Abstract 2318: The microtubule cytoskeleton in the dynamic regulation of translation: implications for taxane therapy.

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Abstract Microtubule (MT)-targeting drugs, and in particular the MT-stabilizing drug Taxol, are the most widely used and most effective class of drugs in clinical oncology. However, certain tumor types are refractory to MT-based therapy, and even responsive tumors often acquire drug resistance. Today, 20 years following Taxol's FDA approval we still fail to understand the molecular basis of taxane clinical response. Traditionally, taxane activity has been attributed solely to their anti-mitotic effects; however, we and others have shown that interphase MT functions are the “true” taxane targets mediating clinical activity. In support of this hypothesis we identified a novel mechanism of MT-dependent RNA translation. We found that MTs are intricately involved in regulating hypoxia inducible factor (HIF-1α) translation. Taxol-induced MT stabilization released HIF-1α mRNA from polysomes, and enriched HIF-1α mRNA in Argonaute 2 (Ago2)-containing P-bodes, where HIF-targeting miRNAs were also recruited. MT repolymerization allowed HIF mRNA to re-enter active translation, suggesting that MTs exert a tight control over HIF-1α. Our data have revealed a previously unexplored role for MTs in cancer cell biology and identified an unconventional link between the MT cytoskeleton and HIF protein synthesis. Interestingly, our work suggests that other mRNAs are also regulated in a MT-dependent manner. Polysome profiles in several cancer cell lines consistently showed a striking increase in the 80S monosomal peak following MT disruption, a hallmark of translation inhibition. In addition, taxane treatment resulted in P-body recruitment of two miRNAs not predicted to target HIF-1α, suggesting that other mRNAs are also repressed following MT disruption. Therefore, it is crucial to determine which other mRNAs, important for cancer cell survival, are affected by taxane treatment. To identify MT-dependent messages, we are currently performing polysome profiling coupled with RNA-Seq to identify which mRNAs change translational status following taxane treatment. In parallel, we are also performing RNA-Seq of Ago2-associated mRNAs to identify which mRNAs are enriched in P-bodies following taxane treatment. Preliminary polysome profiling experiments in MDA-MB-231 breast cancer cells yielded 116 transcripts with 2-fold or greater repression of translation upon Taxol treatment, including Aurora A kinase and several cyclin-dependent kinases, which has been validated at the protein level. Importantly, we found that a substantial fraction of the mRNAs that become untranslated are also among those most highly bound by Ago2 upon Taxol treatment, assessed by gene set enrichment analysis (GSEA). Our work has the potential to identify additional mRNAs whose translation is MT-dependent, and therefore lead to novel target discovery whose therapeutic exploitation can synergize with existing taxane-based chemotherapy. Citation Format: Marisa Carbonaro, Duane Hassane, Paraskevi Giannakakou. The microtubule cytoskeleton in the dynamic regulation of translation: implications for taxane therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2318. doi:10.1158/1538-7445.AM2013-2318