Abstract 5162: Microtubule stabilization alters tumor secretome and fibroblast activation.

Abstract

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, drugs targeting MTs, like the taxanes, often fail in the metastatic setting. Therefore it is of utmost importance to fully understand the roles of MTs and MT-targeting agents in tumor biology. An important process during tumor development and metastasis involves the remodeling of the 3D microenvironment surrounding tumor cells. In order to successfully proliferate and metastasize, tumor cells need to dynamically respond and remodel their surrounding microenvironment. We hypothesized that tumor cells may stimulate residential fibroblasts in MT-dependent manner, then activated fibroblasts may play a major role in remodeling of 3D microenvironment. To test this hypothesis, we assessed the MT-dependence of metastatic MDA-MB-231 breast cancer cell activation of HMF3S human mammary fibroblasts. HMF3S fibroblasts were treated with either vehicle- or paclitaxel (PTX)-pretreated MDA-MB-231 cell conditioned media. Vehicle conditioned media activated HMF3S fibroblasts as shown by increased directional 3D motility towards a serum gradient. In contrast, PTX-pretreatment abolished the increased directional motility of the fibroblasts. Importantly, vehicle condition media from PTX-treated K20T MDA-MB-231 cells, which carry a tubulin mutation at taxane binding site, did not show reduced 3D motility of fibroblasts. Together, these results suggest that breast cancer cells produce fibroblast activation factor(s) in MT dependent manner. In order to identify the factors responsible for this MT-dependent cell-cell communication, we performed a differential proteomic analysis of the conditioned media from vehicle- and PTX-pretreated MDA-MB-231 cells. Using splitless nanoflow chromatography coupled with quadrupole time-of-flight (QTOF) mass spectrometry, we identified a total of 2124 proteins within these tumor secretomes. Using on a 2-fold change threshold, we identified 78 under-represented and 31 over-represented proteins in the secretome of PTX- versus vehicle-pretreated cells. We focused on under-represented proteins based on the hypothesis that MT stabilization would inhibit their secretion. We have identified proteins involved in cell motility, cell-cell communication, or extracellular matrix remodeling. TGF-β, CTGF, c-Met, fibronectin, and lysyl oxidase 2 were the key down-regulated proteins identified in the tumor secretome upon PTX-induced MT stabilization. Among the factors identified by mass spectrometry analyses, TGF-β has been known to play an important role in cell migration. Supporting this idea, treatment of vehicle-pretreated condition media with TGF-β blocking antibody abolished 3D migration of fibroblasts. These findings shed a light on a novel role of interphase MTs in tumor biology and may pave the way to enhance our understanding of taxane clinical efficacy. Citation Format: Andy Tran, Katsuhiro Kita, Alexandre Matov, Duane C. Hassane, Lewis M. Brown, Paraskevi Giannakakou. Microtubule stabilization alters tumor secretome and fibroblast activation. [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 5162. doi:10.1158/1538-7445.AM2013-5162