle disorganizers. Collectively, our results demonstrate that Aurora A inhibition mediated by MLN8054 triggers deleterious events in tumor cells during mitosis.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]