Oncogene (2005) 24, 5005–5015 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc REVIEW Aurora : shining lights on the therapeutic horizon?

Paul D Andrews*,1

1Division of Regulation and Expression, Wellcome Trust Biocentre, University of Dundee, Dundee, DD1 5EH, UK

The Aurora kinases have been implicated in tumorigenesis to ensure accurate segregation and timely and are important regulators of diverse cell cycle events, cytokinesis. The approximate execution points of ranging from the entry into , function, Aurora A and B are shown in Figure 2. Aurora C’s mitotic spindle formation,chromosome biorientation and regulation and localization have remained obscure until segregation,and cytokinesis. The recent identification of recently (Li et al., 2004b; Sasai et al., 2004) and its real novel binding partners and key downstream effectors, function is unknown. together with new small-molecule inhibitors that display With over protein 500 kinases in the efficacy against tumours,heralds an upsurge of interest in grouped into at least 20 structurally related families, the these critical kinases. This review details new develop- specificity of inhibitors is a critical concern when ments in the field and analyses the potential of Aurora administrated in therapy. However, the recognition that kinases as anticancer targets. the selective inhibition of individual protein kinases is Oncogene (2005) 24, 5005–5015. doi:10.1038/sj.onc.1208752 not only possible, but has therapeutic efficacy (as exemplified by the success of the Bcl-Abl kinase Keywords: ; ; spindle inhibitor, Gleevec, for the treatment of chronic myeloid checkpoint; centrosome; ; therapy leukemia) has led to a huge interest in the development of kinase inhibitors for therapy. Here, I review recent advances in our understanding of Aurora kinase function, critically assess how detailed knowledge of their function could be utilized in cancer Introduction therapy and go on to discuss whether the Aurora kinases are indeed suitable targets for therapeutic intervention. Many fundamental processes in cancer cells are regu- lated by protein phosphorylation: kinases regulate the cell’s response to DNA damage, control DNA replica- Aurora A: cancer, and the connection tion, regulate entry into, passage through and exit from mitosis, and ensure accurate chromosome segregation; The human Aurora A gene, STK15, resides in a region they monitor nutrient availability and control cell of the genome (20q13.2) often amplified in certain growth pathways; and they regulate apoptosis and (Bar-Shira et al., 2002). Aurora A overexpres- hence govern a cell’s commitment either to live or to die. sion occurs in a high proportion of breast, colorectal In metazoans, there are three highly related Aurora and gastric cancers (Tanaka et al., 1999; Sakakura kinases: Aurora A, B and C (reviewed in Andrews et al., et al., 2001; Goepfert et al., 2002; Gritsko et al., 2003; 2003; Carmena and Earnshaw, 2003; Brown et al., Moreno-Bueno et al., 2003; Fraizer et al., 2004; Jeng 2004). Despite their relatedness at the sequence level, the et al., 2004; Kamada et al., 2004; Tong et al., 2004). localization and functions of Aurora A and B are largely While these data are largely correlative, more compel- non-overlapping. Aurora A is localized at the centro- ling was the observation that overexpression of a some from the time of centrosome duplication through constitutively active mutant of Aurora A in rat1 cells to mitotic exit and regulates centrosome function. In led to neoplastic transformation, suggesting Aurora A is contrast, Aurora B is localized to inner-centromeric a bona fide oncogene (Bischoff et al., 1998). However, in chromatin from until the – a recent report, controlled overexpression of Aurora A transition, whereupon it is relocalized to both the (STK6) in mice led to mitotic defects and mammary in the spindle midzone (and the cell cortex hyperplasia, but not malignant transformation (Zhang at the cleavage furrow) during telophase, ending up in et al., 2004). Aurora A was also recently shown to be a the midbody throughout cytokinesis (see Figure 1; low-penetrance skin tumour-susceptibility gene, with an reviewed in Andrews et al., 2003; Carmena and Earn- allelic variant (Ile31), frequently found amplified, whose shaw, 2003; Meraldi et al., 2004). Such behaviour marks presence was correlated with in human colon out Aurora B as a canonical ‘chromosome passenger’ tumour cells, with the Ile31 allele transforming rat1 cells protein (Earnshaw and Cooke, 1991), whose function is more potently than the more common Phe31 allele (Ewart-Toland et al., 2003). A separate study also found *Correspondence: PD Andrews; E-mail: [email protected] a correlation between the Ile31 allele and the riskof Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5006

Figure 1 Localization of Aurora A and B. Aurora A and B localize to different cellular structures. Aurora A localization to centrosome/spindle poles in metaphase and telophase (upper panels). Aurora B localization to the inner from prometaphase to metaphase and then the spindle midzone microtubules in late anaphase/telophase (lower panels) and in the midbody during cytokinesis (not shown)

of Aurora A levels (Mao et al., 2004). Further evidence pointing to a complexity in the regulation of Aurora levels comes from a recent study in rats, where estrogen was shown to lead to increases in Aurora A protein and mRNA levels, centrosome amplification, chromosome instability and ultimately to breast cancer (Li et al., 2004a). Aurora A’s role in centrosome function and duplica- tion is well established, yet still poorly understood (reviewed by Andrews et al., 2003; Meraldi et al., 2004). Disruption of Aurora A function delocalizes several components of the centrosome. Aurora A phosphor- ylates and recruits microtubule-associated proteins such as the kinesin Eg5 (Giet et al., 1999) and the acidic coiled-coil protein, TACC (Giet et al., 2002; Bellanger and Gonczy, 2003; Conte et al., 2003) to the centrosome. Recently, the human Lats2 tumour-suppressor protein Figure 2 Aurora kinase cell cycle execution points and substrates. was also shown to be recruited to the centrosome via Schematic diagram illustrating the known execution points and Aurora A-mediated phosphorlyation (Toji et al., 2004). substrates of Aurora A and B across the cell cycle. The substrates Aurora A appears to control entry into mitosis by phosphorylated in each phase of the cell cycle are color coded: regulating either the association of the cell cycle orange circles indicate Aurora A substrates and green circles indicate Aurora B substrates. Ipl1p substrates are indicated as a machinery with centrosomes, or possibly even directly double green and orange line (Hirota et al., 2003; Dutertre et al., 2004). Aurora A binds and phosphorylates the breast cancer-associated gene product, BRCA1, in vitro and in vivo and regulates breast carcinoma (Sun et al., 2004). Aurora A over- its function (Ouchi et al., 2004). Aurora A also regulates expression can occur because of gene amplification, human telomerase reverse transcriptase mRNA levels transcriptional induction or post-translational stabiliza- through c-myc, in ovarian and breast epithelial carcino- tion (Farruggio et al., 1999; Crane et al., 2004). Indeed, mas (Yang et al., 2004b). The inter-relationship bet- in a recent study the p53-dependent loss of the tumour ween all these pathways is, however, still obscure (see suppressor ubiquitin , hCDC4 led to a stabilization Figure 3).

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5007 Aurora A regulation is complex and involves the become attached to microtubules emanating from interplay between the small GTPase Ran and the spindle opposite spindle poles in a process known as biorienta- protein TPX2 as well as regulatory input from the type-1 tion (reviewed by Andrews et al., 2003; see Figure 4). protein phosphatase, PP1 (reviewed by Andrews et al., Errors in biorientation lead to CIN. Commonly 2003; Gruss and Vernos, 2004). Ran releases TPX2 from encountered errors include merotelic attachments (one importins a and b allowing TPX2 to bind Aurora A, kinetochore attached to microtubules from both poles) targeting it to spindle microtubules at the pole. TPX2 and syntelic attachments (both attached to activates Aurora A activity by stimulating Aurora A microtubules from the same pole), as illustrated in autophosphorylation and by protecting it from the Figure 4. The cell’s surveillance mechanisms (check- inhibitory action of PP1 (Eyers et al., 2003; Tsai et al., points; discussed below) that monitor the attachment 2003). The crystal structure of activated Aurora A in and biorientation state of each chromosome give complex with a TPX2 fragment showed TPX2 binding is correction pathways sufficient time to ensure that all sufficient to allow autophosphorylation of the two are configured properly, and thereby activatory T-loop theonines, allowing Aurora A to segregated equally in anaphase. Aurora B plays a key adopt a conformation similar to the ‘active’ conforma- role in this correction process. tion of other Ser/Thr kinases (Bayliss et al., 2003). This Aurora B (Ipl1p) phosphorylates a number of level of detailed knowledge is invaluable for the kinetochore components in budding yeast and is development of new Aurora A inhibitors that may essential for correcting errors in microtubule attachment specifically target the activation step. (see Figures 2–4), thereby ensuring faithful chromosome Recently, an Aurora A–p53 connection has segregation (Biggins et al., 1999; He et al., 2001; Kang been revealed. Previously, parallels had been noticed et al., 2001; Cheeseman et al., 2002; Tanaka et al., 2002; between phenotypes associated with Aurora A gain-of- Shang et al., 2003; Westermann et al., 2003). In function mutations (or its overexpression) and those particular, Dam1p has been extensively characterized caused by p53 loss-of-function (Meraldi et al., 2002). as an Ipl1p substrate acting downstream of Aurora B The activity and stability of p53 is governed by many for microtubule attachment (Kang et al., 2001; Cheese- post-translational modifications, most importantly, man et al., 2002; Shang et al., 2003), although to date no multisite phosphorylation. In an intriguing new deve- clear Dam1p orthologues are known to exist in the lopment, Aurora A has been shown to phosphorylate genomes of higher eukaryotes. However, Aurora B’s Ser315 of p53, leading to its Mdm2-mediated destabi- function appears to be conserved across evolution. In lization (Katayama et al., 2004). Aurora A RNAi led mammalian cells, the Aurora kinase inhibitor Hesper- to a loss of Ser315 phosphorylation, p53 stabilization adin increased the incidence of mal-oriented chromo- andaG2/M cell cycle arrest. In a separate study, somes (Hauf et al., 2003). Furthermore, another live cell phosphorylation of Ser215 of p53 abrogated p53’s study using a different Aurora kinase inhibitor (AKI) DNA-binding and transactivation function (Liu et al., showed Aurora kinases to be required for correcting 2004). To complicate matters further, p53 has been syntelic chromosomes (Lampson et al., 2004). Under- shown to blockAurora A activity, an effect overcome standing the molecular details of these processes and by TPX2 (Eyers and Maller, 2004). How this web how Aurora B achieves biorientation is of prime of interactions plays out in cells remains to be dissec- importance for the future design of inhibitors, since on ted. Aurora A may regulate p53 in vitro, but it is face value inhibiting this kinase would be predicted to still unclear whether this occurs in vivo. Intriguingly, enhance chromosome loss. How might Aurora B correct one report has shown Aurora A-mediated phosphory- biorientation mistakes in human cells? Until recently, lation of CENP-A to be necessary to recruit Aurora B the only known Aurora B substrates in metazoans were to the inner-centromere in prometaphase (Kunitoku histone H3, CENP-A, vimentin, desmin, GFAP, topoi- et al., 2003). This type of dependency between the somerase II, mgcRacGAP and myosin regulatory light two kinases and their substrates (summarized in chain, which have no clear linkwith biorientation Figure 3) will be important to verify in other cellular (reviewed in Andrews et al., 2003). Recently, however, a contexts. major advance came in three reports describing the identification of MCAK, a centromeric microtubule- destabilizing kinesin (reviewed by Ovechkina and Wordeman, 2003; Moore and Wordeman, 2004), as an Aurora B: regulator of kinetochore-microtubule Aurora B substrate in vitro and in vivo, in human cells attachment (Andrews et al., 2004) and Xenopus (Lan et al., 2004; Ohi et al., 2004). MCAK is required for chromosome Cancer cells are often aneuploid, that is, they possess congression, biorientation, bipolar spindle formation aberrant chromosome structure or number (Nowak and anaphase chromosome movement (see Moore and et al., 2002; Rajagopalan et al., 2003). Chromosome Wordeman, 2004). Phosphorylation of MCAK by instability (CIN), one form of aneuploidy, is typified by Aurora B inhibits MCAK’s catalytic activity in vitro the gain or loss of chromosomes and is linked to cancer and in vivo (Andrews et al., 2004; Lan et al., 2004; Ohi progression (Rajagopalan and Lengauer, 2004). To et al., 2004). More importantly, expression of phosphor- ensure each sister is faithfully segregated to ylation-site mutants in cells increased the number of the daughter cells in anaphase, sister kinetochores must incorrectly attached chromosomes (lateral, monotelic

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5008

Figure 3 Aurora kinases: pathways and functions. Functional interaction map of Aurora A and Aurora B. Dotted lines indicate known effects of the kinase on localization and solid lines indicate a functional (often direct) effect

and syntelic microtubule–kinetochore attachments), that dynamic cycles of phosphorylation and depho- configurations very rarely seen in cells expressing wild- sphorylation locally modulating MCAK activity and type MCAK (Andrews et al., 2004). This directly localization may be key (Andrews et al., 2003, 2004). phenocopies loss of Aurora B activity, implying MCAK Whether the recently discovered inner-centromere pro- is a key downstream effector of Aurora B. Subsequently, tein ICIS (which immunoprecipitates with Aurora B, it has been shown that the inhibition of MCAK by the INCENP and MCAK), which appears to recruit Aurora B complex is necessary for bipolar spindle MCAK to the inner-centromere and stimulate its formation around Xenopus chromatin (Gadea and microtubule-destabilizing activity in vitro (Ohi et al., Ruderman, 2004; Sampath et al., 2004). Aurora B also 2003), also contributes to the regulation in vivo remains regulates MCAK localization at the inner centromere an open question. The identification of MCAK as a (Andrews et al., 2004) as well as other kinetochore and bona fide, in vivo Aurora B substrate, whose functions spindle checkpoint proteins to kinetochores (Murata- closely match those of Aurora B, now places MCAK at Hori and Wang, 2002; Ditchfield et al., 2003). Precisely centre stage. The detailed mechanism by which Aurora how Aurora B-mediated modulation of MCAK activity B regulates microtubule dynamics and microtubule– and localization allows error correction to take place is kinetochore attachment will be critical to unravel if we still under investigation. PP1, located at the outer are to target Aurora B and its downstream effectors in a kinetochore (Trinkle-Mulcahy et al., 2003), regulates therapeutic context. MCAK phosphorylation status and Aurora B activity and dephosphorylates Aurora B localization present themselves as attractive biomarkers substrates (Murnion et al., 2001), leading us to postulate for Aurora B inhibition.

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5009 cancer therapies under development are based on reactivation or depletion of survivin (Chen et al., 2004; Ning et al., 2004; Uchida et al., 2004). However, survivin’s proposed anti-apoptotic role is highly con- troversial. Indeed, recent studies have largely ruled out a direct anti-apoptotic role for survivin: in a recent study using Cre-Lox thymocytes lacking survivin, no evidence could be found for a direct role in apoptosis (although induction of p53 and p21, growth arrest and cell death were observed), leading the authors to postulate that survivin prevents ‘mitotic catastrophe’, rather than protects cells from apoptotic stimuli (Okada et al., 2004); a second independent study came to similar conclusions (Xing et al., 2004). Mitotic catastrophe has been partially characterized and appears to possess some of the classical features of apoptosis such as caspase-2 activation (Castedo et al., 2004). A recent study using shRNA in both primary lung fibroblasts and normal epithelial cells revealed that cells could survive in the absence of survivin, but failed to segregated chromo- somes or perform cytokinesis successfully, undergoing a p53-dependent blockto re-replication (Yang et al., 2004a). Two other reports also failed to find apoptotic defects in survivin RNAi HeLa cells (Carvalho et al., 2003; Lens et al., 2003). In contrast, another study reports apoptotic cell death and strong suppression of tumour in a mouse xenograft model using adenovirus-mediated transfer of siRNA against survivin (Uchida et al., 2004). Furthermore, one study recently pinpointed a single residue in survivin (Asp53) that affects survivin’s anti-apoptotic function–mutation of Figure 4 Chromosome biorientation and aurora B. The process of this residue to alanine converted survivin into a p53- biorientation involves several stages during which time errors in independent pro-apoptotic protein (Song et al., 2004). It kinetochore (Kt) attachment to microtubules (MT) can occur. (I) A is difficult to ignore all the evidence that implicates merotelically attached chromosome where a single kinetochore is occupied by microtubules emanating from opposite poles; (II) a survivin as an important anti-apoptotic factor. Perhaps mono-oriented chromosome where a single microtubule from one it is the hypomorphic nature of shRNA/siRNA experi- pole is attached to a single kinetochore; (III) a syntelically attached ments and the potential for adaptation in Cre-Lox chromosome where both sister kinetochores are occupied by experiments which gives credence to the notion that microtubules emanating from the same pole; and (IV) an apoptosis is not directly inhibited by survivin. Alter- amphitelically attached (bioriented) chromosome where each sister kinetochore is occupied by microtubules emanating from opposite natively, given survivin’s known function as part of the pole. Aurora B is critical for correction of syntelic attachments Aurora B complex, does lackof survivin perturb spindle (inset panel) checkpoint function to such an extent that it leads to mitotic catastrophe, which under certain conditions can be read as apoptosis? The importance of apoptotic The Aurora B complex: new subunits and old debates versus non-apoptotic cell death pathways in cancer therapy is extremely important and has been reviewed Aurora B exists in a stable complex with INCENP elsewhere (Okada and Mak, 2004). The true nature of and survivin, all behaving as chromosome passengers survivin’s role in cell survival therefore remains the (Vagnarelli and Earnshaw, 2004). INCENP and survivin subject of much debate, but of critical importance in the have regulatory and targeting functions (see Andrews cancer therapy arena. et al., 2003). Both INCENP and survivin are phos- in vitro phorylated by Aurora B (Knatko and Swedlow, Borealin unpublished; Kang et al., 2001; Cheeseman et al., 2002; Wheatley et al., 2004). Recently, a fourth Aurora B complex subunit was described independently by two groups (Gassmann Survivin et al., 2004; Sampath et al., 2004). This small conserved protein, called Borealin in human cells (Gassmann et al., This essential subunit of the Aurora B complex has been 2004), and Dasra B in Xenopus (Sampath et al., 2004), hailed as a key inhibitor of apoptosis (see Altieri, 2003, immunoprecipitates with Aurora B, INCENP and and references therein). Survivin levels are an often-used survivin. Borealin is weakly related to CSC-1, a protein prognostic marker for cancer progression. A number of shown to be part of the Aurora B complex in

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5010 Caenorhabditis elegans (Romano et al., 2003). Borealin Aurora B function caused a failure to retain checkpoint appears to specifically target the Aurora B complex to proteins at the kinetochore and led to an over-ride of (Gassmann et al., 2004). Borealin RNAi only the taxol-sensitive, ‘tension-sensing’, arm of the leads to biorientation defects – in particular a striking spindle checkpoint (Ditchfield et al., 2003; Hauf et al., increase in merotelic and syntelic microtubule-kineto- 2003). These results mirror those observed when chore attachments – as well as multipolar spindles that survivin is depleted by RNAi (Carvalho et al., 2003; form after biorientation and severely perturb anaphase Lens et al., 2003) and in the Ipl1 mutant in budding chromosome segregation (Gassmann et al., 2004). yeast (Biggins and Murray, 2001). Given Aurora A’s Borealin is an Aurora B substrate in vitro, but unlike apparent role in Aurora B recruitment, the Aurora A’s INCENP and survivin does not stimulate Aurora B effects on the checkpoint may be indirect. activity (Gassmann et al., 2004). Significantly, in Of paramount importance is how Aurora kinase contrast to Aurora B RNAi, Borealin RNAi had little inhibition effects checkpoint function and cell cycle effect on histone H3 Ser10 phosphorylation (Gassmann progression – the treatment of mammalian cells with et al., 2004). The implication of this result is that Aurora inhibitors causes checkpoint over-ride, prema- separate Aurora B subcomplexes exist with distinct ture exit from mitosis during metaphase and cytokinesis substrate specificities, a potentially interesting avenue failure, leading to polyploidization (Ditchfield et al., for future inhibitor design. Kinases and phosphatases 2003; Hauf et al., 2003), a situation not necessarily have multiple substrates; therefore, complete inhibition conducive to tumour killing. What lies at the heart of of activity or loss of mRNA expression by the matter here is the whether a cell maintains a RNAi is predicted to result in a more severe phenotypes checkpoint-triggered mitotic arrest, and whether the cell than depletion of their associated targeting subunits or subsequently enters a cell death pathway or re-enters the specialized regulators. It is for this reason that a cell cycle. The outcome seems to be determined by many complete picture of the makeup of the Aurora kinase factors such as the cause of the checkpoint arrest, its complexes is necessary and desirable. duration, the dose of the inhibitor, the cell type and genetic makeup (for an extensive review, see Rieder and Maiato, 2004). Aurora kinases and the spindle checkpoint

The spindle assembly checkpoint is a critical surveillance Aurora kinase inhibitors mechanism to ensure faithful chromosome segregation at the metaphase–anaphase transition. Defects in this Since the discovery of the Aurora kinases, much effort checkpoint may cause the aneuploidy found in many has been made by pharmaceutical companies to identify cancer cells. The checkpoint monitors both the micro- inhibitors. To date, three Aurora kinase inhibitors have tubule attachment status of the cells’ kinetochores and been fully published (Ditchfield et al., 2003; Hauf et al., the tension that develops across sister centromeres when 2003; Harrington et al., 2004; see Table 1). In addition, a chromosomes are fully bioriented. Only when all derivative of an original parent structure in the patent chromosomes are bioriented and at the metaphase plate database has been synthesized and utilized in two is the checkpoint relieved and the cell cycle then allowed studies (Straight et al., 2003; Lampson et al., 2004), to advance (reviewed by Musacchio and Hardwick, but no data exist on the specificity of this latter 2002). Spindle checkpoint defects generate a CIN compound in vitro or in vivo. phenotype and are linked to tumorigenesis (reviewed et al by Draviam ., 2004; Lengauer and Wang, 2004). ZM447439 Aurora kinases have been implicated in the spindle checkpoint in yeast and metazoans. The single Aurora ZM447439 (Ditchfield et al., 2003) is one of a number of in Schizosacchromyses pombe, ark1, has been shown to compounds developed by AstraZeneca, generated by function in the attachment arm of the checkpoint screening a 250 000 compound library using Aurora A (Petersen and Hagan, 2003). However, in budding yeast, kinase and a model peptide substrate. ZM447439 Ipl1p may have a role in the tension arm of the inhibits Aurora A and B, but is at least 10-fold more checkpoint (Biggins and Murray, 2001). Aurora A may potent against Aurora kinases than against the other 13 alter spindle checkpoint function in metazoans, since its kinases tested (see Table 1). ZM447439 caused growth overexpression leads to polyploidization in the presence inhibition in a colony-forming assay, a small increase in of the chemotherapeutic agent paclitaxel, suggestive of the level of apoptotic cells and p53-dependent poly- an over-ride of the spindle checkpoint, an effect ploidization (see Table 1). dependent on the BUB1 checkpoint gene (Anand et al., 2003). In another study, Aurora A caused Hesperadin checkpoint override in and disrupted the binding of BubR1 to Cdc20 leading to a CIN phenotype Hesperadin (Hauf et al., 2003) was discovered by (Jiang et al., 2003). Whether these result indicate a direct Boehringer Ingelheim by virtue of its effects on ploidy. effect of Aurora A on spindle checkpoint function Hesperadin specificity was tested against Aurora B (but remains to be determined, since two other reports not Aurora A) and a diverse panel of 25 kinases in vitro, implicate Aurora B in the spindle checkpoint. Loss of seven of which were inhibited with reasonable Kis (see

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5011 Table 1 Aurora Kinase Inhibitors: a comparison of the three well-characterized and published AKIs, showing their structure and a summary of the phenotypes of cells treated with the drugs Company Generic name Compound name Boehringer Hesperadin Sulphonylamino substituted 3-(aminomethylide)-2-indolinone Ingelheim Specificity Aurora kinases inhibited (IC50) Structure Aurora B (50 nM in cells; >200 mM in vitro) Off target kinases inhibited (IC50) AMPK, LCK, MKK1, MAPKAP-K1, CHK1 and PHK (1 mM) CycB/CDK1 (2.8 mM) (25 tested) Phenotype Decreased H3 phosphorylation Chromosome segregation and cytokinesis defects lead- ing to polyploidization to >32N content Increased syntelic Kt–MT attachments Overcame spindle checkpoint arrest triggered by taxol but not nocodazole

Company Generic name Compound name Astra Zeneca ZM447439 (4-(4-(N-benzoylamino)anilino)-6-methoxy-7-(3-(1-morpholino) propoxy)quinazoline Specificity Aurora kinases inhibited (IC50) Structure Aurora A (0.11 mM) Aurora B (0.13 mM) Off-target kinases inhibited (IC50) MEK (1.79 mM), SRC (1.03 mM) and LCK (0.88 mM) (13 tested) Phenotype Decreased H3 phosphorylation Endoreduplication then arrested with 4N/8N DNA content Arrest was p53-dependent. Inhibited colony formation in proliferation assay Compromises spindle checkpoint, causing failure to recruit BubRI, Mad2 and CENP-E

Company Generic name Compound name Vertex VX-680 Cyclopropane carboxylic acid {4-[4-(4-methyl-piperazin-1-yl)- pyrimidin-2ylsulphanyl]-phenyl}-amide Specificity Aurora kinases inhibited (IC50) Structure Aurora A (0.6 nM) Aurora B (18 nM) Aurora C (4.6 nM) Off-target kinases inhibited (IC50) FLT-3; 30 nM (55 tested) Phenotype Accumulation of cells with >4N content Inhibited the proliferation of a panel of cancer cell lines Loss of H3 phosphorylation in vivo Fourfold increase in apoptosis in vivo Effective killing of AML tumour cells from patients Tumour killing in xenograft models

Table 1). It is unclear whether Hesperadin is a useful effective in killing three different tumour cell types drug for therapeutics since it generated highly polyploid (leukemia, colon and pancreas) in xenograft models. cells with no apparent loss of viability. VX-680 also potently killed treatment-refractory pri- mary AML cells from patients. This efficacy was VX-680 apparently achieved with some level of toxicity to bone-marrow cells, as the authors reported a degree of VX-680 is the most recently published drug (Harrington neutropenia and some loss in body weight, although et al., 2004), coming out of a knowledge-based chemical these recovered after drug withdrawal. Unfortunately, synthesis programme undertaken by Vertex Pharma- no evidence was presented to indicate the presence or ceuticals, drawing on ‘unique structural features’ in absence of other toxic side effects and/or long-term Aurora A. VX-680 potently inhibited all three Aurora survival rates in treated mice, a necessary next step kinases but had greater than 100-fold selectivity for towards clinical trials. A key unanswered question is Auroras over a diverse panel of 55 kinases tested in vitro. exactly how VX-680 leads to tumour cell death, since The phenotypes associated with VX-680 treatment are only a small proportion of cells were detectable summarized in Table 1. Significantly, VX-680 is high apoptotic. As has been pointed out recently (Sausville,

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5012 2004), tumour growth rates in patients rarely match essential to assess phenotypic changes when different those of the rapidly growing xenograft tumours in inhibitors and combinations of inhibitors are used. model systems. A critical question, which applies to the Equally, an in-depth understanding of the molecular use of all kinase inhibitors uses in oncology, is whether basis of genetic instability in tumours will be essential. the efficacy of these ostensibly reversible Aurora kinase Pharmaceutical companies indisputably have the re- inhibitors, when administered over short periods, sources to enable these types of large pharmacogenomic translates into clinically useful diminution in tumour and proteomic studies, but will profit-led drug discovery growth in situ. It is noteworthy that the clinically highly take sufficient time to dissect the inter-relationships effective taxanes bind to their target () essentially between signalling pathways? Indeed, there has been irreversibly. Chemotherapeutic agents that target micro- vocal criticism of the current modus operandi of pharma- tubules have been used effectively to kill cancer cells for led drug discovery (Dancey and Sausville, 2003; Sager over 35 years (reviewed by Mollinedo and Gajate, 2003). and Lengauer, 2003). The taxanes (e.g. paclitaxel/taxol), the epothilones and So, do the Aurora kinases represent good cancer the discodermolides, all stabilize microtubules by therapy targets? Aurora A overexpression is correlated inhibiting their dynamics and potently induce p53- with tumour progression. Aurora A is also mutated in independent apoptosis in cancer cells. The Vinca certain cancers and behaves, at least in certain circum- alkaloids (vincristine, vinblastine and vindestine), noco- stances, as an oncogene. Aurora A’s interaction with dazole and colchicine all destabilize microtubules, tumour suppressors such as p53, BRCA1 and is leading to cell cycle arrest and apoptosis. Many other also suggestive of a real connection to oncogenesis. On naturally occurring antimitotic compounds also target the other hand, Aurora B is intimately involved in tubulin (see Mollinedo and Gajate, 2003). While these preventing CIN, a phenomenon frequently implicated in classes of therapeutic drug are effective, they do not the genetic heterogeneity of cancers and may even drive appear to discriminate between dividing and nondivid- oncogenesis. Survivin, part of the Aurora B complex, ing cells — paclitaxel, for instance, causes peripheral may well be a key protector against apoptosis and/or neuropathy due to its effects on the neuronal micro- mitotic catastrophe. Given these facts, as well as their tubule network. The recent revelation that Aurora B in known roles in so many fundamentally important particular affects microtubule dynamics, spindle assem- aspects of a range of cell cycle events (e.g. centrosome bly and spindle checkpoint function is therefore of function, mitotic entry, spindle assembly, kinetochore considerable interest in this context. We may be able to function, chromosome segregation, microtubule dyna- exploit these inter-relationships in order to produce new mics, spindle checkpoint function and cytokinesis), therapeutic reagents, to inform the design of treatment pharmaceutical companies clearly believe Aurora regimens (e.g. combination therapies) and thereby arrest kinases are hot property. mitotic cancer cells, triggering their death. For clinicians, the demonstration that small molecule Aurora kinase inhibitors are effective at killing cancer cells has brought these kinases under the spotlight; Anticancer targets for therapy however, it seems appropriate to voice a cautionary note as to the overall efficacy of such inhibitors in cancer What makes a good target for cancer therapy? Ideally, treatment. Aurora inhibitors may trigger apoptosis in a the target, whether it is a specific gene, or protein, or proportion of cells and lead to the arrest of tumour process, would be different between the cancer cell and growth in model systems, but it is noticeable that these the normal cell. Defining differences between cancer inhibitor treatments only increased the proportion of cells and normal cells is a critical yet daunting challenge. apoptotic cells slightly. Nothing is known about how the Techniques as diverse as classical human genetics, inhibitors cause cell death (e.g. is it the result of microarrays (genome and proteome), cell biology and polyploidization), to what extent this happens in vivo biochemistry are currently being used to pinpoint these and whether the long-term outcome of their inhibition is differences. In the case of protein kinases as targets, it is favourable for maintaining a long-term remission. On important to differentiate between kinases proven to be face value, inhibition of any kinase required for stable pivotal in the cancer phenotype (e.g. mutationally chromosome inheritance is dangerous, because of a activated) and other kinases that are might be periph- greater probability of genetic heterogeneity and hence erally involved (e.g. overexpressed in cancer cells) by the potential for tumour evolution. Undoubtedly, non-specifically allowing deregulated cell growth and massive chromosome loss does, in the majority of cells, division. With the development of techniques to analyse lead to cell death, but at what point does increased the phosphoproteomes of cell populations, drug speci- chromosome instability trigger cell death pathways? In ficity questions may now be addressable in normal addition, Aurora B is required for cytokinesis and its versus tumour cells, an approach complementary to inhibition leads to polyploidization – a condition that potentially uninformative in vitro biochemical assays. may result in the survival of a severely aneuploidy Genetic variations between patients and between differ- cancerous cell. Nothing is understood of how this is ent cell populations within an individual’s cancer, can sensed in the cell. More studies are required to ascertain influence drug efficacy and could represent considerable the long-term effects of Aurora kinase inhibitor admin- challenges to overcome for drug use in the future. The istration, in a suitable model organism. Rushing Aurora availability of a tailored panel of biomarkers will be kinase inhibitors to the clinic would seem to be a rash

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5013 move at this juncture. A more detailed understanding of centric, ‘knowledge-based’ drug design strategy used the interplay between Aurora kinases, the microtubule commonly at present, generate useful selective inhibi- and apoptosis/mitotic catastrophe path- tors? Indeed, will ATP-competitive inhibitors in general ways will allow us to decide on strategies to treat cancer be of sufficient selectivity and of high enough affinity to cells effectively and achieve the holy grail: an anti- have efficacy in the context of a tumour, inside a mitotic drug that is tumour cell-specific and triggers cell patient? Adaptive mutations can and do arise in target death pathways. proteins, leading to drug resistance – a clear problem in the treatment of certain cancers with tyrosine kinase inhibitors (Pao et al., 2005). An alternative school of Horizons thought advocates the use of either less selective, kinase family-specific inhibitors or cocktails of drugs, to deliver While much is known of Aurora A regulation, structure the lethal blow to the cancer. An area somewhat in its and its role in oncogenesis, little is understood about its infancy is to screen for compounds that disrupt protein– key in vivo substrates and their cellular functions. protein interactions (reviewed by Pagliaro et al., 2004), Equally, while a great deal is understood about Aurora rather than against the . Small-molecule B’s in vivo function, detailed knowledge of its regulation inhibitors remain the vogue; other reagents such as is only just emerging and structural knowledge is cell-permeant peptides have had technical limitations. currently absent. Significantly, the structure of the Cell permeability, bioavailability, catabolic rate, off- Aurora B in complex with an INCENP fragment target toxicity and drug resistance are still critical issues 7 ( inhibitor) will soon be available in the public domain in the use of drugs in cancer therapy. (Mussachio, A and Mapelli M, personal communica- RNAi-based therapeutics could have an important tion), which will go some way to redressing the balance, role to play in the fight against cancer. In this context, given the wealth of structural information on Aurora A. RNAi of Aurora kinases’ downstream effectors or If the Aurora kinases are bona fide targets for cancer targeting subunits or specific splice variants, if applic- therapy, an integrated molecular and cellular under- able, may allow the highly selective targeting of a standing of their function, interactions, dynamics, up- defined subset of kinase functions. The drawback of stream regulators and downstream effectors in different using RNAi is its hypomorphic nature, the potential for cell types will be required to properly inform drug cell adaptation and the possibility of an off-target effect. screening programmes and therapies. Many key ques- An alternative to RNAi is the use of gene therapy using tions remain unanswered. What signalling pathways viral delivery of dominant-negative mutants – an activate the kinase activity of the Auroras in vivo? How approach employed recently using a pro-apoptotic allele does Aurora kinase structure and/or subunit composi- of survivin in the treatment of colon cancer (Tu et al., tion dictate its localization and substrate specificity in 2005). vivo? Indeed, how many substrates are phosphorylated An emerging theme in the Aurora kinase field is that by each Aurora kinase in different cell types? To what the phosphorylation status of a target protein deter- extent do the Aurora kinase substrates overlap and what mines its location. Therefore, a clearly important future functional interactions exist between family members? strategy will be to use cell-based screens, tailored to What is the function of Aurora C? What are the monitor loss of specific Aurora functions (e.g. screening molecular details underlying the connections between using panels of phospho-specific antibodies and/or using these kinases and the spindle and the spindle checkpoint substrate localization as readouts). This may work and can we readily exploit these connections to kill alongside, or even supercede, conventional biochemical tumour cells specifically? Of great interest would be to high-throughput screens. ascertain if mutations in encoding Aurora B With all the new data emerging on Aurora kinases, complex components are associated with cancer. the great excitement in the field is probably justified. A critical issue is how our knowledge of Aurora Hopefully, the Aurora kinases will prove to be genuinely kinase structure and function will inform the strategies bright lights on the therapeutic horizon and not just a employed in the search for new anticancer therapeutics. case of now you see them now you don’t. In the case of small-molecule kinase inhibitors, will the key drugs come from ‘blindly’ screening compound Acknowledgements libraries, be they the small natural compound libraries I thankJason Swedlow for critical reading of the manuscript or the huge synthetic libraries? Will the structure- and support. I acknowledge The Wellcome Trust for support.

References

Altieri DC. (2003). Adv. Cancer Res., 88, 31–52. Bar-Shira A, Pinthus JH, Rozovsky U, Goldstein M, Sellers Anand S, Penrhyn-Lowe S and Venkitaraman AR. (2003). WR, Yaron Y, Eshhar Z and Orr-Urtreger A. (2002). Cancer Cancer Cell, 3, 51–62. Res., 62, 6803–6807. Andrews PD, Knatko E, Moore WJ and Swedlow JR. (2003). Bayliss R, Sardon T, Vernos I and Conti E. (2003). Mol. Cell., Curr. Opin. Cell. Biol., 15, 672–683. 12, 851–862. Andrews PD, Ovechkina Y, Morrice N, Wagenbach M, Bellanger JM and Gonczy P. (2003). Curr. Biol., 13, Duncan K, Wordeman L and Swedlow JR. (2004). Dev. 1488–1498. Cell., 6, 253–268. Biggins S and Murray AW. (2001). Genes Dev., 15, 3118–3129.

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5014 Biggins S, Severin FF, Bhalla N, Sassoon I, Hyman AA and Harrington EA, Bebbington D, Moore J, Rasmussen RK, Murray AW. (1999). Genes Dev., 13, 532–544. Ajose-Adeogun AO, Nakayama T, Graham JA, Demur C, Bischoff JR, Anderson L, Zhu Y, Mossie K, Ng L, Souza B, Hercend T, Diu-Hercend A, Su M, Golec JM and Miller Schryver B, Flanagan P, Clairvoyant F, Ginther C, Chan KM. (2004). Nat. Med., 10, 262–267. CS, Novotny M, Slamon DJ and Plowman GD. (1998). Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter EMBO J., 17, 3052–3065. R, Heckel A, Van Meel J, Rieder CL and Peters JM. (2003). Brown JR, Koretke KK, Birkeland ML, Sanseau P and J. Cell. Biol., 161, 281–294. PatrickDR. (2004). BMC Evol. Biol., 4, 39. He X, Rines DR, Espelin CW and Sorger PK. (2001). Cell, Carmena M and Earnshaw WC. (2003). Nat. Rev. Mol. Cell. 106, 195–206. Biol., 4, 842–854. Hirota T, Kunitoku N, Sasayama T, Marumoto T, Zhang Carvalho A, Carmena M, Sambade C, Earnshaw WC and D, Nitta M, Hatakeyama K and Saya H. (2003). Cell, 114, Wheatley SP. (2003). J. Cell. Sci., 116, 2987–2998. 585–598. Castedo M, Perfettini JL, Roumier T, Andreau K, Medema R Jeng YM, Peng SY, Lin CY and Hsu HC. (2004). Clin. Cancer and Kroemer G. (2004). Oncogene, 23, 2825–2837. Res., 10, 2065–2071. Cheeseman IM, Anderson S, Jwa M, Green EM, Kang J, Jiang Y, Zhang Y, Lees E and Seghezzi W. (2003). Oncogene, Yates III JR, Chan CS, Drubin DG and Barnes G. (2002). 22, 8293–8301. Cell, 111, 163–172. Kamada K, Yamada Y, Hirao T, Fujimoto H, Takahama Y, Chen JS, Liu JC, Shen L, Rau KM, Kuo HP, Li YM, Shi D, Ueno M, Takayama T, Naito A, Hirao S and Nakajima Y. Lee YC, Chang KJ and Hung MC. (2004). Cancer Gene (2004). Oncol. Rep., 12, 593–599. Ther., 11, 740–747. Kang J, Cheeseman IM, Kallstrom G, Velmurugan S, Barnes Conte N, Delaval B, Ginestier C, Ferrand A, Isnardon D, G and Chan CS. (2001). J. Cell. Biol., 155, 763–774. Larroque C, Prigent C, Seraphin B, Jacquemier J and Katayama H, Sasai K, Kawai H, Yuan ZM, BondarukJ, Birnbaum D. (2003). Oncogene, 22, 8102–8116. Suzuki F, Fujii S, Arlinghaus RB, Czerniak BA and Sen S. Crane R, Kloepfer A and Ruderman JV. (2004). J. Cell. Sci., (2004). Nat. Genet., 36, 55–62. 117, 5975–5983. Kunitoku N, Sasayama T, Marumoto T, Zhang D, Honda S, Dancey J and Sausville EA. (2003). Nat. Rev. Drug Discov., 2, Kobayashi O, Hatakeyama K, Ushio Y, Saya H and Hirota 296–313. T. (2003). Dev. Cell., 5, 853–864. Ditchfield C, Johnson VL, Tighe A, Ellston R, Haworth C, Lampson MA, Renduchitala K, Khodjakov A and Kapoor Johnson T, MortlockA, Keen N and Taylor SS. (2003). TM. (2004). Nat. Cell. Biol., 6, 232–237. J. Cell. Biol., 161, 267–280. Lan W, Zhang X, Kline-Smith SL, Rosasco SE, Barrett-Wilt Draviam VM, Xie S and Sorger PK. (2004). Curr. Opin. Genet. GA, Shabanowitz J, Hunt DF, WalczakCE and Stukenberg Dev., 14, 120–125. PT. (2004). Curr. Biol., 14, 273–286. Dutertre S, Cazales M, Quaranta M, Froment C, Trabut V, Lengauer C and Wang Z. (2004). Nat. Genet., 36, 1144–1145. Dozier C, Mirey G, Bouche JP, Theis-Febvre N, Schmitt E, Lens SM, Wolthuis RM, Klompmaker R, Kauw J, Agami R, Monsarrat B, Prigent C and Ducommun B. (2004). J. Cell. Brummelkamp T, Kops G and Medema RH. (2003). EMBO Sci., 117, 2523–2531. J., 22, 2934–2947. Earnshaw WC and Cooke CA. (1991). J. Cell. Sci., 98, Li JJ, Weroha SJ, Lingle WL, Papa D, Salisbury JL and Li SA. 443–461. (2004a). Proc. Natl. Acad. Sci. USA, 101, 18123–18128. Ewart-Toland A, Briassouli P, de Koning JP, Mao JH, Yuan Li X, Sakashita G, Matsuzaki H, Sugimoto K, Kimura K, J, Chan F, MacCarthy-Morrogh L, Ponder BA, Nagase H, Hanaoka F, Taniguchi H, Furukawa K and Urano T. Burn J, Ball S, Almeida M, Linardopoulos S and Balmain A. (2004b). J. Biol. Chem., 279, 47201–47211. (2003). Nat. Genet., 34, 403–412. Liu Q, Kaneko S, Yang L, Feldman RI, Nicosia SV, Chen J Eyers PA, Erikson E, Chen LG and Maller JL. (2003). Curr. and Cheng JQ. (2004). J. Biol. Chem., 279, 52175–52182. Biol., 13, 691–697. Mao JH, Perez-Losada J, Wu D, Delrosario R, Tsunematsu R, Eyers PA and Maller JL. (2004). J. Biol. Chem., 279, Nakayama KI, Brown K, Bryson S and Balmain A. (2004). 9008–9015. Nature, 432, 775–779. Farruggio DC, Townsley FM and Ruderman JV. (1999). Proc. Meraldi P, Honda R and Nigg EA. (2002). EMBO J., 21, Natl. Acad. Sci. USA, 96, 7306–7311. 483–492. Fraizer GC, Diaz MF, Lee IL, Grossman HB and Sen S. Meraldi P, Honda R and Nigg EA. (2004). Curr. Opin. Genet. (2004). Int. J. Oncol., 25, 1631–1639. Dev., 14, 29–36. Gadea BB and Ruderman JV. (2004). Mol. Biol. Cell, 16, Mollinedo F and Gajate C. (2003). Apoptosis, 8, 413–450. 1305–1318. Moore A and Wordeman L. (2004). Trends Cell Biol., 14, Gassmann R, Carvalho A, Henzing AJ, Ruchaud S, Hudson 537–546. DF, Honda R, Nigg EA, Gerloff DL and Earnshaw WC. Moreno-Bueno G, Sanchez-Estevez C, Cassia R, Rodriguez- (2004). J. Cell. Biol., 166, 179–191. Perales S, Diaz-Uriarte R, Dominguez O, Hardisson D, Giet R, McLean D, Descamps S, Lee MJ, Raff JW, Andujar M, Prat J, Matias-Guiu X, Cigudosa JC and Prigent C and Glover DM. (2002). J. Cell. Biol., 156, Palacios J. (2003). Cancer Res., 63, 5697–5702. 437–451. Murata-Hori M and Wang Yl. (2002). Curr. Biol., 12, 894–899. Giet R, Uzbekov R, Cubizolles F, Le Guellec K and Prigent C. Murnion ME, Adams RR, Callister DM, Allis CD, Earnshaw (1999). J. Biol. Chem., 274, 15005–15013. WC and Swedlow JR. (2001). J. Biol. Chem., 276, Goepfert TM, Adigun YE, Zhong L, Gay J, Medina D and 26656–26665. Brinkley WR. (2002). Cancer Res., 62, 4115–4122. Musacchio A and HardwickKG. (2002). Nat. Rev. Mol. Cell. Gritsko TM, Coppola D, Paciga JE, Yang L, Sun M, Shelley Biol., 3, 731–741. SA, Fiorica JV, Nicosia SV and Cheng JQ. (2003). Clin. Ning S, Fuessel S, Kotzsch M, Kraemer K, Kappler M, Cancer Res., 9, 1420–1426. Schmidt U, Taubert H, Wirth MP and Meye A. (2004). Int. Gruss OJ and Vernos I. (2004). J. Cell. Biol., 166, 949–955. J. Oncol., 25, 1065–1071.

Oncogene Aurora kinases: shining lights on the therapeutic horizon? PD Andrews 5015 NowakMA, Komarova NL, Sengupta A, Jallepalli PV, Shih Straight AF, Cheung A, Limouze J, Chen I, Westwood Ie M, Vogelstein B and Lengauer C. (2002). Proc. Natl. NJ, Sellers JR and Mitchison TJ. (2003). Science, 299, Acad. Sci. USA, 99, 16226–16231. 1743–1747. Ohi R, Coughlin ML, Lane WS and Mitchison TJ. (2003). Sun T, Miao X, Wang J, Tan W, Zhou Y, Yu C and Lin D. Dev. Cell., 5, 309–321. (2004). Carcinogenesis, 25, 2225–2230. Ohi R, Sapra T, Howard J and Mitchison TJ. (2004). Mol. Tanaka T, Kimura M, Matsunaga K, Fukada D, Mori H and Cell. Biol., 15, 2895–2906. Okano Y. (1999). Cancer Res., 59, 2041–2044. Okada H, Bakal C, Shahinian A, Elia A, Wakeham A, Suh Tanaka TU, Rachidi N, Janke C, Pereira G, Galova M, WK, Duncan GS, Ciofani M, Rottapel R, Zuniga-Pflucker Schiebel E, StarkMJ and Nasmyth K. (2002). Cell, 108, JC and MakTW. (2004). J. Exp. Med., 199, 399–410. 317–329. Okada H and Mak TW. (2004). Nat. Rev. Cancer, 4, 592–603. Toji S, Yabuta N, Hosomi T, Nishihara S, Kobayashi T, Ouchi M, Fujiuchi N, Sasai K, Katayama H, Minamishima Suzuki S, Tamai K and Nojima H. (2004). Genes Cells, 9, YA, Ongusaha PP, Deng C, Sen S, Lee SW and Ouchi T. 383–397. (2004). J. Biol. Chem., 279, 19643–19648. Tong T, Zhong Y, Kong J, Dong L, Song Y, Fu M, Liu Z, Ovechkina Y and Wordeman L. (2003). Traffic, 4, 1–9. Wang M, Guo L and Lu S et al. (2004). Clin. Cancer Res., Pagliaro L, Felding J, Audouze K, Nielsen SJ, Terry RB, 10, 7304–7310. Krog-Jensen C and Butcher S. (2004). Curr. Opin. Chem. Trinkle-Mulcahy L, Andrews PD, Wickramasinghe S, Sleeman Biol., 8, 442–449. J, Prescott A, Lam YW, Lyon C, Swedlow JR and Lamond Pao W, Miller VA, Politi KA, Riely GJ, Somwar R, Zakowski AI. (2003). Mol. Cell. Biol., 14, 107–117. MF, Kris MG and Varmus H. (2005). PLoS Med., 2, e73. Tsai MY, Wiese C, Cao K, Martin O, Donovan P, Ruderman Petersen J and Hagan IM. (2003). Curr. Biol., 13, 590–597. J, Prigent C and Zheng Y. (2003). Nat. Cell. Biol., 5, Rajagopalan H and Lengauer C. (2004). Nature, 432, 338–341. 242–248. Rajagopalan H, NovakMA, Vogelstein B and Lengauer C. Tu SP, Cui JT, Liston P, Huajiang X, Xu R, Lin MC, Zhu YB, (2003). Nat. Canc. Rev., 3, 659–701. Zou B, Ng SS and Jiang SH et al. (2005). Gastroenterology, Rieder CL and Maiato H. (2004). Dev. Cell, 7, 637–651. 128, 361–375. Romano A, Guse A, Krascenicova I, Schnabel H, Schnabel R Uchida H, Tanaka T, Sasaki K, Kato K, Dehari H, Ito Y, and Glotzer M. (2003). J. Cell. Biol., 161, 229–236. Kobune M, Miyagishi M, Taira K, Tahara H and Hamada Sager JA and Lengauer C. (2003). Cancer Biol. Ther., 2, H. (2004). Mol. Ther., 10, 162–171. 452–455. Vagnarelli P and Earnshaw WC. (2004). Chromosoma, 113, Sakakura C, Hagiwara A, Yasuoka R, Fujita Y, Nakanishi M, 211–222. Masuda K, Shimomura K, Nakamura Y, Inazawa J, Abe T Westermann S, Cheeseman IM, Anderson S, Yates III JR, and Yamagishi H. (2001). Br. J. Cancer, 84, 824–831. Drubin DG and Barnes G. (2003). J. Cell. Biol., 163, Sampath SC, Ohi R, Leismann O, Salic A, Pozniakovski A 215–222. and Funabiki H. (2004). Cell, 118, 187–202. Wheatley SP, Henzing AJ, Dodson H, Khaled W and Sasai K, Katayama H, Stenoien DL, Fujii S, Honda R, Earnshaw WC. (2004). J. Biol. Chem., 279, 5655–5660. Kimura M, Okano Y, Tatsuka M, Suzuki F, Nigg EA, Xing Z, Conway EM, Kang C and Winoto A. (2004). J. Exp. Earnshaw WC, Brinkley WR and Sen S. (2004). Cell Motil. Med., 199, 69–80. Cytoskeleton, 59, 249–263. Yang D, Welm A and Bishop JM. (2004a). Proc. Natl. Acad. Sausville EA. (2004). Nat. Med., 10, 234–235. Sci. USA, 101, 15100–15105. Shang C, Hazbun TR, Cheeseman IM, Aranda J, Fields Yang H, Ou CC, Feldman RI, Nicosia SV, KrukPA and S, Drubin DG and Barnes G. (2003). Mol. Cell. Biol., 14, Cheng JQ. (2004b). Cancer Res., 64, 463–467. 3342–3355. Zhang D, Hirota T, Marumoto T, Shimizu M, Kunitoku N, Song Z, Liu S, He H, Hoti N, Wang Y, Feng S and Wu M. Sasayama T, Arima Y, Feng L, Suzuki M, Takeya M and (2004). Mol. Cell. Biol., 15, 1287–1296. Saya H. (2004). Oncogene, 23, 8720–8730.

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