Abstract C217: Aneuploidy induction by the microtubule-stabilizing agent peloruside A involves the cell cycle checkpoint proteins MAD2 and p55CDC.

Peloruside A (PelA) is a novel secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. It is a potent microtubule-stabilizer and binds to a distinct site on -tubulin compared to the widely used anti-cancer drug paclitaxel. PelA has clear potential benefits over paclitaxel, including increased solubility, reduced sensitivity to multiple drug resistance in cancer cell lines, and improved tolerability and efficacy in non-small cell lung cancer xenografts in mice. Previous chemical genetics studies have indicated that MAD2 (mitotic arrest deficiency-2), a spindle-assembly checkpoint (SAC) protein, could potentially be a secondary target of PelA. Premature dissociation of MAD2 from the SAC protein, p55CDC, has been implicated in the induction of aneuploidy in response to low concentrations of paclitaxel; thus, we sought to validate and investigate MAD2 as an additional target of PelA. Flow cytometric analysis of 1A9 (human ovarian carcinoma) cells confirmed that low concentrations of PelA (< 40 nM) induced chromosome mis-segregation in up to 15% of cells. Western blot analyses detected minimal changes in the abundance levels of MAD2, BUBR1 and p55CDC at both high (100 nM) and low (40 nM) PelA concentrations. However, differences in the association between MAD2 and BUBR1 with p55CDC were observed after cells were released from synchronization and assessed by co-immunoprecipitation. The mitotic arrest seen at 100 nM PelA was accompanied by a sustained formation of MAD2/p55CDC and BUBR1/p55CDC complexes; whereas, 40 nM PelA resulted in a similar dissociation profile of these complexes compared to controls. Given that PelA-induced aneuploidy coincides with G₂-M progression, it is likely that dissociation of these complexes occurs despite aberrant microtubule-kinetochore attachments. Studies are currently underway to determine whether this increase in aneuploidy is due to the presence of erroneous attachments that are not detected by the SAC, such as merotelically oriented chromosomes. Aneuploidy is important because of its link to apoptosis and cancer cell death, and these experiments will help to elucidate some of the underlying signalling pathways. Additionally, these studies improve our understanding of the molecular actions of PelA during the cell cycle and highlight the importance of characterizing the concentration-dependent effects of drug interactions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C217.