Cancer-associated centrosomal transforming acidic coiled coil (TACC) proteins are involved in mitotic spindle function. By employing gene targeting, we have recently described a nonredundant and essential role of TACC3 in regulating cell proliferation. In this study, we used an inducible RNA interference approach to characterize the molecular function of TACC3 and its role in mitotic progression and cell survival. Our data demonstrate that a TACC3 knockdown arrests G 1 checkpoint-compromised HeLa cells prior to anaphase with aberrant spindle morphology and severely misaligned chromosomes. Interestingly, TACC3-depleted cells fail to accumulate the mitotic kinase Aurora B and the checkpoint protein BubR1 to normal levels at kinetochores. Moreover, localization of the structural protein Ndc80 at outer kinetochores is reduced, indicating a defective kinetochore-microtubule attachment in TACC3-deficient cells. As a consequence of prolonged TACC3 depletion, cells undergo caspase-dependent cell death that relies on a spindle checkpoint-dependent mitotic arrest. TACC3 knockdown cells that escape from this arrest by mitotic slippage become highly polyploid and accumulate supernumerary centrosomes. Similarly, deficiency of the post-mitotic cell cycle inhibitor p21 WAF exacerbates the effects of TACC3 depletion. Our findings therefore point to an essential role of TACC3 in spindle assembly and cellular survival and identify TACC3 as a potential therapeutic target in cancer cells.The centrosome organizes the bipolar mitotic spindle to ensure faithful separation of chromosomes during mitosis (1). Spindle poles, kinetochores, and various microtubule-associated proteins are involved in the regulation of microtubule dynamics (2). The assembly of the mitotic spindle is a highly dynamic process and tightly controlled by the cell cycle. On the other hand, alterations in centrosome and mitotic spindle architecture have profound consequences for cell cycle progression and lead to chromosomal instability, aneuploidy, and cell death (1, 3-6). The finding that many cancer cells have genetic instability and centrosomal abnormalities has generated much interest in studying the role of chromosomal missegregation and aneuploidy for tumorigenesis.Members of the transforming acidic coiled-coil (TACC) 3 family function as important structural components of the centrosome/spindle apparatus (7). TACC proteins are evolutionarily conserved and share a 200-amino acid coiled coil motif at their C terminus but have only limited homology outside this domain (8). TACC proteins interact with the microtubule-stabilizing protein ch-TOG/Msps/XMAP215 (9) that is important for centrosome integrity, centrosome-dependent assembly of microtubules, and spindle stability (10 -12). Moreover, the Xenopus TACC homologue Maskin is involved in translational mRNA regulation during oocyte development (13), a TACC function so far not observed in mammals. Although there is only one TACC gene in Drosophila and Xenopus, the mammalian TACC family consists of three genes. In...

Microtubule-interfering cancer drugs such as paclitaxel (PTX) often cause chemoresistance and severe side effects, including neurotoxicity. To explore potentially novel antineoplastic molecular targets, we investigated the cellular response of breast carcinoma cells to short hairpin(sh)RNA-mediated depletion of the centrosomal protein transforming acidic coiled coil (TACC) 3, an Aurora A kinase target expressed during mitosis. Unlike PTX, knockdown of TACC3 did not trigger a cell death response, but instead resulted in a progressive loss of the pro-apoptotic Bcl-2 protein Bim that links microtubule integrity to spindle poison-induced cell death. Interestingly, TACC3-depleted cells arrested in G 1 through a cellular senescence program characterized by the upregulation of nuclear p21 WAF , downregulation of the retinoblastoma protein and extracellular signal-regulated kinase 1/2, formation of HP1c (phospho-Ser83)-positive senescence-associated heterochromatic foci and increased senescence-associated b-galactosidase activity. Remarkably, the onset of senescence following TACC3 knockdown was strongly accelerated in the presence of non-toxic PTX concentrations. Thus, we conclude that mitotic spindle stress is a major trigger of premature senescence and propose that the combined targeting of the centrosomal Aurora A-TACC3 axis together with drugs interfering with microtubule dynamics may efficiently improve the chemosensitivity of cancer cells.

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Regulators of the mitotic spindle apparatus are attractive cellular targets for antitumor therapy. The centrosomal protein transforming acidic coiled coil (TACC) 3 is required for spindle assembly and proper chromosome segregation. In this study, we employed an inducible RNA interference approach to downregulate TACC3 expression. We show that TACC3 knock-down in NIH3T3 fibroblasts caused aneuploidy, but failed to overtly impair mitotic progression. TACC3 depletion rather triggered a postmitotic p53-p21 WAF pathway and led to a reversible cell cycle arrest. Similar effects were induced by low concentrations of paclitaxel, a spindle poison used in antitumor therapy. Interestingly, however, and unlike in TACC3-proficient cells, paclitaxel was able to induce strong polyploidy and subsequent apoptosis in TACC3-depleted cells. Even though paclitaxel treatment was associated with the activation of the survival kinase Akt and an antiapoptotic expression of cytoplasmic p21 WAF and cyclin D1, this inhibition of cell death was abrogated by depletion of TACC3. Thus, our data identify TACC3 as a potential target to overcome p21 WAF -associated protection of transformed cells against paclitaxel-induced cell death.