Abstract 2988: Regulation of mitosis and taxane response by Daxx

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Current theories suggest that mitotic proteins are essential for cellular response to taxanes (paclitaxel and docetaxel), a widely-used family of chemotherapeutic compounds. We found that absence or depletion of protein Daxx increases cellular resistance to paclitaxel _a common trait of patients diagnosed with breast cancer and other malignancies. Particularly, we observed that 1) paclitaxel induced cell death, in both human breast cancer and mouse cell lines, depends on the level of Daxx; 2) cells become paclitaxel-resistant upon experimental depletion of Daxx; 3) breast cancer specimens accumulate differential level of Daxx; 4) docetaxel response in breast cancer patients correlates with level of Daxx mRNA. We also found that Daxx is important for the proper progression of mitosis in human cells: duration of mitotic stages is altered in cells stably depleted of Daxx which also show increased stability of cyclin B1. Thus, protein Daxx, that was previously characterized as a transcription repressor and an apoptotic-related protein, may also have a novel function in mitosis progression as a prometaphase checkpoint release protein in control conditions and upon mitotic stress. In order to understand the mechanism by which Daxx orchestrates mitosis progression and identify new potential targets responsible for taxane resistance, we adopted a functional proteomic approach to identify Daxx-associated mitotic complex. We optimized the protocol for Tag based affinity purification in a way to obtain Daxx expressed downstream of FLAG Tag in tandem with HA Tag (FH) and a Thrombin Cleavage Site (TCS). HEp2 cells transduced with pOZ-FH-TCS-Daxx and the empty pOZ-FH plasmid (control), synchronized by double thymidine block and next arrested in pro-metaphase with paclitaxel, were used to purify Daxx containing mitotic complex. Mitotic specific Daxx interacting proteins were identified by SDS-PAGE followed by mass spectrometry. The functional characterization of newly identified Daxx mitotic partners will shed lights on the complex mechanism/phenomena of taxanes resistance and ultimately offer the molecular basis for successful treatment strategies of breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2011-2988