Studies have shown that transforming acidic coiled-coil protein 3 (TACO), a key component of centrosome-microtubule dynamic networks, is significantly associated with various types of human cancer. We have recently reported that high levels of TACO are found in breast cancer, lead to the accumulation of spontaneous DNA damage due to defective DNA damage response signaling, and confer cellular sensitivity to radiation and poly(ADP-ribose) polymerase (PARP) inhibitors. Although our study suggests a potential role of TACO as a biomarker in breast cancer detection and prediction of therapy outcome, its role as a therapeutic target in breast cancer is not well studied. In this study, we show that a small molecule TACC3 inhibitor, KHS101, suppresses cell growth, motility, epithelial-mesenchymal transition (EMT), and breast cancer cell sternness while it induces apoptotic cell death. Quantitative multiplexed proteomic analysis using tandem mass tags (TMTs) revealed that KHS101 alters multiple biological processes and signaling pathways, and significantly reduces the expression of mitotic kinases Aurora A and Polo-like kinase 1 (PLK1), which are closely associated with TACO. Our findings therefore provide a new insight into the potential mechanisms of the action of KHS101 and suggest its possible use as a dual or multi-targeting mitotic inhibitor in breast cancer. (C) 2018 Elsevier Inc. All rights reserved.