Establishing An Experimental Paradigm To Study The Interphase Effects Of Microtubule Targeting Agents

Abstract Microtubule targeting agents (MTAs) are highly effective anticancer drugs. While these drugs were traditionally classified as antimitotics, compelling evidence suggests that the ability of MTAs to interrupt microtubule-dependent trafficking and signaling in interphase cells contributes to their anticancer efficacies. Previous studies of the interphase effects of MTAs on oncogenic signaling pathways have led to an important reevaluation of their mechanisms of anticancer actions. These effects have been primarily reported in cells that have been treated with MTAs for extended periods of time. However, MTAs rapidly alter microtubule dynamics which results, within a few hours, in global changes to gene expression and cellular signaling. We propose that evaluating the effects of MTAs on oncogenic pathways at times and concentrations that are associated with early microtubule disruption will allow analysis of the initiating events that link the direct action of MTAs on microtubule structure and dynamics to effects on interphase signaling that contribute to anticancer efficacy. The concentrations and treatment times that were optimal for the study of clinically used MTAs on cellular trafficking and signaling events were first determined. Our experimental paradigm of a 2 h treatment of breast cancer cells with clinically relevant concentrations of MTAs eliminated contributions due to mitotic accumulation and changes in gene expression associated with longer treatments. While all MTAs disrupt microtubule dynamics, the differences among MTAs in their rapid downstream effects on cellular signaling have not been systematically evaluated. A goal of this project was to identify differences in the effects of diverse MTAs on interphase signaling pathways that may underlie their differential efficacy in patient populations. This short term treatment paradigm led to the identification of profound differences among MTAs in their ability to disrupt Src-dependent E-cadherin re-localization, canonical and non-canonical TGF-β signaling, and β-catenin localization. These findings demonstrate the ability of diverse MTAs to rapidly impact interphase oncogenic signaling and trafficking pathways. This experimental design sets forth a method to evaluate the initial effects of diverse MTAs to gain critical insight into their differential abilities to inhibit key oncogenic signaling pathways. These types of studies might, in the future, help facilitate the rational selection of specific MTAs for patients depending on tumor characteristics. Funding for this work was provided by Eisai Inc. Citation Format: April L. Risinger, Nicholas F. Dybdal-Hargreaves, Roma Kaul, Allison D. Clark, Susan L. Mooberry. Establishing an experimental paradigm to study the interphase effects of microtubule targeting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5115. doi:10.1158/1538-7445.AM2017-5115