Oncogene (2003) 22, 8102–8116 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc TACC1–chTOG–Aurora A protein complex in breast cancer Nathalie Conte1,Be´ ne´ dicte Delaval1, Christophe Ginestier1, Alexia Ferrand1, Daniel Isnardon2, Christian Larroque3, Claude Prigent4, Bertrand Se´ raphin5, Jocelyne Jacquemier1 and Daniel Birnbaum*,1 1Department of Molecular Oncology, U119 Inserm, Institut Paoli-Calmettes, IFR57, Marseille, France; 2Imaging Core Facility, Institut Paoli-Calmettes, Marseille, France; 3E229 Inserm, CRLC Val d’Aurelle/Paul Lamarque, Montpellier, France; 4Laboratoire du cycle cellulaire, UMR 6061 CNRS, IFR 97, Faculte´ de Me´decine, Rennes, France; 5Centre de Ge´ne´tique Mole´culaire, Gif-sur-Yvette, France The three human TACC (transforming acidic coiled-coil) metabolism, including mitosis and intracellular trans- genes encode a family of proteins with poorly defined port of molecules, is progressing but many components functions that are suspected to play a role in oncogenesis. remain to be discovered and characterized. We describe A Xenopus TACC homolog called Maskin is involved in here the interaction of the TACC1 protein with several translational control, while Drosophila D-TACC interacts protein partners that makes it a good candidate to with the microtubule-associated protein MSPS (Mini participate in microtubule-associated processes in nor- SPindleS) to ensure proper dynamics of spindle pole mal and tumoral cells. microtubules during cell division. We have delineated here In mammals, TACC1 (Still et al., 1999a) belongs to a the interactions of TACC1 with four proteins, namely the family of three paralogous (Popovici et al., 2001) genes microtubule-associated chTOG (colonic and hepatic that also includes TACC2/AZU1/ECTACC (Chen et al., tumor-overexpressed gene) protein (ortholog of Drosophi- 2000; Pu et al., 2001; Lauffart et al., 2003) and TACC3/ la MSPS), the adaptor protein TRAP (tudor repeat ERIC1 (Still et al., 1999b; Sadek et al., 2000; McKeve- associator with PCTAIRE2), the mitotic serine/threonine ney et al., 2001; Hao et al., 2002; Sadek et al., 2003). kinase Aurora A and the mRNA regulator LSM7 Mammalian TACC have known orthologs in Drosophila (Like-Sm protein 7). To measure the relevance of the melanogaster, named tacc (Gergely et al., 2000b), and in TACC1-associated complex in human cancer we have Xenopus laevis, named Maskin (Stebbins-Boaz et al., examined the expression of the three TACC, chTOG and 1999). Drosophila tacc encodes D-TACC, a centrosomal Aurora A in breast cancer using immunohistochemistry on protein required for normal spindle function in the early tissue microarrays. We show that expressions of TACC1, embryo (Gergely et al., 2000b); D-TACC interacts with TACC2, TACC3 and Aurora A are significantly corre- the microtubule-associated MSPS (Mini SPindleS) lated and downregulated in a subset of breast tumors. protein to regulate microtubule behavior (Gergely Using siRNAs, we further show that depletion of chTOG et al., 2000b; Cullen and Ohkura, 2001; Lee et al., and, to a lesser extent of TACC1, perturbates cell 2001; Theurkauf, 2001). This complex is regulated by division. We propose that TACC proteins, which we also the Aurora A serine/threonine kinase (Giet et al., 2002). named ‘Taxins’, control mRNA translation and cell D-TACC and TACC3 associate with fly and human division in conjunction with microtubule organization Aurora A kinase, respectively (Giet et al., 2002). The and in association with chTOG and Aurora A, and that msps gene has orthologs in other species named zyg-9 in these complexes and cell processes may be affected during Caenorhabditis elegans, XMAP215 in X. laevis, and mammary gland oncogenesis. chTOG (colonic and hepatic tumor overexpressed gene) Oncogene (2003) 22, 8102–8116. doi:10.1038/sj.onc.1206972 in humans (Charrasse et al., 1995, 1998; Charrasse and Larroque, 2000; Spittle et al., 2000). The TACC1– Keywords: Aurora kinase; breast cancer; cell division; chTOG interaction has been recently described in a two- chTOG protein; microtubule; SM proteins; TACC hybrid screen in yeast (Lauffart et al., 2002). protein; tissue microarray; RNAi Maskin associates with both the 30UTR-CPE binding protein (CPEB) and the 50UTR CAP-binding protein eIF-4E to regulate mRNAs translation during oocyte Introduction maturation in Xenopus (Stebbins-Boaz et al., 1999); CPEB and Maskin are present on the mitotic apparatus The identification of protein complexes involved in of animal pole blastomeres in embryos (Groisman et al., processes associated with microtubule structure and 2000). We have recently shown that TACC1 interacts with LSM7 and SmG (Conte et al., 2002), two proteins related to the yeast Sm small nuclear ribonucleoproteins *Correspondence: D Birnbaum, U119 Inserm, 27 Bd Lei Roure, 13009 Marseille, France; E-mail: [email protected] (snRNPs). The Sm/LSM proteins associate with small The first two authors contributed equally to the work nuclear RNAs to form the core of snRNPs required Received 25 April 2003; revised 2 July 2003; accepted 10 July 2003 for a large variety of RNA maturation processes and TACC1–chTOG–Aurora protein complex N Conte et al 8103 mRNA degradation (Salgado-Garrido et al., 1999; Bouveret et al., 2000; Tharun et al., 2000). To date, few other mammalian TACC-interacting partners have been identified (Sadek et al., 2000; Lauffart et al., 2002, 2003; Steadman et al., 2002; Aitola et al., 2003). TACC proteins (designated hereafter as ‘Taxins’), under regulation by kinases, could intervene in micro- tubule-associated processes such as mitotic spindle formation (reviewed in Gergely, 2002; Lappin et al., 2002) and transport of RNAs specifically to spindles and centrosomes and to subcellular areas necessary for cell polarity (Richter, 2001). Variation in the structure and/ or expression of Taxins and Taxins partners may have consequences on cellular growth (Raff, 2002). Human TACC were initially characterized as potential cancer genes (Still et al., 1999a, b; Chen et al., 2000). TACC1 is downregulated in breast cancer (Conte et al., 2002) and has been characterized as a gastric tumor antigen (Line et al., 2002a, b). TACC2/AZU1 is downregulated in transformed cell lines (Chen et al., 2000). The knockout of the mouse Tacc3 gene causes embryonic lethality associated with a high rate of apoptosis in several cell lineages (Piekorz et al., 2002). Overexpression of chTOG occurs in human hepatomas and colonic tumors (Charrasse et al., 1995). The STK6 gene, encoding the Aurora A kinase, is localized in the 20q13 chromosomal region, which is frequently amplified in cancers, and Figure 1 Conservation of the Taxin family in metazoans. An overexpression of Aurora A leads to cell transformation unrooted phylogenetic tree (neighbor-joining method, Poisson correction, 225 sites from C-terminal region) of 10 TACC proteins (for reviews, see Bischoff and Plowman, 1999; Giet and (Taxins) is shown (Cel: C. elegans, Dme: D. melanogaster, Cin: C. Prigent, 1999; Nigg, 2001; Dutertre et al., 2002). intestinalis, Hsa: H. sapiens, Mmu: M. musculus, Xla: X. laevis, We have demonstrated and delineated here the Ocu: Oryctolagus cuniculus). Significant bootstrap values (out of interaction between TACC1 and protein partners, 500 replicates) are indicated at each node. Rabbit TACC4 (TACC4 Ocu) is actually a wrongly named TACC3 (Steadman et al., 2002). including LSM7, chTOG, Aurora A kinase and Branch lengths are proportional to time (0.1 Byr). Protostomians TRAP/PCTAIRE2BP. We have measured the relevance (i.e. Drosophila and C. elegans) and nonvertebrate chordates of the potential TACC1-associated complex in human (urochordate ascidian C. intestinalis) have only one TACC cancer by determining the simultaneous expression of member. The topology of the tree is the same using the maximum the three Taxins and of two Taxin-interactor proteins in parsimony method around 400 breast cancer samples using immunohisto- chemistry on tissue microarrays. We have further shown serine- and proline-rich motifs named SPAZ. We that inhibition of TACC1 or chTOG transcription leads screened a human mammary gland cDNA expression to abnormal cell division. Finally, in view of these library with two different TACC1 proteins (TACC1s results, we propose a preliminary model of TACC1 and TACC1spaz region) fused to the GAL4-binding function. domain. We isolated four clones using TACC1s as bait (Figure 2a). Two of them corresponded to the C- terminal region (aa 1776–1980) of the chTOG protein and the two others to LSM7, which we had already Results found in a previous screen using TACC1l as bait (Conte et al., 2002). We isolated seven clones using TACC1spaz Phylogenetic analysis of the TACC family region as bait; two of them encoded the C-terminal Through searches in databases, we identified one tacc region (aa 520–708) of TRAP(tudor repeat associator gene in the chordate nonvertebrate Ciona intestinalis with PCTAIRE 2). The TRAP gene, also called and built a phylogenetic tree of the Taxins (Figure 1). PCTAIRE2BP, encodes a protein that contains five The tree shows the relationships of the three human Tudor domains. TRAPassociates with serine/threonine Taxins and orthologs. kinase PCTAIRE2 (Hirose et al., 2000), which is expressed in terminally differentiated neurons (Hirose et al., 1997). Identification of TACC1-interactors by in vitro methods To document TACC1 interactions with these poten- We used the two-hybrid system in yeast to identify tial partners, we produced GST-fusion proteins that potential TACC1-interacting partners. TACC1 encodes were used in pull-down experiments using HA–TACC1l at least two protein isoforms, a long form, TACC1l, and (Figure 2b) or myc-TACC1s (Figure 2c). GST–chTOG, a short form, TACC1s. The latter lacks two series of GST–TRAPand GST–SmG precipitated TACC1l Oncogene TACC1–chTOG–Aurora protein complex N Conte et al 8104 Figure 2 Identification of TACC1 protein partners using a two-hybrid screen in yeast. (a) The TACC proteins used as baits in a previous (TACC1l – Conte et al., 2002) and present (TACC1s and TACC1spaz) yeast two-hybrid screens are depicted.