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Mitotic spindle multipolarity without centrosome amplificationHelder Maiato and Elsa Logarinho
Mitotic spindle bipolarity is essential for faithful segregation of chromosomes during cell division. Multipolar spindles are often seen in human cancers and are usually associated with supernumerary centrosomes that result from centrosome overduplication or cytokinesis failure. A less-understood path to multipolar spindle formation may arise due to loss of spindle pole integrity in response to spindle and/or chromosomal forces. Here we discuss the different routes leading to multipolar spindle formation, focusing on spindle multipolarity without centrosome amplification. We also present the distinct and common features between these pathways and discuss their therapeutic implications.
The mitotic spindle pole is normally established by one centrosome containing a pair of centrioles embedded in pericentriolar material (PCM) containing γ-tubulin ring complexes (γ-TuRCs) from which microtubules nucleate1. Similarly to DNA, centrosomes normally rep-licate only once every cell cycle. Thus, during mitosis, two centrosomes ensure the bipolar nature of the mitotic spindle that is essential for the bi-orientation and accurate segregation of chromosomes to two daughter cells. Ever since the work of Hansemann, Boveri and Galeotti around the beginning of the twentieth century, mitotic spindle multipolarity has been used to diagnose pathologic mitosis in human cancers2–4. The presence of multipolar spindles is often associated with supernumer-ary centrosomes and chromosomal instability5–7, and may result from the activation of oncogenic kinases that control centrosome duplica-tion8 and/or the loss of tumour-suppressor genes9,10. Paradoxically, the frequency of multipolar anaphases is considerably lower than that of multipolar ‘metaphases’11, and multipolar divisions are rare events in tumours, which grow primarily by typical bipolar mitosis12. Thus, multipolar mitoses may be transient entities rather than perpetuators of the cancer cell genome, implying that proliferating cancer cells with supernumerary centrosomes either eliminate and/or inactivate the function of excess centrosomes, or promote the coalescence of multiple centrosomes at the poles to form a functional bipolar spindle13. Whereas evidence for centrosome elimination or inactivation in tumour cells is lacking, centrosome clustering seems to be the prevailing mechanism14,15, and several proteins and forces have been established to play a role in this process15–18.
An alternative but much less understood mechanism leading to multipolar spindle formation is independent of centrosome amplifica-tion and is associated with loss of spindle pole integrity. In this Review we discuss the multiple routes leading to loss of spindle pole integrity in
mammalian cells, with sporadic incursions into other animal models. We also shed light on the molecular players of this process, the func-tional perturbation of which makes mitotic spindle poles susceptible to the action of spindle- and chromosome-mediated forces leading to irreversible multipolar spindle formation. Finally, we conclude with a comparative analysis of genetic and pharmacological approaches that prevent supernumerary centrosome clustering and/or promote loss of spindle pole integrity.
Mitotic spindle multipolarity due to centrosome amplificationSeveral abnormal conditions — including centriole overduplication, de novo centriole assembly, cytokinesis failure, mitotic slippage (the escape from mitotic arrest) and cell fusion — may lead to centrosome amplification and the formation of multipolar spindles, potentially com-promising mitotic fidelity (Fig. 1). The mechanisms behind these condi-tions and their implications for genomic stability have been extensively reviewed19–22, and so here we will concentrate on aspects that are relevant for a comparative analysis with the mechanisms behind multipolar spin-dle formation without centrosome amplification.
The genesis of multipolar spindle formation can be determined by ultrastructural analysis of the poles or the use of centriole markers (for example, centrin-2) in addition to PCM components (Fig. 2a). These methods allow the quantification of the number of centrioles at each pole and the number of poles in a cell, and indicate whether or not centrosome amplification is the underlying cause of multipolarity. Typically, spindles from cells with centrosome amplification exhibit more than two poles, each with at least two centrioles. As mentioned above, supernumerary centrosomes normally cluster into functional bipolar spindles14,15, suggesting that most multipolar mitoses associ-ated with supernumerary centrosomes are transient and do not give
Helder Maiato and Elsa Logarinho are in the Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal, and in the Cell Division Unit, Department of Experimental Biology, Faculdade de Medicina, Universidade do Porto, 4200-319 Porto, Portugal. e-mail: [email protected]; [email protected]
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rise to multiple (>2) aneuploid daughter cells. As remarked by Boveri in his visionary treatise on the origin of malignant tumours, “the essential element in my hypothesis is not the abnormal mitoses, but a particular abnormal composition of the chromatin, irrespective of how it arises”4, which is in agreement with recent work demonstrating that the transient formation of multipolar spindles with supernumer-ary centrosomes might lead to chromosomal instability23,24. Normally the kinetochore on each sister chromatid is attached to microtubules emanating from one of the spindle poles. However, erroneous merotelic kinetochore-microtubule attachments (a single kinetochore attached to microtubules oriented to more than one spindle pole) can cause chro-mosomal instability in cells that ultimately undergo bipolar division23,24. Because merotelic attachments are not detectable by the spindle assem-bly checkpoint (SAC), which normally blocks anaphase in the presence of unattached kinetochores, they can cause lagging chromosomes in anaphase25. Importantly, the progeny derived from the few cells that do not cluster the extra centrosomes and instead undergo multipolar divisions mostly die or undergo cell cycle arrest, probably because of massive chromosome missegregation23.
Mitotic spindle multipolarity due to loss of spindle pole integrityLoss of spindle pole integrity in cells with a normal number of cen-trosomes might also lead to the formation of additional spindle poles. However, these spindle poles are somewhat atypical in the sense that centriole number may vary depending on the route(s) leading to the loss of spindle pole integrity. These include premature centriole dis-engagement, which normally leads to multipolar spindles with single centrioles at individual poles, and PCM fragmentation, which leads to an accumulation of acentriolar poles in addition to two normal poles (Fig. 2b).
The connection between centrioles is established during S phase and persists until late mitosis and G1 phase. Centriole disengagement is crucial for the licensing of centrioles for duplication and for limiting duplication to one event per cell cycle22. Interestingly, centriole disen-gagement at the end of mitosis is dependent on the activity of sepa-rase26,27, a protease that also controls the resolution of sister chromatin cohesion through cleavage of cohesin, and is inhibited by the SAC. Thus, inefficient inhibition of separase during a mitotic delay or arrest might result in both unscheduled sister chromatid separation and premature
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Figure 1 A schematic summary of the main causes of mitotic spindle multipolarity with and without centrosome amplification. The centrosome consists of a pair of centrioles surrounded by the pericentriolar material (PCM). (a) Two major mechanisms by which cells acquire supernumerary centrosomes are: centriole overduplication, caused by defects in the processes controlling centriole replication (curved arrow); and cytokinesis failure, which leads to both centrosome amplification and tetraploidy. (b) Two alternative mechanisms by which cells make multipolar spindles without extra centrosomes but with
additional microtubule nucleating foci (poles) are centriole disengagement and PCM fragmentation. Centriole disengagement arises from defects in centriole cohesion that lead to separation of paired centrioles before completion of chromosome segregation. PCM fragmentation caused by loss of centrosome structural integrity also generates acentriolar fragments that can nucleate microtubules. Both centriole disengagement and PCM fragmentation result in multipolar spindle formation following the action of spindle forces produced during chromosome alignment.
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centriole disengagement followed by formation of multipolar spindles. However, it remains unclear whether centriole disengagement relies on a local effect by separase or is merely a consequence of sister-chromatid separation and mitotic exit. Several conditions that delay the exit from mitosis with a completely or near-completely established metaphase plate — such as SKA3 depletion, expression of a SPDL1 (spindly) mutant, depletion of the APC/C activator CDC20, expression of non-degradable cyclin B1, CENP-E depletion and proteasome inhibition — were recently shown to cause uncoordinated sister chromatid separation and scat-tering, typically after a two-hour arrest. This was followed by centriole disengagement and multipolar spindle formation28,29, and the condition became known as ‘cohesion fatigue’. Curiously, it is well known that sev-eral treatments that impose a mitotic delay or arrest in the presence of microtubules (for example, recovery from low doses of colcemid or nocodazole, nitrous oxide, heat shock or proteasome inhibition) lead to multipolar spindle formation with atypical centriole distribution at each pole, including single or no centrioles, and the presence of mis-aligned chromosomes with ‘abnormal’ kinetochore–microtubule attach-ments30–34. In one of these studies it was indeed suggested that the longer the cell is blocked in mitosis, the greater the chance of it developing a tri- or tetrapolar spindle32.
How mitotic delay leads to loss of spindle pole integrity remains unclear, but it might be related to the presence of misaligned chromo-somes (or chromatids) and an inability to satisfy the SAC in a timely
manner (discussed in the section ‘Role of spindle and chromosomal forces’). Importantly, loss of spindle pole integrity can be delayed, but not prevented, if sister chromatid cohesion is manipulated or separase activity is compromised, suggesting that separase activity and cohesin cleavage are rate-limiting but not critical to triggering centriole disen-gagement28,29. In agreement with this idea, neither separase nor cohesin cleavage are required or sufficient for centriole disengagement in Drosophila melanogaster and Caenorhabditis elegans embryonic divi-sions35,36. Instead, Cdk1 inactivation seems to be the trigger for centri-ole disengagement, at least during Drosophila embryogenesis35. These observations have implications for the phenotypic analysis of mitotic spindle multipolarity without centrosome amplification, which might reflect the loss of function of a structural protein required for centriole engagement (or for spindle pole integrity in general), or an unspecific effect associated with a mitotic delay. We suspect that the latter might be the rule rather than the exception, given the strong correlation between mitotic delay imposed by apparently unrelated perturbations and loss of spindle pole integrity (see Supplementary Table 1). As an example, cells exposed to DNA damage or with incompletely replicated DNA
have a compromised capacity to satisfy the SAC (ref. 37), and also form multipolar spindles with acentriolar poles or split centrioles during mito-sis38,39. This does not necessarily imply that damaged or unreplicated DNA directly leads to multipolar spindle formation, which might occur simply as a consequence of a mitotic delay.
Figure 2 Images illustrating mitotic spindle multipolarity with and without centrosome amplification. (a) Fixed-cell analysis of HeLa cells stably co-expressing EGFP–centrin-2 (green) and stained for α-tubulin (red) and DNA (blue) for quantification of the number of centrioles per pole in a multipolar mitotic cell and inference of the causing mechanism behind multipolar spindle formation. Representative images are shown for centriole overduplication, cytokinesis failure, centriole disengagement and PCM fragmentation. Insets are magnifications of
the EGFP–centrin-2 signal in the indicated poles (numbered). (b) Live imaging of CLASPs-depleted HeLa cells stably co-expressing EGFP–centrin-2 (green) and α-tubulin–mRFP (red) as an essential experimental tool to unequivocally distinguish between centriole disengagement and PCM fragmentation. Arrowheads indicate the loss of spindle pole integrity. Scale bars, 5 μm. Time is in h:min:sec from nuclear envelope breakdown. Images generated by E.L. using unpublished data from experiments described in ref. 46.
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Independently of centriole disengagement, spindle pole integ-rity might be lost due to PCM fragmentation. Recent studies using super-resolution microscopy have established that the PCM, previously thought to be amorphous, actually forms highly ordered structures (reviewed in ref. 1). Additionally, centrosomes are surrounded by cytoplasmic virus-like granules known as centriolar or pericentriolar satellites that are involved in the recruitment and turnover of centro-somal proteins40–42. Several centrosomal proteins including PCM-1, centrin-2 and ninein aggregate at pericentriolar satellites, which are transported along microtubules by the minus-end-directed dynein–dynactin motor complex40,42. Ninein at pericentriolar satellites may work as a microtubule anchoring factor during interphase43,44. More recently, depletion of ninein or other pericentriolar satellite proteins was found to result in PCM fragmentation, generating multipolar spindles18,42,45–49 (Fig. 3a and Supplementary Table 1). Therefore, a role for pericentriolar satellites during mitosis is emerging as part of the intricate structural and molecular network that ensures spindle pole integrity. It will be important to investigate how pericentriolar satellites
are organized with other structural PCM components to ensure spindle pole integrity during mitosis.
Loss of function of the mitotic kinases PLK1 and aurora A has also been linked to mitotic spindle multipolarity without centrosome ampli-fication through a functional interplay between the centrosome- and spindle-associated proteins kizuna, TPX2, chTOG, TACC3 and clath-rin, which are required for spindle pole integrity47,50–59. Additionally, defects in RanGTP-dependent factors such as TPX2, RanBP1, RCC1 and importin-β, which are associated with centrosome-independent spindle assembly, may cause multipolar spindle formation due to PCM fragmentation and/or premature centriole disengagement, and this is usually associated with misaligned chromosomes60–64. However, it remains unclear whether all these loss-of-function phenotypes reflect a specific role at spindle poles or result from cohesion fatigue after a mitotic delay. One way to dissect these possibilities would be to use live cell imaging with appropriate markers46 to determine whether loss of spindle pole integrity is caused by centriole disengagement (Fig. 2b). Centriole disengagement could be preceded, or not, by sister chromatid
Kid/kinesin-10 PCM protein(e.g. CLASPs)
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Figure 3 The role of misaligned chromosomes and chromatids in multipolar spindle formation due to loss of spindle pole integrity. (a) A hypothetical schematic representation of the Eg5 (kinesin-5) counteracting forces generated by kinetochore- (red arrow) and arm-associated (yellow arrow) motors on misaligned chromosomes leading to irreversible spindle multipolarity. (i) Functional centrosomes (orange) normally resist Eg5/kinesin-5 counteracting forces owing to their structural integrity ensured by a complex network of both PCM (e.g. CLASPs) and pericentriollar satellite (e.g. ninein) proteins. (ii) Structurally compromised centrosomes (brown) lacking PCM or centriole components become fragmented (red curved arrow) in response to chromosome alignment traction forces. (iii) Multipolar spindle
conformation generated by loss of spindle pole integrity is not transient. (iv) Cells often enter anaphase producing aneuploid daughter cells. (b) A hypothetical schematic representation of the forces mediated by misaligned chromatids during cohesion fatigue and their tension over the poles. (i) Single scattered chromatids generate Eg5/kinesin-5 and dynein (green arrow) counteracting forces through the action of kinetochore- and arm-associated motors. (ii) Centrioles loose integrity during prolonged mitotic delay and become fragmented in response to chromosome alignment traction forces. (iii) Scattered chromatids and pole fragmentation become more severe during the mitotic arrest. (iv) This eventually leads to cell death in mitosis or mitotic slippage.
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scattering after metaphase plate formation and mitotic delay as dis-cussed above.
Role of spindle and chromosomal forcesMultipolar spindle formation due to loss of spindle pole integrity occurs after bipolarization at a time when kinetochore-microtubule attachments start to become established, indicating that spindle and/or chromosomal forces are required for this process. During spindle bipolarization cen-trioles must resist the pulling and pushing forces mediated by dynein and Eg5 (KIF11; also known as kinesin-5), respectively, acting on micro-tubules to drive centrosome separation65. Subsequently, the NuMA–dynein–dynactin complex and HSET (KIFC1; also known as kinesin-14) help to focus microtubules at spindle poles and counteract Eg5-mediated pushing forces which generate kinetochore tension on bi-oriented chro-mosomes66,67. When end-on kinetochore–microtubule attachments are disrupted or chromosome-arm-mediated forces are inhibited, the motor-driven activities of NuMA–dynein–dynactin and HSET are no longer required for spindle pole focusing, which relies essentially on the microtubule crosslinking activity of TPX2 (refs 68,69). Therefore, spin-dle pole organization must be coordinated with kinetochore- and chro-mosome-arm-mediated forces. The literature describes many instances where molecular perturbations that lead to loss of spindle pole integrity are associated with misaligned chromosomes (Supplementary Table 1), leading to the conclusion that spindle pole organization is required for chromosome alignment. We suggest instead that the spindle pole defects associated with such molecular perturbations do not cause chromosome misalignment, but rather are a consequence of misaligned chromosomes. In other words, the loss of particular structural proteins required for spindle pole integrity and focusing is insufficient per se to cause mitotic spindle multipolarity, and the latter is caused by forces required to align chromosomes at the metaphase plate (Fig. 3a).
Chromosome alignment in human cells relies on the coordinated action of the kinetochore-associated plus-end-directed motor CENP-E (also known as kinesin-7) and the chromokinesins Kid (KIF22; also known askinesin-10) and KIF4a (also known as kinesin-4). CENP-E slides misaligned chromosomes along the lattice of pre-existing micro-tubule bundles (through so-called lateral kinetochore–microtubule attachments)70,71 and chromokinesins mediate polar ejection forces that push chromosome arms away from spindle poles72,73. Both kine-tochore- and arm-mediated forces required for chromosome alignment play an active role in the genesis of spindle multipolarity by disrupt-ing spindle pole integrity46,58 (Fig. 3a). Spindle pole integrity requires the recruitment of ninein to residual pericentriolar satellites, which is mediated by CLASPs, and both CLASPs and ninein are required to resist kinetochore- and arm-generated forces independently of end-on microtubule–kinetochore attachments46 (Fig. 3a).
Similarly to ninein, other pericentriolar satellite proteins prevent multipolarity by ensuring spindle pole resistance to forces exerted during chromosome alignment. CEP72 regulates the centrosomal localization of kizuna, a PLK1 target required for spindle pole integrity in response to Kid-mediated forces58. CEP90 also prevents PCM fragmentation in response to mechanical forces from spindle microtubules during promet-aphase48. Interestingly, a similar dependence on CENP-E-mediated forces leading to loss of spindle pole integrity due to centriole disengagement was observed following expression of a non-functional mutant of Hec1 at kinetochores74 (Fig. 3b), which causes cohesion fatigue after completion of
chromosome alignment and a mitotic delay or arrest (Francesca Degrassi, personal communication). Depletion of CENP-E further rescues mitotic spindle multipolarity and premature centriole disengagement after astrin depletion46, likely to be caused by cohesion fatigue75. Importantly, despite sister chromatid scattering after a prolonged metaphase delay caused by loss of CENP-E function28, mitotic spindles remain bipolar46, indicating that premature centriole disengagement can be functionally separated from cohesion fatigue. Moreover, the CENP-E orthologue in Drosophila (CENP-meta) facilitates extra centrosome clustering in S2 cells16, which might explain the small number of multipolar spindles upon inhibition of human CENP-E in human cancer cell lines76,77. These results suggest that CENP-E-mediated forces can either rescue or promote spindle multipolarity depending on the presence or absence of supernumerary centrosomes, respectively.
Based on the observations outlined above, we propose that kine-tochore- and arm-mediated forces generated by misaligned chromo-somes (Fig. 3a) or scattered chromatids resulting from cohesion fatigue (Fig. 3b) oppose spindle pushing forces mediated by Eg5, and are respon-sible for the formation of multipolar spindles due to loss of spindle pole integrity. Whereas it may seem intuitive that traction forces during chromosome alignment can disrupt structurally compromised spindle poles, it remains unclear how forces mediated by single misaligned chro-matids that result from cohesion fatigue impinge on tension over the poles. Unstable end-on and/or lateral kinetochore–microtubule attach-ments predominate following cohesion fatigue, and therefore CENP-E, together with polar ejection forces mediated by chromokinesins, might counteract dynein-mediated kinetochore poleward motion78 and/or Eg5-mediated pushing forces. These opposite forces could result in suf-ficient tension to break the poles and cause centriole disengagement (Fig. 3b). In agreement with this model, Kid-mediated ejection forces are required to expel chromosome arms from the spindle region, favouring early lateral kinetochore–microtubule attachments, which were shown to produce significant levels of centromere stretching79. Understanding how the different kinetochore- and arm-associated motors are func-tionally coordinated during mitosis will be critical to clarify the role of chromosome-mediated forces in determining mitotic spindle architec-ture. Importantly, because cells with single scattered chromatids prevent efficient SAC satisfaction28,29,80, their fate following different molecular perturbations might be used to distinguish between loss of spindle pole integrity due to a specific role in the process (with cells often entering a multipolar anaphase; Fig. 3a) and a downstream effect caused by cohe-sion fatigue (with cells often dying in mitosis or undergoing mitotic slippage; Fig. 3b).
Extra centrosome clustering and spindle pole integrity are ensured by common molecular mechanismsRecent genome-wide studies in Drosophila S2 cells and human cancer cells have shed light on the molecular mechanisms behind extra cen-trosome clustering16,18. These studies have highlighted the relevance of SAC-dependent control of mitotic duration, the requirement for several microtubule-associated and motor proteins, spindle anchoring to the cell cortex and the formation of functional end-on kinetochore–micro-tubule attachments to cluster extra centrosomes into bipolar spindles. An analysis of the molecular players required for spindle pole integrity and their involvement in extra centrosome clustering (Supplementary Table 1) indicates that, with the exception of SAC-dependent extension
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of mitotic duration and CENP-E (both of which are specifically required for extra centrosome clustering16,46,81,82), similar molecular mechanisms are responsible for managing extra centrosomes and preventing loss of pole integrity, as previously suggested15. Specifically, proteins con-tributing to microtubule anchorage and coupling at spindle poles — including the minus-end-directed motors dynein and kinesin-14, the microtubule-associated proteins TPX2, chTOG, NuMA and TACC3, components of the Ran pathway and the Augmin complex — all prevent mitotic spindle multipolarity in cells with and without extra centrosomes (see Supplementary Table 1 and references therein). Moreover, proteins implicated in the establishment and regulation of end-on microtubule–kinetochore attachments, including the chromosomal passenger com-plex (CPC) and the NDC80 complex, as well as proteins required for the establishment of sister chromatid cohesion, are important for extra centrosome clustering and spindle pole integrity18,46,74,83 (Supplementary Table 1). These spindle intrinsic mechanisms seem to cooperate with actin-dependent cortical forces mediated by myosin 10A and cell-matrix adhesion proteins to cluster multiple centrosomes and/or ensure bipolar spindle pole integrity16,17,84,85. Interestingly, spindle pole integrity defects caused by preventing mitotic cell rounding can be rescued by experi-mentally increasing microtubule length85, which may affect the extent of microtubule crosslinking and/or microtubule-dependent forces that are important to preserve spindle pole integrity.
Spindle multipolarity and cancerIt remains unknown what fraction of multipolar spindles in cancer are caused by centrosome amplification. This is primarily because the use of antibodies against PCM proteins (for example, γ-tubulin and pericen-trin) — the main tool to investigate centrosome amplification associated with mitotic spindle abnormalities in human cancer samples and cancer-derived cell lines — does not provide information about centrosome structural integrity and composition. However, in a few studies, centriole markers and/or electron microscopy revealed not only structural and numerical alterations of the centrosomes86 but also the absence of cen-trioles from such supernumerary γ-tubulin- and pericentrin-positive structures in cancer cells87. These findings raise caution regarding the aetiology of mitotic spindle multipolarity and chromosomal instability observed in human cancers.
Cytokinesis failure, telomere damage or virus-mediated cell fusion can result in supernumerary centrosomes and/or a tetraploid state. The so-called tetraploid intermediates can induce chromosomal instability and tumour formation in mice in a manner dependent on p53 loss88–90, and recent genomics data support the idea that tetraploid intermedi-ates are common in cancer91,92, but it remains unknown whether these are associated with supernumerary centrosomes. Curiously, despite several reported examples of live born tetraploid human individuals93, it is well established that tetraploidy in cells with functional p53 leads to cell cycle arrest in G1 phase (reviewed in ref. 94). Indeed, the forma-tion of tetraploid intermediates through repeated cleavage failure during cytokinesis of human cells in culture is not associated with centrosome amplification and impairs cell viability95. Nevertheless, proliferation of some tetraploid cells might be assured after re-establishment of normal centrosome number23,96 or prevented by the recently discovered phe-nomenon of interphase cytofission, in which binuclear cells can parti-tion its nuclei by fission97. Interestingly, pioneer studies of female genital carcinomas reported that multipolarity is not always connected with
polyploidy and that the first, and also most common, multipolar divi-sions to appear during cancer progression generate tripolar spindles98. It is therefore tempting to speculate that loss of pole integrity, which leads mostly to tripolar spindles without centrosome amplification, might be an early step in tumorigenesis and chromosomal instability, at least in some forms of cancer.
Therapeutic potential of multipolar mitosesRegardless of the potential role played by multipolar spindles in the aetiology of cancer, this feature is starting to be explored therapeutically. The notion of ‘anaphase catastrophe’99 has recently been proposed as a pro-apoptotic death mechanism resulting from multipolar anaphases and with anti-neoplastic potential, the rationale being that cancer cells with supernumerary centrosomes would undergo a multipolar ana-phase if centrosome clustering is prevented16,99. This would spare nor-mal mitotic cells with just two centrosomes and therefore selectively kill cancer cells16,100. A particularly attractive target is HSET because of the correlation in cancer cells between centrosome amplification and increased sensitivity to HSET inhibition either by RNA interference16 or by recently developed small-molecule inhibitors101,102.
Although anaphase catastrophe was initially formulated for cancer cells that were prevented from clustering supernumerary centrosomes, there is compelling evidence that irreversible multipolar spindle forma-tion caused by loss of spindle pole integrity has the same outcome46. As we conclude from this review, proteins required for extra centro-some clustering are also important to preserve spindle pole integrity in cells with normal centrosome number (Supplementary Table 1), rais-ing concerns about the potential of anaphase catastrophe to selectively kill cancer cells. For example, it was shown recently that, in addition to its well-established role in centrosome clustering, HSET is required for proper bipolar spindle assembly, stable pole-focusing and survival of at least some cancer cells with normal centrosome number103. Finally, it is worth noting that the Drosophila kinesin-14 orthologue Ncd is required for both meiotic (acentrosomal) and mitotic spindle assembly during early embryonic divisions104,105. Together, these data suggest that the observed sensitivity of some cancer cells to kinesin-14 inhibition may not be exclusively due to the presence of extra centrosomes. In this regard, cancer cells might be more sensitive to kinesin-14 inhibition also because of the importance of kinesin-14 in centrosome-independent mitotic spindle formation103,106,107, which, as in Drosophila108, may be hyperactivated in cancer cells109.
Several chemical, physical and biological treatments have been reported to cause multipolar spindles by preventing extra centrosome clustering and/or disrupting spindle pole integrity (Supplementary Table 1). These include the widely used fungicide drugs methyl 2-benzi-midazolecarbamate, griseofulvin and more potent derivatives, as well as heat shock, X-ray radiation or viral infections32,34,38,110–120. More recently, the microtubule-stabilizing agent epothilone B and the aurora A inhibi-tors tripolin A, MLN8237 and MLN8054, were shown to cause the for-mation of multipolar spindles with acentrosomal poles121,122. Finally, the microtubule-stabilizing taxanes, which are amongst the most successful drugs routinely used in the treatment of several solid tumours123, have been shown to cause mitotic spindle multipolarity in cultured trans-formed and untransformed cells or in vivo, especially after a short pulse with clinically relevant concentrations124–130. Most strikingly, multipolar-ity induced by taxanes is not associated with centrosome amplification
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and is caused by loss of bipolar spindle pole integrity, or arises by de novo acentrosomal spindle formation124,127,128,130. Curiously, taxanes preferen-tially associate with centrosomes and spindle pole microtubules125, which might explain the observed loss of spindle bipolarity. Also relevant is the fact that although progression through mitosis is slowed, cells treated with taxanes are not blocked in mitosis but instead enter a multipo-lar anaphase, often complete cytokinesis and give rise to an aneuploid progeny with reduced viability124,126,128,131. Similar results have recently been obtained in a pilot clinical trial with breast cancer patients, show-ing additionally that the patients’ response to taxol treatment did not correlate with mitotic arrest132. Thus, the cytotoxic effect associated with short exposure to clinically relevant doses of taxanes does not seem to be caused by mitotic arrest, but rather by a catastrophic exit from mitosis after SAC satisfaction.
ConclusionsWe still know very little about the aetiology of multipolar spindle forma-tion and its relevance in health and disease. Although the predominant view that supernumerary centrosomes are important drivers of chro-mosomal instability is not disputed here, it may have contributed to the overlooking of the relevance of spindle multipolarity without centro-some amplification. The inclusion of centriole markers in fixed prepara-tions supported by live-cell analysis of mitotic spindles in cultured tissues and/or in vivo will be fundamental to evaluate the real penetrance of loss of spindle pole integrity in human cancers. Importantly, the find-ings summarized here support the idea that the same molecular mecha-nisms involved in the clustering of supernumerary centrosomes might also account for normal mitotic spindle bipolarity. We draw attention to the role played by misaligned chromosomes during early mitosis as important force-generating bodies that can influence mitotic spindle architecture, especially in conditions of fragile spindle poles (for example due to the action of drugs). We also highlight the integrated nature of mitotic spindles, where all components must work in concert to achieve the proper conformation and function. Finally, the correlation between mitotic delay and the development of multipolarity without centrosome amplification clearly justifies in-depth investigation, with important clinical implications towards the control of human cancers.
ACKNOWLEDGMENTSWe thank Beth Weaver and Francesca Degrassi for communicating results before publication. We apologise to all colleagues whose primary work could not be cited due to length restrictions. E.L. is supported by Programa Operacional Regional do Norte (ON.2) and grants NORTE-07-0124-FEDER-000003 and PTDC/SAU-OBD/100261/2008 from Fundação para a Ciência e a Tecnologia (FCT) of Portugal (COMPETE-FEDER). Work in the laboratory of H.M is funded by FEDER through the Operational Competitiveness Programme – COMPETE and by National Funds through FCT – Fundação para a Ciência e a Tecnologia under the project FCOMP-01-0124-FEDER-015941 (PTDC/SAU-ONC/112917/2009), the Human Frontier Science Program and the 7th framework program grant PRECISE from the European Research Council.
Note: Supplementary Information is available in the online version of the paper
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*Including cohesion fatigue. †Live imaging not performed or inconclusive, and/or lack of centriole marker. ND, not determined; NC, not clear. When determined, the table also lists the correlation of this phenotype with the presence of misaligned chromosomes, the forces involved, whether or not extra centrosome clustering was seen and the subsequent fate of the cells.
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Biol. 192, 959–968 (2011).135. Lawo, S. et al. HAUS, the 8-subunit human Augmin complex, regulates centrosome and spindle integrity. Curr. Biol. 19, 816–826 (2009).136. Holmfeldt, P., Zhang, X., Stenmark, S., Walczak, C. E. & Gullberg, M. CaMKIIgamma-mediated inactivation of the Kin I kinesin MCAK is essential for bipolar spindle formation.
EMBO J. 24, 1256–1266 (2005).137. Jeffery, J. M., Urquhart, A. J., Subramaniam, V. N., Parton, R. G. & Khanna, K. K. Centrobin regulates the assembly of functional mitotic spindles. Oncogene 29, 2649–2658
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