Abstract 1585: Loss of CDK5 sensitizes ovarian cancer cells to paclitaxel by enhancing intracellular retention

Abstract

Abstract In order to improve the efficacy of paclitaxel in treatment of ovarian cancer, we have performed a kinome siRNA library screen to identify kinases that regulate paclitaxel sensitivity. CDK5 was one of kinase targets that could modulate paclitaxel sensitivity in SKOv3 cells. Unlike typical cyclin dependent kinases, CDK5 does not have a clearly defined role in the regulation of the cell cycle. CDK5 is required for the regulation of neuronal synaptic vesicular transport in the brain through phosphorylation of Munc 18, a key regulator of vesicular exocytosis. In this report, we aimed to explore the mechanisms by which CDK5 potentiate paclitaxel activity. We found that eighteen ovarian cancer cell lines expressed variable CDK5 levels measured by quantitative real time PCR and Western blotting. Basal levels of CDK5 in ovarian cancer cell lines appeared not to correlate with paclitaxel sensitivity. We used immunoprecipitation of CDK5 and in vitro kinase assays using 32P-ATP and observed that paclitaxel treatment of SKOv3 cells resulted in a profound (>20 fold) increase in CDK5 activity. Selective depletion of CDK5 using 4 individual siRNAs in SKOv3, TOV112 and DOV13 ovarian cancer cells lines resulted in a significant increase in paclitaxel-induced cytotoxicity as measured by cell viability assays. In addition, loss of CDK5 resulted in a significant increase in paclitaxel-mediated induction of caspase 3/7 activation in the ovarian cancer cell lines SKOv3, ES2, OVAR429 and DOV13. Remarkably, the magnitude of sensitization was directly proportional (p<0.001) to the dose of paclitaxel used. Knockdown of CDK5 did not enhance microtubule stabilization or increase paclitaxel-induced mitotic arrest. Instead, CDK5 depletion in SKOv3 cells resulted in a significant (p<0.001) increase in retention of fluorescent-tagged paclitaxel. This effect was similar to but independent from the effect of inhibition of trans-membrane pumps using verapamil, suggesting that the two mechanisms act in parallel. Indeed, knockdown of CDK5 in combination with verapamil resulted in greater intracellular retention of paclitaxel in SKOv3 cells. Consequently, we conclude that depletion of CDK5 results in decreased paclitaxel exocytosis leading to increased cytotoxicity to ovarian cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1585.