Abstract 696: BRCA1-mediated ubiquitination of tubulin may serve as a signal for taxol-induced apoptosis
Authors: Matthew Sung, Paraskevi Giannakakou
Journal: Cancer Research (2011)

Abstract The taxanes are among the most effective chemotherapeutic agents used for the treatment of solid tumors, yet eventually most tumors become resistant to taxane chemotherapy. Mechanisms responsible for clinical resistance to taxanes have not been fully elucidated. Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene, whose expression has been clearly correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer (NSCLC). However, the molecular mechanism underlying the relationship between BRCA1 expression and taxane activity remains unclear. BRCA1 is a nuclear shuffling protein with many more described nuclear actions than cytoplasmic. BRCA1 has been shown to regulate the cell's ability to undergo apoptosis and that increased BRCA1 cytoplasmic localization correlates with increased apoptotic functions. In association with BARD1, the BRCA1/BARD1 complex functions as an E3 ubiquitin ligase. Recent literature suggests γ-tubulin is a target of this complex resulting in mono-ubiquitination of γ-tubulin which regulates its centrosomal localization and function. Here, we test the hypothesis that either α- or β-tubulin also serve as a target for the BRCA1/BARD1 E3 ubiquitin ligase activity which regulates the cell's sensitivity to Taxol. To approach this, we generated a stable-BRCA1 knockdown (B1-KD) of the NSCLC cell line A549 in which an 80% knockdown of BRCA1 results in resistance to Taxol when compared to the parental line, phenocopying the clinical data correlating low BRCA1 expression with taxane resistance. The B1-KD line exhibited aberrant centrosomal amplification and activity likely due to the γ-tubulin mislocalization. Then, we looked at the ability of BRCA1 to ubiquinate α- or β- tubulin subunits that form the microtubule cytoskeleton. Tubulin immunoprecipitation experiments revealed that the B1-KD results in a loss of tubulin ubiquitination when compared to the parental line. Taxol treatment in both cell lines seems to increase tubulin ubiquitination suggesting that the residual BRCA1 in the knockdown line is enough to ubiquitinate tubulin. Currently, we are looking at mechanisms linking tubulin ubiquitination to cell death, mainly capthesin-B-, calpain- or caspase-mediated apoptosis. Initial experiments suggest that capthesin-B inhibition does not inhibit Taxol-induced cell death in both the parental and B1-KD A549 cell line. We propose a model in which Taxol-induced tubulin ubiquitination, via the BRCA1/BARD1 complex, serves as the signal to initiate downstream cell death signaling pathways in the presence of Taxol treatment. This model would assign a strong cytoplasmic role for BRCA1 and show BRCA1 regulation of apoptosis may exist outside of the nucleus. It might also provide insight into how lung cancer cells become resistant to taxanes with the potential to disrupt these resistance mechanisms and enhance therapeutic efficacy. 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 696. doi:10.1158/1538-7445.AM2011-696