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A Small Compound Targeting TACC3 Revealed Its Different Spatiotemporal Contributions for Spindle Assembly in Cancer Cells

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Oncogene (2014) 33, 4242–4252 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc ORIGINAL ARTICLE A small compound targeting TACC3 revealed its different spatiotemporal contributions for spindle assembly in cancer cells RYao1, Y Kondoh2, Y Natsume1, H Yamanaka1, M Inoue1,3,HToki3, R Takagi2, T Shimizu2, T Yamori4, H Osada2 and T Noda1,3 The mitotic spindle is assembled by the coordinated action of centrosomes and kinetochore microtubules. An evolutionally conserved protein family, transforming acidic coiled-coil (TACC), has been shown to be involved in this process. In humans, TACC3 is aberrantly expressed in a variety of human cancers, but its biological significance remains to be elucidated. Here, using a novel compound targeting TACC3, spindlactone (SPL), we show that the perturbation of TACC3 selectively inhibited the nucleation of centrosome microtubules in ovarian cancer cells. In contrast to centrosome microtubules, the kinetochore microtubules were robustly assembled, forming ectopic spindle poles that resulted in multipolar spindles. Interestingly, the extensive inhibition of TACC3 partially suppressed the nucleation of kinetochore microtubules. These dose-dependent effects of SPL were consistent with the results observed by the depletion of TACC3 and its binding partner, colonic and hepatic tumor overexpressed gene protein (TOGp). Although these proteins both have roles in the assembly of centrosome and kinetochore microtubules, their contributions were spatiotemporally different. Notably, SPL did not affect spindle assembly in normal cells. Furthermore, the oral administration of SPL significantly suppressed tumor growth in vivo. The unique mechanism of action of SPL not only enables it to be used as a tool to dissect the molecular basis of spindle assembly but also to provide a rationale for the use of TACC3 as a molecular target for cancer treatment. This rationale offers an opportunity to develop new strategies for cancer chemotherapy that overcome the limitations of microtubule toxins and expand their scope and clinical efficacy. Oncogene (2014) 33, 4242–4252; doi:10.1038/onc.2013.382; published online 30 September 2013 Keywords: TACC3; TOGp; spindle assembly INTRODUCTION one of the sister kinetochores on a chromosome.7 Although this The mitotic spindle is a well-established molecular target for ‘search and capture’ mechanism appears to be the predominant cancer chemotherapy, and clinically, drugs targeting microtubules means by which chromosomes become attached and aligned on are used to treat hematological and solid malignancies. In tumor the spindle, mathematical modeling studies have indicated that cells, the anti-mitotic effects of these drugs are thought to result unbiased search and capture alone is not sufficient to align all the from the suppression of the dynamic instability of microtubules, chromosomes on the mitotic spindle within a normal timeframe.8 which is required for spindle assembly during mitosis. However, Growing evidence indicates that in addition to centrosomes, these drugs also inhibit microtubule function in non-dividing cells, chromosomes also drive spindle assembly. This chromosome- which results in serious side effects, such as peripheral neuro- driven mitotic spindle formation was originally thought to function pathy. In addition, resistance to microtubule-targeted drugs is only in meiosis, a process in which centrosomes are not present. acquired by the overexpression of specific tubulin isoforms.1 To However, subsequent studies have indicated that this process overcome these problems, new therapeutic agents are being works cooperatively with centrosome microtubules to assemble developed to target the mitotic proteins involved in spindle bipolar mitotic spindles in somatic cells.9–11 In these systems, assembly rather than the tubulin itself.2 Among these mitotic kinetochore microtubules support effective spindle assembly by proteins, the motor protein KIF-11, which is also called Eg5; the guiding centrosome microtubule growth toward the kinetochores. Aurora kinases; and the Polo-like kinases have been extensively However, the direct evaluation of kinetochore microtubules in studied as potential drug targets. Although the antitumor somatic cells is hampered by the mass of centrosome microtubules activities of these agents have been demonstrated both in vitro that rapidly grow after nuclear envelope breakdown (NEB). Thus, and in xenograft models,3–5 the scope of their clinical efficacy the relative contributions of centrosome and kinetochore appears to be limited.6 Thus, novel therapeutic targets that microtubules to spindle assembly remain to be elucidated. selectively perturb spindle microtubules without affecting Human transforming acidic coiled-coil (TACC3) was originally microtubule functions in post-mitotic cells are beneficial to the identified within a translocation breakpoint region that was development of effective therapeutics for cancer treatment. associated with multiple myelomas,12 and subsequent studies In vertebrate somatic cells, spindle microtubule nucleation occurs have indicated that it is aberrantly expressed in various cancers.13–16 at the centrosome, which contains plus ends that dynamically grow TACC proteins are evolutionally conserved, from yeasts to humans. and shrink, probing space until they are captured and stabilized by In Drosophila, a microtubule-associated protein, termed msps, was 1Department of Cell Biology, Cancer Institute, The Japanese Foundation for Cancer Research, Tokyo, Japan; 2Chemical Biology Core Facility, RIKEN Advanced Science Institute, Saitama, Japan; 3Team for the Advanced Development and Evaluation of Human Disease Models, Bioresource Center, RIKEN, Tsukuba, Japan and 4Division of Molecular Pharmacology, Cancer Chemotherapy Center, The Japanese Foundation for Cancer Research, Tokyo, Japan. Correspondence: Dr R Yao, Department of Cell Biology, Cancer Institute, The Japanese Foundation for Cancer Research, 3-8-31, Ariake, Koto-ku, Tokyo 135-8550, Japan. E-mail: [email protected] Received 11 March 2013; revised 17 July 2013; accepted 9 August 2013; published online 30 September 2013 Spindle aberration by the TACC3 inhibitor R Yao et al 4243 identified as a binding partner of the D-TACC protein, and their the investigation of the molecular mechanisms involved in spindle interaction is thought to be critical for the function of TACC proteins assembly and for the development of new cancer chemotherapy in promoting microtubule assembly.17,18 A Xenopus homolog of strategies. msps, XMAP215, regulates microtubule plus-end assembly and is a processive microtubule polymerase.19 On the basis of these findings, the current model suggests that TACC proteins recruit msps/XMAP215 to the centrosome, enhancing microtubule RESULTS polymerization.20 Interestingly, tumor overexpressed gene protein The Identification of small compounds that inhibit TACC3 (TOGp), the human homolog of msps/XMAP215, which was To explore the role of TACC3 in spindle assembly and to develop a originally identified as a gene that was overexpressed in colon novel cancer chemotherapeutic targeting TACC3, we searched for and hepatic cancers,21 has been shown to physically interact with small compounds that were able to inhibit TACC3 function. We human TACC3.18 Furthermore, TOGp depletion has been shown to utilized a chemical array onto which small compounds obtained induce multipolar spindles, but the molecular basis of this action from the Natural Products Depository (NPDepo) chemical library remains under debate.22–24 The depletion of TACC3 leads to a range had been immobilized (Figure 1a).28 Using a cell lysate that of phenotypes, including a minor decrease in microtubule expressed a TACC3-DsRed fusion protein as a probe, we screened polymerization,22 chromosome misalignment leading to caspase- 6800 compounds and identified 70 as candidate TACC3- dependent apoptosis25 and declustered centrosomes in cancer cells interacting molecules. with supernumerary centrosomes.26 Using conditional knockout To determine the effects of the candidate compounds on mice, we previously demonstrated that disrupting Tacc3 in mitosis, we performed a visual screen using single cell-based lymphoma cells induced the formation of multipolar spindles.27 analysis. We used ovarian cancer cells because previous studies Interestingly, an in vivo analysis demonstrated that the thymic have indicated that TACC3 is aberrantly expressed in this type of lymphoma tissues underwent massive apoptosis, leading to tumor cancer.14,29 Cells expressing enhanced green fluorescent protein regression, whereas the normal thymus exhibited no overt (EGFP)-a-tubulin were synchronized using a double thymidine abnormalities. These observations indicate that Tacc3 is an block, and the candidate compounds were added to the medium important component of spindle assembly in cancer cells and is a at the release from the second block. Fluorescence images were potential target for cancer chemotherapy. In this study, we collected, and the image sequences were analyzed to determine developed a novel chemical compound that perturbed TACC3 the duration of mitosis. function. The pharmacological inhibition of TACC3 revealed Among the 70 candidates, four small molecules, NPD3574, different spatiotemporal contributions of TACC3 in spindle NPD2568, NPD4089 and NPD2448, induced mitotic arrest in a dose- formation. This unique mechanism of action provides a tool for dependent manner (Supplementary Figures S1a, b). Interestingly,
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