9PD - Aurora-B mediated snail phosphorylation is essential for mitotic spindle checkpoint and for preventing chromosomal instability in breast cancer

Abstract

BackgroundSnail is a transcriptional repressor that promotes epithelial-to-mesenchymal transition (EMT). Snail is upregulated in breast cancer and it is associated with chromosomal instability (CIN) and metastasis. The mitotic spindle assembly checkpoint (SAC) is a critical mechanism to prevent CIN. Extensive studies have investigated signaling pathways that involve in protein-protein interactions and post translational modifications. However, it not known how mitosis might be impacted by transcriptional regulation. Due to the fact that Snail is a transcriptional regulator and that Snail is upregulated in breast cancer with CIN, it is critical to understand whether and how abnormally regulated Snail contributes to breast cancer CIN via transcriptional regulation. MethodsWe used siRNA to knockdown Snail in breast caner cell lines to look for a change in SAC. We developed a phosphor-specific antibody to study Aurora-B mediated phosphorylation. Site specific mutagenesis was used to generate phosphorylation site mutants, and exogenous proteins were expressed in cells to look for an effect in SAC and CIN. ResultsWe found that Snail is an essential element that is required for the SAC. Genetic depletion of Snail in breast cancer cells by siRNA led to a defect in activation of SAC (measured by accumulation of Histone Serine 10 phosphorylation in response to spindle damaging agents). Interestingly we also found that the Snail protein level was significantly enhanced in the presence of nocodazole. Further, we found that Snail upregulation was through phosphorylation in an Aurora-B dependent manner. We identified a serine site that can be phosphorylated by Aurora-B. Using a phosphor-specific antibody we have developed, we further proved that the phosphorylation event was initiated in mitosis. Functional studies reveal a critical role for Snail phosphorylation in activation of the SAC and prevention of CIN. Conclusions1. Snail is required for mitotic spindle checkpoint activation; 2. Snail is phosphorylated by Aurora B in mitosis; 3. Aurora-B medicated Snail phosphorylation is required for optinal activation of the spindle checkpoint and prevention of chromosomal instability in breast cancer. Legal entity responsible for the studyThe authors. FundingTianjin Medical University Cancer Institute and Hospital. DisclosureAll authors have declared no conflicts of interest.