Abstract 1158: Microtubule perturbation regulates remodeling of tumor microenvironment by modulating the composition of tumor cell secretome
Authors: Kita Katsuhiro , Tran Andy , Brown Lewis M. , Hassane Duane C. , Carey Shawn , Matov Alexandre , Reinhart-King Cynthia A. , Giannakakou Paraskevi 
Published: May 2015

Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, MT-targeting drugs, such as the taxanes, often fail in the metastatic setting and mechanisms underlying drug resistance are poorly elucidated. An important process during tumor development and metastasis involves the dynamic remodeling of the 3D microenvironment surrounding the tumor cells which enables them to successfully proliferate and metastasize. We hypothesized that tumor cell MTs may regulate the cellular signaling process that controls tumor-mediated remodeling of the microenvironment by paracrine activation of fibroblasts. To assess the role of MT dynamics in the secretion of factors from the tumor cells that mediate communication with the microenvironment, we collected conditioned medium (CM) from untreated or paclitaxel (PTX)-pre-treated metastatic breast cancer MDA-MB-231 cells and applied it to HMF3S human mammary fibroblasts. We observed that CM from untreated 231 cells resulted in fibroblast activation as evidenced by their increased directional 3D cell motility towards a serum gradient. CM from PTX-pretreated 231 cells, at concentrations that suppress MT dynamics, but not from the PTX-resistant β-tubulin mutation 231 clone, 231K20T cells, did not result in activation of fibroblasts' 3D motility, suggesting that MT-mediated secretion of soluble factor(s) (secretome) underlies fibroblast activation. Mass-spectrometry analysis of CM derived from untreated or PTX-pretreated 231 cells identified proteins whose secretion was diminished following suppression of MT dynamics and which are involved in cell motility, cell-cell communication, or ECM remodeling, such as TGFβ, CTGF, c-Met, fibronectin, and lysyl oxidase 2. Treatment of 231-derived CM with an anti-TGFβ blocking antibody diminished HMF3S fibroblast motility in 3D, suggesting that the presence of TGFβ in the tumor cell secretome mediated the activation of tumor associated fibroblasts. To further probe the MT-dependent regulation of TGFβ secretion we showed that PTX treatment prevented trafficking of intracellular TGFβ to the cell surface in 231 cells, likely underlying its reduced secretion. Confocal reflectance and two-photon microscopic imaging revealed that CM treatment of HMF3S fibroblasts induced significant collagen remodeling comparable to treatment with exogenous TGFβ alone. Our findings point to a novel role of interphase MTs in tumor cell secretome and raise the possibility that MTs may regulate secretion of distinct soluble factors in different types of tumor cells, which then mediate the communication between tumor cell compartment and the tumor microenvironment. These novel insights have significant implications for the mechanism of action and resistance to MT inhibitors.

Citation Format: Katsuhiro Kita, Andy Tran, Lewis M. Brown, Duane C. Hassane, Shawn Carey, Alexandre Matov, Cynthia A. Reinhart-King, Paraskevi Giannakakou. Microtubule perturbation regulates remodeling of tumor microenvironment by modulating the composition of tumor cell secretome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2014-1158