Oncogene (2011) 30, 521–534 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE A critical role of integrin-linked kinase, ch-TOG and TACC3 in centrosome clustering in cancer cells AB Fielding1, S Lim1, K Montgomery1, I Dobreva1 and S Dedhar1,2 1Department of Integrative Oncology, British Columbia Cancer Research Centre of the BC Cancer Agency, Vancouver, British Columbia, Canada and 2Department of Biochemistry and Molecular Biology, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada Many cancer cells contain more than two centrosomes, Introduction which imposes a potential for multipolar mitoses, leading to cell death. To circumvent this, cancer cells develop Supernumerary centrosomes, a state in which cells mechanisms to cluster supernumerary centrosomes to contain more than two centrosomes, are a common form bipolar spindles, enabling successful mitosis. Dis- characteristic of human cancer cells. Supernumerary ruption of centrosome clustering thus provides a selective centrosomes were first observed in cancer cells nearly means of killing supernumerary centrosome-harboring 100 years ago (Boveri, 1914) and have now been cancer cells. Although the mechanisms of centrosome reported in many malignancies in vivo, including clustering are poorly understood, recent genetic analyses bladder, brain, breast, bile duct, cervical, colon, head have identified requirements for both actin and tubulin and neck, liver, lung, ovarian, pancreas and prostate regulating proteins. In this study, we demonstrate that the tumors, as well as myeloma, lymphomas and leukemias integrin-linked kinase (ILK), a protein critically involved (Lingle and Salisbury, 1999; Kramer et al., 2002; Nigg, in actin and mitotic microtubule organization, is required 2002; Pihan et al., 2003; Zyss and Gergely, 2009). for centrosome clustering. Inhibition of ILK expression or Although supernumerary centrosomes can poten- activity inhibits centrosome clustering in several breast tially facilitate tumorigenesis by disrupting cellular and prostate cancer cell lines that have centrosome polarity (Nigg, 2006; Basto et al., 2008) and increasing amplification. Furthermore, cancer cells with supernumer- the likelihood of aneuploidy (Lingle et al., 2002; Ganem ary centrosomes are significantly more sensitive to ILK et al., 2009; Silkworth et al., 2009), they also impose a inhibition than cells with two centrosomes, demonstrating potential for multipolar mitoses leading to cell death that inhibiting ILK offers a selective means of targeting (Brinkley, 2001; Nigg, 2002; Ganem et al., 2009). To cancer cells. Live cell analysis shows ILK perturbation circumvent this, many cancer cells develop mechanisms leads cancer cells to undergo multipolar anaphases, to cluster supernumerary centrosomes to form bipolar mitotic arrest and cell death in mitosis. We also show spindles and hence, ensure bipolar mitosis and cell that ILK performs its centrosome clustering activity in a survival (Ring et al., 1982; Brinkley, 2001; Quintyne focal adhesion-independent, but centrosome-dependent, et al., 2005; Rebacz et al., 2007; Kwon et al., 2008). manner through the microtubule regulating proteins Disruption of centrosome clustering and induction of TACC3 and ch-TOG. In addition, we identify a specific multipolar spindle formation thus provides a selective TACC3 phosphorylation site that is required for centro- means of killing supernumerary centrosome-harboring some clustering and demonstrate that ILK regulates this cancer cells (Kwon et al., 2008; Xu and Saunders, 2008). phosphorylation in an Aurora-A-dependent manner. Although details remain poorly understood, recent Oncogene (2011) 30, 521–534; doi:10.1038/onc.2010.431; genetic analyses have identified three broad, overlapping published online 13 September 2010 mechanisms that regulate centrosome clustering (Kwon et al., 2008). First, several microtubule-interacting/ Keywords: integrin-linked kinase; centrosome cluster- regulating genes were identified, including Ncd (human ing; multipolar mitosis; TACC3; ch-TOG/XMAP215; spleen, embryo, testes protein (HSET)) and D-TACC cancer (Kwon et al., 2008). D-TACC, a protein involved in stabilizing the minus and plus ends of microtubules at centrosomes in concert with the microtubule-associated protein ch-TOG/XMAP-215/CKAP5 (Lee et al., 2001; Barros et al., 2005), was not investigated further in this report. The second broad group of genes identified to be Correspondence: Dr S Dedhar, Department of Biochemistry, Uni- required for the prevention of multipolar mitoses were versity of British Columbia, BC Cancer Research Centre, 675 West those involved either directly in actin cytoskeleton 10th Avenue, Vancouver, British Columbia, Canada V5Z1L3. E-mail: [email protected] organization, or those which contribute to interphase Received 22 January 2010; revised 4 August 2010; accepted 9 August cell shape by linking the extra-cellular-matrix to the 2010; published online 13 September 2010 actin cytoskeleton. Centrosome clustering by ILK, ch-TOG and TACC3 AB Fielding et al 522 Integrin-linked kinase (ILK) localizes to focal adhe- markers of microtubules, centrosomes and DNA to sions and is critically involved in both actin and cell enable the analysis of multipolar spindle formation. The adhesion regulation (for reviews see Legate et al., 2006; resulting phenotypes were scored as illustrated in McDonald et al., 2008). Moreover, several reports Figure 1a and the percentage of ‘de-clustered’ centro- demonstrate that ILK has a role in mitosis by regulation somes calculated for each treatment. Approximately of the microtubule cytoskeleton. Mitotic defects have 10% of control-treated BT549s contained de-clustered been detected upon ILK depletion in Drosophila S2 cells centrosomes and multipolar spindles. This is in accor- (Bettencourt-Dias et al., 2004), mouse hepatocytes dance with data from Kwon et al, who reported 14% of (Gkretsi et al., 2007) and human glioblastoma cells control-treated BT549 cells showed multipolar spindles (Koul et al., 2005; Edwards et al., 2008). It is interesting (Kwon et al., 2008). However, ILK siRNA treatment that, although the cellular phenotypes were not reported resulted in greater than 36% of the cells displaying de- in detail in these studies, all of these systems contain clustered centrosomes (Figures 1a and c). A similar supernumerary centrosomes (Weber et al., 1998; Gui- pattern was observed in MDA-MB-231 cells and also, as dotti et al., 2003; Margall-Ducos et al., 2007; Kwon an example of a different cancer type, prostate-derived et al., 2008). ILK has also been shown to localize to PC3 cells (Figures 1b and c). These results imply that centrosomes and regulate mitotic microtubule organiza- ILK participates in the clustering of supernumerary tion, likely through its influences on Aurora-A/ch-TOG/ centrosomes in both breast and prostate cancer cells. TACC3 complex formation (Fielding et al., 2008). It These experiments were performed using pericentrin to is interesting that the ILK-interacting proteins at stain for centrosomes because of the availability of a both focal adhesions (Mig-2 (Tu et al., 2003)) and the highly specific antibody against this protein. However, centrosomes (TACC3 (Fielding et al., 2008)) have been because of the potential difficulties of distinguishing implicated in centrosome clustering (Kwon et al., 2008) between true centriole-containing centrosomes and and ILK itself has been suggested as a candidate for other sites of microtubule nucleation that may recruit regulating this process (Xu and Saunders, 2008). There- components of the soluble pericentriolar-material, such fore, we sought to test whether ILK is required for as pericentrin, multipolar spindles induced by ILK centrosome clustering and if so whether inhibiting ILK perturbation were also examined with an antibody can selectively inhibit the growth of cancer cells. against centrin 2, one of the core structural components In this study, we demonstrate that ILK is required for of centrioles. This indicated that nearly all spindle poles centrosome clustering, and inhibiting ILK expression examined in ILK-depleted cells were due to the presence with small interfering RNA (siRNA) or activity with a of true centrosomes as each stained for a pair of centrin small molecule inhibitor, induces multipolar spindles in 2 foci (Supplementary Figure 1). Quantification of the several breast and prostate cancer cell lines that have percentage of de-clustered centrosomes in ILK-depleted centrosome amplification. Furthermore, cancer cells cells as judged by centrin 2 staining gave very similar with supernumerary centrosomes are more sensitive to results to the pericentrin quantifications (Supplementary ILK inhibition than cells with two centrosomes and Figure 1c). show defects in cell division resulting in decreased cell Next, we sought to determine whether inhibiting ILK proliferation. We also found that the centrosomal kinase activity using a pharmacological ILK inhibitor proteins TACC3 and ch-TOG, which co-localize, inter- could also prevent centrosome clustering and whether act with and are regulated by ILK (Dobreva et al., 2008; this effect was specific to cancer cells. Fielding et al., 2008) are required for centrosome clustering in mammalian cells, whereas key focal adhesion binding partners of ILK are not. ILK per- Pharmacological inhibition of ILK leads to multipolar forms its centrosome-clustering functions independently