Mitotic chromosomal instability and cancer: mouse modelling of the human disease (original) (raw)

. Author manuscript; available in PMC: 2017 Jul 25.

Published in final edited form as: Nat Rev Cancer. 2010 Feb;10(2):102–115. doi: 10.1038/nrc2781

Abstract

The stepwise progression from an early dysplastic lesion to full-blown metastatic malignancy is associated with increases in genomic instability. Mitotic chromosomal instability — the inability to faithfully segregate equal chromosome complements to two daughter cells during mitosis — is a widespread phenomenon in solid tumours that is thought to serve as the fuel for tumorigenic progression. How chromosome instability (CIN) arises in tumours and what consequences it has are still, however, hotly debated issues. Here we review the recent literature with an emphasis on models that recapitulate observations from human disease.

Since Boveri observed abnormal chromosome complements in tumour cells at the beginning of the twentieth century1,2, the role of chromosome instability (CIN) in tumour initiation and progression has been a central issue in cancer biology. Only recently, using sophisticated mouse modelling approaches, is it becoming clear that CIN is not simply a passenger phenotype but probably plays a causative part in a substantial proportion of malignancies. However, several questions and controversies still remain. Here we review these issues through the analysis of recent findings and their relevance to human disease. We focus on two crucial questions: first, how is aneuploidy generated? Second, what is the role of CIN in tumour initiation and/or progression?

Throughout this Review we will concentrate on the CIN that arises as a result of an abnormal mitosis. This CIN can occur because of alterations in mitotic timing, mitotic checkpoint control, or of microtubule or centrosome dynamics. Abnormalities in double-strand break repair or telomere maintenance can also eventually lead to CIN as a result of repeated chromosome breakage–fusion–bridge cycles. Because of space constraints, however, we will not cover the deregulation of these pathways, although we note that they may ultimately lead to mitotic abnormalities. Failure of the mitotic checkpoint machinery, which blocks the separation of sister chromatids before microtubule attachment (described below), has been an obvious candidate mechanism involved in the generation of CIN during mitosis. Disappointingly, the mitotic checkpoint is rarely found to be compromised in human tumours. It is, however, frequently hyperactivated in chromosomally unstable lesions and, importantly, this overactivation of the mitotic checkpoint is intricately linked to the inhibition of major tumour suppressive pathways and the acquisition of CIN.

As a point of nomenclature, distinctions have previously been made between whole chromosome instability (W-CIN) and CIN that includes translocations, interstitial deletions and amplifications (segmental chromosome instability or S-CIN)3. S-CIN is observed in several model systems into which mitotic defects have been introduced4–6. Some models of CIN only show W-CIN changes, yet human tumours often contain both abnormal chromosome complements and structural changes, and so we prefer to use the global definition of CIN in this Review. Where indicated, we will distinguish between the two forms as W-CIN and S-CIN. Other defects in genome integrity, such as microsatellite instability, defects in nucleotide excision repair or base excision repair, defects in telomere maintenance or the stability of larger repeats and abnormalities in the G2/M DNA damage checkpoint, also play a part in tumour initiation and progression and have been reviewed extensively elsewhere7–11.

Many of the studies that model mitotic CIN in mice are based on perturbations in the mitotic checkpoint pathway, which ensures the accurate segregation of chromosomes during mitosis. Because of its relevance to understanding genomic stability, a brief overview of the history of the identification of checkpoints is included in BOX 1. We reiterate, however, that although the identification of a DNA damage checkpoint and a mitotic checkpoint in yeast primed the cancer field to search for checkpoint mutations in human tumours, the requirement for the mitotic checkpoint in every cell division and the lethality observed in its absence make its loss in tumours an unlikely mechanism for the generation of CIN.

Box 1. A brief history of the identification of checkpoints.

In the late 1980s Ted Weinert and Leland Hartwell identified the existence of a DNA damage checkpoint in budding yeast (Saccharomyces cerevisiae)98. The Rad9 mutant strain they characterized was shown to be defective in the ability to arrest cell division as a result of irradiation-induced DNA damage. Importantly, Rad9 mutants were viable in the absence of DNA damage; only when irradiated with ionizing radiation did the lethality become evident. Moreover, ionizing radiation-induced lethality could be rescued by providing sufficient time for repair by growing the cells in the presence of the microtubule poison benzimidazole, which we now know leads to activation of the mitotic checkpoint. These studies provided experimental evidence for the existence of cell cycle checkpoints99. These signalling pathways are postulated to be non-essential in the absence of damage, and their main function is to arrest cell division until the damage is repaired or, in the case of mammalian cells, undergo programmed cell death when repair is not completed. Soon after these findings, two laboratories simultaneously identified an equivalent checkpoint responsible for arresting cell division in S. cerevisiae in response to mitotic spindle abnormalities induced by microtubule poisons. The mitotic arrest deficient (Mad)100 and budding uninhibited by benzimidazole (Bub)101 genes were identified in screens for sensitivity to spindle poisons and, as such, loss-of-function mutants were viable so long as cell division proceeded normally. These studies provided direct evidence for the existence of a spindle assembly checkpoint or mitotic checkpoint in budding yeast, the function of which is to arrest cell division at metaphase until all kinetochores are attached to microtubules from opposite spindle poles.

The later characterization of the mitotic checkpoint in mammalian cells102 revealed important differences relative to budding yeast. First, the mitotic checkpoint is essential in all normal or transformed mammalian cells examined49,53,103,104. This unexpected result may be due to karyotypic complexity and the transient requirement of the mitotic checkpoint during an unperturbed cell cycle to prevent an intolerable level of chromosome instability. In addition, non-kinetochore-bound human MAD2 and BUBR1 have checkpoint-independent functions that are required to prevent premature exit from mitosis, perhaps by blocking the degradation of key substrates early in the mitotic cycle23,28,105. Therefore, unlike the case in budding yeast, mitotic checkpoint genes are generally essential in each mammalian cell division and, in contrast to the DNA damage checkpoint, their complete loss is unlikely to account for the accumulation of genomic damage in human tumours.

An outline of the mitotic checkpoint

The molecular mechanisms responsible for the mitotic checkpoint have been thoroughly reviewed elsewhere12,13. Here, we present a broad outline (FIG. 1), emphasizing some of the recent controversies.

Figure 1. The mitotic checkpoint.

a | Outline of the mitotic checkpoint. An unattached kinetochore is shown on the left with the inner complex in purple, MAD1 in grey and MAD2 in its open and closed forms in purple. The mitotic checkpoint complex (MCC) is shown to inhibit the anaphase promoting complex/cyclosome (APC/C), which after attachment of the last kinetochore is activated and ubiquitylates securin and cyclin B1. More details of this pathway are described in the main text. b | The amplification of the unoccupied kinetochore signal is thought to depend on the conversion of MAD2 open complexes (MAD2(O)) to closed complexes (MAD2(C)) that bind to cell division cycle 20 (CDC20) and deliver it to the APC/C for ubiquitylation (Ub). The nature of the MCC is still debated, as indicated by question marks. A separate APC/C is shown to indicate its role in timing, independent of the kinetochore-derived signal. CDK1, cyclin-dependent kinase 1.

In its simplest form, the mitotic checkpoint is a mechanism by which eukaryotic cells arrest cell division at metaphase until all sister kinetochores are attached to microtubules from opposite spindle poles. In prometaphase, sister chromatids are topologically linked by the ring-like cohesin complexes14. In addition, the activity of cyclin-dependent kinase 1 (CDK1, also known as cell division cycle 2; CDC2), the main mitotic kinase, is high and maintains the mitotic state. As mammalian cells proceed from prometaphase to metaphase, a signalling complex that contains mitotic arrest deficient 1 (MAD1), MAD2, MPS1 (also known as TTK), BUB1, BUB3 and BUBR1 assembles at unoccupied kinetochores. This in turn leads to the generation of a diffusible signal that is dependent on MAD2 and BUBR1 (REFS 15–19), which prevents the E3 ubiquitin ligase complex anaphase promoting complex/cyclosome (APC/C)20 from degrading its mitotic targets cyclin B1 and securin (also known as pituitary tumour-transforming 1; PTTG1). In this state, exit from mitosis and the separation of sister chromatids are inhibited. As soon as the last kinetochore pair is attached to the microtubules at opposite spindle poles, the inhibitory diffusible signal is extinguished and the APC/C is fully activated through the release of inhibition of its cofactor, CDC20. This leads to the ubiquitylation of cyclin B1 and securin, the two crucial partners of CDK1 and the cysteine protease separase (also known as ESPL1), respectively. Degradation of cyclin B1 by the 26S proteasome leads to a rapid decline in CDK1 activity, allowing exit from mitosis. Securin is a small inhibitory chaperone of separase, the activity of which is essential for the dissolution of cohesin complexes at and near sister chromatid kinetochores. Degradation of securin by the 26S proteasome and release from inhibition by CDK1–cyclin B1 phosphorylation owing to cyclin B1 degradation leads conjunctly to activation of separase and cleavage of the SCC1 (also known as RAD21) component of cohesin; the net effect of this is the separation of sister chromatids. Both of these events, inhibition of CDK1 and activation of separase, are necessary for a correct metaphase-to-anaphase transition and faithful segregation of chromosomes.

Although it is clear that a diffusible inhibitory signal is generated at kinetochores and prevents APC/C acting on cyclin B1 and securin15, the nature of this event remains unclear. Musacchio and others have proposed a prion-like model based on two structural conformations adopted by MAD2 (REFS 21,22): open and closed. At this point the evidence for this model is biochemical but it accounts for the role of MAD2 at kinetochores, its interaction with CDC20 and the signal amplification required for the inhibition of the cell cycle by a single unoccupied kinetochore. Unoccupied kinetochores are known to recruit MAD1, which in turn binds with high affinity to MAD2 in its closed conformation. This MAD1–MAD2 complex is then thought to catalyse the conversion of open MAD2 monomers (the predominant form in the cytosol) to closed MAD2 forms that then bind to CDC20. This interaction serves a dual purpose: it inhibits the activity of APC/C (at least with regard to cyclin B1 and securin) and catalyses the further conversion of MAD2 open monomers to closed MAD2–CDC20 complexes, accounting for the required signal amplification.

Several observations substantially complicate this model. First, it is unclear what the role of BUBR1 is with regard to APC/C inhibition. BUBR1 is necessary for mitotic checkpoint function and is recruited to unoccupied kinetochores23. Its kinase domain is, however, dispensable for APC/C inhibition, and its amino (N)-terminal domain is sufficient to act as a pseudo-substrate inhibitor of the APC/C. Moreover, this APC/C inhibitory function is not dependent on the presence of BUBR1 at kinetochores. These findings have led to a modified MAD2 template model in which the heterodimeric CDC20–MAD2 closed conformer is required to deliver BUBR1 and perhaps BUB3 to the APC/C, where BUBR1 can then inhibit CDC20 function. The proposed mitotic checkpoint complex (MCC) composed of MAD2, CDC20, BUBR1 and BUB3 may be transient. MAD2 may depart, leaving behind a CDC20–BUBR1–BUB3 complex bound to the APC/C, although the duration of MAD2 persistence is still not completely resolved (for example, REFS 19,24).

Structural studies25 suggest that CDC20 is displaced from its active location on the APC/C when MCC components are present; an event that may in turn facilitate CDC20 ubiquitylation26, thereby maintaining a ‘checkpoint-on’ state. Here, ubiquitylation of CDC20 would continue so long as BUBR1–BUB3 (and perhaps MAD2) are still bound to the APC/C and would cease once the checkpoint is satisfied. Phosphorylation of MAD2 (REF. 27) and, recently, acetylation of BUBR1 (REF. 28) have been proposed to extinguish the binding to the APC/C and/or the stability of these components. This in turn would allow CDC20 to reoccupy its site on the APC/C, where it is protected from ubiquitylation. CDC20 would then assume its role in directing the APC/C to its principal downstream targets, securin and cyclin B1. Remarkably, cyclin A and NEK2 are ubiquitylated by the APC/C in the checkpoint-on state, adding further complexity to the inhibition of the APC/C. Substrate specificity and therefore substrate ordering seem to be key events in the different stages of mitosis but a molecular understanding of how substrate specificity arises remains limited.

In contrast to these findings, two independent reports have proposed that ubiquitylation of CDC20 by the E2 enzyme UBCH10 inactivates the checkpoint by blocking the association of CDC20 with MCC components29,30. The strongest evidence against this last model and in favour of one in which CDC20 ubiquitylation leads to its degradation during the checkpoint-on state comes from the finding that a form of CDC20 that lacks lysine and therefore cannot be ubiquitylated shows premature escape from mitotic arrest26.

Reconciling all of these findings and integrating them into the MAD2 template model will certainly require more complex biochemical models but, more importantly, these models will need to be tested in vivo to understand how a single unoccupied kinetochore can maintain a cell, at least for a certain period of time (as we discuss below), in mitosis.

Aneuploidy and CIN in tumours

The notion that CIN contributes to tumour initiation and/or progression is as old as our understanding of chromosomes. As mentioned, Boveri postulated more than 100 years ago that abnormalities in chromosome segregation could promote tumour formation1,2,26. Although some arguments can still be made for aneuploidy as simply a passenger event in tumours, three lines of observation argue otherwise.

First, in vitro transformation of cell lines through various genetic alterations that lead to CIN suggests aneuploidy has a direct causal role in tumorigenesis. Transformation of cells in culture has been a standard assay to determine the oncogenic or tumour suppressive nature of a gene for more than two decades31,32. Although the genetic events that must occur for a primary cell to become transformed may differ substantially from those that occur in human tumours, several now-established oncogenes and tumour suppressors were identified by transformation assays33–36. Among the mitotic checkpoint genes with roles that have been explored in in vitro transformation, securin overexpression in primary cells leads to marked aneuploidy and is sufficient for transformation37. Overexpression of aurora kinase A (AURKA), the function of which is required for centrosome maturation, bipolar spindle assembly and mitotic entry38, similarly leads to aneuploidy and transformation in human and rodent cells39,40 as a result of abnormal mitoses.

Second, perhaps the most robust causative data linking CIN to tumorigenesis comes from the study of mouse models of aneuploidy. Several laboratories have generated mouse models of aneuploidy based on mutations or transcriptional changes of mitotic checkpoint genes observed in tumours. An obvious caveat of all these individual studies is that these genes have non-mitotic functions that might explain their tumorigenic potential. In addition to their accepted mitotic functions, MAD2 (REF. 41) and RANBP2, a RAN GTPase binding protein that localizes to kinetochores during mitosis42, have been implicated in nuclear trafficking. Securin has also been linked to modification of p53 function43, and BUB1 and BUBR1 have been shown to play a part in the response to DNA damage44,45. Nevertheless, data from studies that have analysed various different genes involved in mitotic checkpoint control argue strongly for a contributory role of aneuploidy itself in tumour initiation and progression. TABLE 1 summarizes some of the mouse models of aneuploidy and their contributions to our understanding of CIN in human tumours. As we will describe in the next section, some of these models are more faithful to the mechanisms that are associated with CIN in human tumours than others but the message is the same: in general, CIN favours tumour formation.

Table 1.

Cancer models of CIN

Third, a large amount of data collected from human tumours suggests that aneuploidy has a causative role in tumorigenesis by showing that CIN and chromosomal aberrations correlate with tumour grade and prognosis46,47. Further supporting this argument, genes involved in maintaining chromosome stability are frequently deregulated in human tumours48, as we will discuss in the next section. Finally, transcriptional expression profiles of aneuploid tumours have revealed a CIN signature that can be used to stratify lesions according to prognosis in an unbiased manner46. The fact that this CIN signature, which contains genes that are involved in a wide range of pathways, can predict clinical outcome even if genes that are regulated by the cell cycle are omitted provides further evidence that CIN plays a contributory part in the progression of these human tumours.

How is aneuploidy generated in human tumours?

Inevitably, most of the mechanistic studies that aim to answer this question have been carried out in mice and their results are summarized in TABLE 1. Many of these models have been generated based on the hypothesis that loss or downregulation of the mitotic checkpoint is responsible for CIN. Although this is largely the case in vitro and in model systems in vivo, as we discuss below, if these perturbations are to explain the mechanisms by which aneuploidy is generated in human cancer, there must be evidence for such changes in aneuploid human tumours. In other words, sufficiency for a cancer phenotype in mice or any other model cannot by itself be interpreted as an explanation for human disease without direct experimental evidence.

In mammalian cells a weakened mitotic checkpoint would be predicted to facilitate W-CIN as a result of premature exit from mitosis and premature separation of sister chromatids (FIG. 2). An overview of mouse models of aneuploidy reveals that this prediction is correct. Fibroblasts or lymphocytes derived from mice heterozygous for Mad2l1 (which encodes MAD2)49, Bub1b (which encodes BUBR1)50,51, Bub3 (REFS 52,53), Bub1 (REF. 54) and centromere protein E (Cenpe)55 or a _Cdc20_AAA mutant that cannot bind MAD2 (REF. 56) show varied levels of aneuploidy. In addition, several animal strains that have these genetic lesions develop tumours in various organs at late stages of life or are more prone to tumours in sensitized backgrounds. Nevertheless, several separate lines of evidence argue against the loss of mitotic checkpoint gene function as the main causative mechanism for aneuploidy in human tumours.

Figure 2. Multiple mechanisms leading to aneuploidy.

The normal mitotic checkpoint (MC) events from prometaphase to anaphase are shown in the centre. An absent checkpoint leads to mitotic cell death. A weak checkpoint (left) leads to premature sister chromatid separation and near-diploid aneuploidy. An overactive checkpoint (right) can lead either to mitotic cell death or lagging chromosomes and subsequent near-diploid aneuploidy or tetraploidy. Multiple centrosomes can have similar consequences to an overactive checkpoint. A multipolar mitosis leads to cell death unless centrosomes cluster, in which case the likelihood of lagging chromosomes is high.

Through extensive analyses of aneuploid human tumours, it is now increasingly clear that mutations in mitotic checkpoint genes are rare (TABLE 1 and reviewed in REF. 57). As we have mentioned, complete loss of the mitotic checkpoint is lethal at the cellular and organismal levels. Note that conditional inactivation of Bub1 in adult male mice58 impairs fertility without any decrease in viability but no other tissues were examined, and it is unclear what the penetrance of inactivation was in this case. It therefore remains possible that actively proliferating tissues were primarily affected in this model and that the conditional Bub1 mice survived because of incomplete penetrance. Downregulation of mitotic checkpoint genes, which is another putative mechanism for weakening the mitotic checkpoint, is also extremely rare. Importantly, the observations of decreased levels of mitotic checkpoint genes in cancer cell lines are often confounded by the lack of adequate controls, such as comparing the levels of MAD2 in various cancer cell lines with those of HeLa cells59–62. The expression of several genes that are required for mitosis and the mitotic checkpoint (MAD2L1, BUB3, polo-like kinases, CDC20, F-box protein 5 (FBXO5, also known as EMI1), NDC80 (also known as HEC1), PTTG1, cyclin B1 (CCNB1), CENPE and CENPA, among others63) is under control of the E2f family of transcription factors and therefore can vary depending on the level of inhibition of the Rb pathway, the number of cells in G2/M phase of the cell cycle in an asynchronous population and the number of quiescent cells. These aspects all vary markedly between cell lines. How one defines a normal level of expression is also a key point here. It is reasonable to assume that the only adequate normal value is that of normal adjacent tissue to the primary tumour, provided the adjacent tissue is proliferating (which is seldom the case). In the case of cancer cell lines, normal adjacent tissue cannot be procured. Further confounding this issue, the levels of a mitotic checkpoint protein in a non-primary cell line relative to HeLa cells say little about the mitotic checkpoint status of that cell line. Indeed, there are few well-documented examples of robust functional mitotic checkpoint defects in tumour cells that have reduced expression of checkpoint proteins.

Several heritable cancer predisposition syndromes result from loss-of-function mutations in genes essential for the DNA damage checkpoint and DNA repair pathways. Li–Fraumeni syndrome (TP53), hereditary non-polyposis colorectal cancer (MLH1 and MSH2), xeroderma pigmentosum (Xp family) and ataxia–telangiectasia (ataxia–telangiectasia mutated; ATM) are a few of the well-recognized ones. The existence of a range of these syndromes underscores two important points: first, that mutation of genes that control the DNA damage checkpoint and DNA repair pathways can be viable at a cellular, and often at an organismal level, and second, the accumulating DNA damage contributes to tumorigenesis. In the case of the mitotic checkpoint, only one genetic disorder has been associated with a mitotic checkpoint gene. Mosaic variegated aneuploidy (MVA) is an autosomal recessive disorder characterized by growth retardation, microcephaly and mosaic aneuploidies, predominantly monosomies and trisomies. Patients also show a high incidence of childhood tumours (Wilms’ tumour, rhabdomyosarcoma and leukaemia). The disease has been genetically mapped to BUBR1 (REF. 64), and CIN is thought to be the driving force for developmental defects and tumour formation. The severity of the phenotype in patients with MVA and the lack of other related syndromes reinforce the notion that the mitotic checkpoint is crucial for normal organism growth and not just for the prevention of genomic abnormalities that result from external stress.

Finally, several cancer cell lines with marked CIN have a robust mitotic checkpoint when treated with microtubule-stabilizing drugs65,66. The strongest evidence against loss or downregulation of the mitotic checkpoint as a cause of aneuploidy in tumours is evident by looking at the transcriptional profiles of aneuploid tumours. In most cases, genes essential for the mitotic checkpoint are upregulated, sometimes to very high levels (Oncomine48,67 and REF. 46). Although this may be the result of unrestricted proliferation in the absence of a functional Rb pathway, the consequence is not an absent or weakened checkpoint but, most likely, an overactive one66. Moreover, in retinoblastoma tissue samples, high levels of MAD2 are not confined to mitotic cells but are also found in interphase cells68, arguing that it is not only the high mitotic index that contributes to MAD2 overexpression after Rb pathway inhibition.

CIN has long been known to be a dominant phenotype in cancer cell lines69 and overactivation of the mitotic checkpoint in cancer cell lines fits this observation readily. It has also been proposed that aneuploidy might be an early event in cancer evolution, which induces a quasi-stable karyotypic state that is balanced by selection towards tumorigenesis70. Inhibition of the Rb pathway and the consequent overexpression of key mitotic checkpoint genes may efficiently initiate tumours because of this coupling of a loss of a tumour suppressor pathway to karyotypic instability.

Overexpression of MAD2 and HEC1 in inducible mouse models has already been shown to be sufficient for generating aneuploidy and initiating tumour formation6,71. In these models, hyperactivation of the mitotic checkpoint is predicted to lead to prolonged mitosis and the failure of one or more sister chromatids to separate on schedule. This would then increase the likelihood of merotelic attachments and lagging whole chromosomes or, in the extreme case, tetraploidy following mitotic slippage (also known as adaptation; FIG. 2). These events are readily seen in cells that overexpress MAD2L1 or NDC80. Although overexpression of these mitotic genes might have nonmitotic consequences or off-target effects, a recent elegant study72 has shown that prolonged activation of the mitotic checkpoint using spindle-stabilizing agents or the mitotic kinesin family member 11 (KIF11, also known as EG5) inhibitor monastrol also leads to lagging sister chromatids, merotelic attachments and aneuploidy after mitotic slippage. Given that mitotic slippage is a well-recognized response to prolonged mitotic arrest73, mitotic checkpoint overactivation (that is, prolonged inhibition of the APC/C and, consequently, abnormal stabilization of cyclin B1 and securin) could lead to an increased rate of aneuploidy by allowing lagging sister chromatids and merotelic attachments to accumulate. Eventually, mitotic slippage would occur, generating potentially tumorigenic aneuploid progeny. Interestingly, although the molecular events leading to mitotic slippage are still unclear, it is thought to result from the degradation of cyclin B1 in an APC/C-dependent manner despite the activation of the mitotic checkpoint74.

Therefore, overactivation of the mitotic checkpoint could be a widespread phenomenon in tumours with CIN. In vitro studies using nocodazole and monastrol, together with in vivo studies overexpressing mitotic checkpoint genes, favour the hypothesis that aneuploidy in tumours is largely a consequence of the upregulation of mitotic genes and subsequent mitotic checkpoint overactivation. It is of interest that, as mentioned above, several mitotic checkpoint genes are direct E2f targets, indicating that loss of a major tumour suppressor pathway (that is, Rb inhibition of E2f) leads not only to uncontrolled proliferation but is also directly associated with the generation of mitotic CIN75–79.

Centrosome amplification has also been tightly associated with aneuploidy as a result of aberrant mitoses, and recent studies have shed light on the mechanistic connection between the two. By looking at how cells that have more than two centrosomes and multipolar spindles survive, several cell lines have been found to preferentially cluster multiple centrosomes to two poles, thereby generating a functional bipolar spindle80,81. Inhibition of centrosome clustering by short-hairpin RNA (shRNA) targeting of KIFC1, which encodes a non-essential kinesin motor protein, led to lethality of multipolar cells81. Moreover, clustered bipolar spindles (in tetraploid cells that had more than two centrosomes) were shown to have an increased frequency of merotely, leading to lagging chromosomes and segregation errors82,83. Tetraploid cells that had two centrosomes were not observed to have an increased number of lagging chromatids compared with diploid cells, arguing that it is the initial microtubule attachment and subsequent clustering of multiple centrosomes that is conducive to the generation of aneuploidy. An interesting possibility then is that overexpression of AURKA, among other genes that regulate mitotic entry and centrosome homeostasis, results in mitotic abnormalities through centrosome amplifications. One can therefore propose that clustering of centrosomes in cells that have multipolar spindles leads to merotelic attachments and that these are conducive to aneuploidy; similar to what is observed during mitotic checkpoint overactivation.

Consequences of CIN

The notion that CIN serves as a tumorigenic driving force has been expanded by a series of observations from mouse models to include the idea that CIN might also be tumour suppressive in certain contexts. In this section we discuss the evidence from mouse models regarding the consequences of CIN at the cellular and organismal levels. These studies underscore the importance of generating mouse models that faithfully recapitulate the biology of human disease to draw physiologically meaningful conclusions.

As noted above, both mitotic checkpoint weakness and mitotic checkpoint overactivation can lead to CIN through different mechanisms. Haploinsufficiency for Cenpe55, Mad2l1 [REF. 49], Mad1l1 (REF. 84), Fzr1 (which encodes CDH1)5, Plk4 (REF. 85) and a hypomorphic allele of Bub1 (REF. 54) all lead to moderate levels of aneuploidy and an increase in the incidence of spontaneous late-onset tumours of lymphoid origin and tumours in some epithelial tissues (especially lung and liver) in mice. This is also the case for the Cdc20AAA mutant, the product of which fails to interact with MAD2; homozygous Cdc20AAA mice are embryonic lethal yet heterozygous adults are tumour prone56. Although spontaneous tumour onset does not necessarily result from CIN, as indicated by the lack of such a phenotype in Bub1b+/− (REFS 50,51), Bub3+/− (REFS 52,53) and heterozygous RNA export 1 (_Rae1+/_−)52 mice, CIN in these cases often increases sensitivity to carcinogen-induced tumours. Why spontaneous tumour onset differs between the different animal models of CIN is unclear but it does not seem to be related to the degree of CIN86. Other mitotic checkpoint-independent functions of these genes could account for these differences between the models but this has yet to be tested86.

Underscoring the previously mentioned observations from human tumours, spontaneous tumorigenesis is also a consequence of checkpoint overactivation, as seen when MAD2 (REF. 6) or HEC1 (REF. 71) is overexpressed, or in the presence of centrosome amplifications as seen when AURKA is overexpressed87. In the case of ubiquitous overexpression of MAD2 or HEC1, spontaneous tumour onset occurs earlier and in a wider range of tissues than that seen for partial loss-of-function mutations. AURKA was only overexpressed in transplanted mammary epithelial cells and so it is unclear whether ubiquitous overexpression of AURKA would show similar phenotypes to MAD2 and HEC1 overexpression in mice. As discussed above, both mitotic checkpoint overactivation and centrosome amplifications lead to lagging chromosomes and merotelic attachments, which, as discussed above, facilitate aneuploidy.

W-CIN and other collateral forms of DNA damage acquired during mitosis, such as chromosome breaks, deletions and amplifications (S-CIN), might together lead to more robust tumour penetrance. Interestingly, gene expression signatures derived from human tumours with CIN have shown that genes involved in DNA damage repair pathways are overexpressed in aneuploid tumours88. Overexpression of these genes seems to be necessary for resistance to chemotherapeutic agents that target microtubules, both for chromosomally unstable cell lines and in a subset of ovarian and breast tumours. These results suggest that the DNA damage repair pathway may be activated during the generation of CIN and is required for subsequent viability, although this hypothesis remains to be tested. MAD2 overexpression, which leads to transient mitotic arrest, has been shown to lead not only to W-CIN but also to double-strand breaks, interstitial deletions and amplifications6. The prevalence of mitotic checkpoint pathway hyperactivation through the overexpression of MAD2 or other components might therefore explain the common appearance of the DNA damage response in a wide spectrum of aneuploid tumours. Prolonged mitotic arrest through the chemical inhibition of microtubule function also leads to a high incidence of lagging chromatids, merotely and chromosome bridges, all of which could lead to DNA breaks as the cleavage furrow progresses during cytokinesis. Whether DNA damage also occurs in other models of aneuploidy has not been determined, but it might underlie some of the differences in tumour phenotypes observed between different models of CIN.

It is important to note that none of the genetic mechanisms used to generate aneuploidy in animal models results in as rapid an onset in tumorigenesis as seen with activating mutations of classic oncogenes, such as Ras family members89 and MYC90, or the loss of classic tumour suppressor genes, such as TP53 (REF. 91) and RB1 (REF. 92). This could be owing to the fact that the induced genomic instability is sufficient to induce transformation but is held in check by an uncharacterized surveillance mechanism that efficiently destroys transformed cells and persists for many months. Alternatively, low-level genomic instability may require multiple events to first establish the transformed state.

Three studies suggest that low-level aneuploidy such as that generated in the above-mentioned models has detrimental effects on the viability of primary cells. Thompson and Compton72 studied the effects of CIN generated by transient mitotic checkpoint overactivation using microtubule-stabilizing agents or monastrol on single-cell colonies of two sets of primary cell lines. Single-cell colonies were then analysed by chromosome-specific fluorescence in situ hybridization (FISH) to measure aneuploidy. Although mitotic checkpoint overactivation clearly increased the fraction of aneuploid cells in the first few passages, cells in later passages were remarkably euploid. It is still unclear whether the increase in the proportion of euploid cells is a result of a decrease in proliferation rate or increased cell death of aneuploid cells. Nevertheless, it is reasonable to conclude from these studies that aneuploidy is detrimental to the fitness of primary cells.

Williams et al.93 used a different strategy to generate isogenic lines of murine fibroblasts that carried trisomies for chromosomes 1, 13, 16 and 19 in the background of Robertsonian translocations. The decreased growth rates, immortalization rates and metabolic activity in most of the trisomic lines led the authors to conclude that low-level aneuploidy in primary cells has detrimental effects not only on organismal fitness but also on cellular fitness and viability. Although the Robertsonian translocations by themselves had no effect on immortalization times, it is possible that the combination of trisomies and Robertsonian translocations were both required for the observed properties of the cells.

Finally, although inducible MAD2 overexpression in mice leads to the appearance of tumours in a range of different organs, MAD2 overexpression in fibroblasts has a marked negative effect on cellular viability6. The overall conclusion from these studies is that in primary cells CIN is detrimental to viability and is therefore selected against. Whether a specific ‘aneuploidy sensor’ is responsible for this impaired fitness or whether it results from an alteration in global transcription remains to be determined. Evidently, this aneuploidy sensor is not 100% efficient, as some trisomies or monosomies are carried to term and, in the cases of Down’s, Turner’s and Klinefelter syndromes, tolerated with few global abnormalities. Nor is this sensor a ubiquitous property of primary cells, as mouse embryonic fibroblasts that lack any Rb family members (_Rb1_−/−, _Rbl1_−/− (which encodes p107) and _Rbl2_−/− (which encodes p130)) rapidly tend towards tetraploidy in the first 20 passages94, although the Rb pathway may be necessary for this sensor. Nevertheless, these findings may explain why mouse models of aneuploidy take so long to develop tumours. Most abnormal mitoses will not generate aneuploid cells that have a substantial growth advantage, and it is only after a particularly long period of time that transformed progeny arise.

A study in 2007 by the Cleveland lab55 suggested that aneuploidy could both promote and suppress tumorigenesis depending on the tissue and genetic contexts. Cenpe+/− mice developed CIN and spontaneous tumours in a similar pattern to other mitotic checkpoint genes. The evidence for the tumour suppressive role of aneuploidy was a 50% reduction in the incidence of liver tumours and a reduction in DMBA-induced tumours, neither of which were statistically significant and could therefore be due to chance alone. In addition, there was a statistically significant but only slight delay in tumour-free survival in _Cdkn2aARF-_null mice, which is a common tumour-prone background used to explore tumour suppressor effects. Nonetheless, other reports have since shown similar results. The incidence of small intestinal tumours was reduced twofold by Bub1b haploinsufficiency in the adenomatosis polyposis coli (Apc)Min/+ mouse model95. Remarkably, the incidence of colon tumours in this model was increased tenfold. The Malumbres group5 showed that, although Fzr1+/− animals have increased rates of aneuploidy and an increased incidence of spontaneous tumours compared with wild-type controls, treatment with the carcinogen DMBA results in fewer lung tumours.

If CIN has a tumour suppressive role as a result of excessive genomic damage and subsequent apoptosis or other forms of cell death, one would expect to see such events in the corresponding tissues. Except for the presence of apoptotic cells in the areas surrounding the small intestinal tumours of Bub1b+/−;ApcMin/+ mice, no such evidence exists. Perhaps more importantly, it is unclear whether it is CIN itself that contributes to the decreased tumour incidence. The ploidy status of tumours or earlier preneoplastic lesions needs to be examined carefully to draw such a conclusion. This type of analysis is confounded by the difficulty of growing tumour cells in vitro for metaphase chromosome counts, and the extrapolation of karyotypes from fibroblasts or lymphocytes from animals that develop intestinal (in the case of Bub1b+/−;ApcMin/+ mice) or lung and skin tumours (using DMBA) is not sufficient evidence for aneuploidy in tumour lesions. One way to obtain these data would be to use FISH or array-comparative genomic hybridization (CGH) to determine the extent of aneuploidy in the normal tissues and in the early lesions that arise in these models. However, CGH results are often confounded by the fact that only clonally expanded genomic lesions can be detected from a population of unstable cells and so in these cases only methods that offer single-cell resolution (such as FISH) would identify random CIN.

Finally, the possibility of non-cell-autonomous effects of adjacent tissues or infiltrating bone marrow-derived cells on tumour growth needs to be addressed. It is possible that tumour suppression is a result of non-cell-autonomous effects through which CIN in cells that compose the tumour microenvironment modulates the host response to the primary tumour. Conditional inactivation studies of selected cell types in mice and careful examination of ploidy changes early in the tumorigenic process need to be performed. Such non-cell-autonomous effects could also be responsible for the generation of tumours that result from organism-wide mitotic checkpoint partial loss-of-function or overactivation but, in general, changes in the tumour microenvironment that promote tumorigenesis on their own are much less common than those that lead to tumour suppression.

Ultimately, it is possible and even likely that excessive CIN inhibits cell viability and, as a result, tumour formation. Cell lethality is a consequence of complete loss of the mitotic checkpoint. Nevertheless, much like the case with ionizing radiation, it seems clear that moderate levels of genomic instability can be the evolutionary fuel that generates pro-tumorigenic changes. Most human tumours show clear evidence of CIN and, in these lesions, the tumour suppressive role that this level of genomic instability initially conferred is eventually overcome. The idea that moderate levels of CIN may have tumour suppressive effects remains an important observation in a few mouse models but a more careful analysis will be required to establish whether this is a general principle that is likely to apply to human disease.

Drugging the mitotic checkpoint pathway

Many chemotherapeutic agents result in activation of the mitotic checkpoint. Microtubule-stabilizing drugs (such as taxanes) and depolymerizing drugs (such as vinka alkaloids) are regularly used as mainstay therapy in several solid tumours, often having marked efficacy96. Nevertheless, the substantial side effects of these drugs, which result from myelosuppression and neurotoxicity, have spawned a search for newer, more specific drugs that might target cells that have an abnormal mitotic checkpoint. TABLE 2 summarizes some of the classic chemotherapeutic agents that target mitosis and some of the more recently developed anti-mitotic agents in clinical trials. Remarkably, a recent study65 has shown that the responses of cells to both classical anti-mitotic drugs and newer agents (such as EG5 inhibitors) show substantial variation, not only between different cell lines but also between cells of the same cell line. Irrespective of whether the cancer cell lines studied showed CIN or not, several responses were elicited following exposure to the drug, ranging from death during mitosis to mitotic slippage, death in the following interphase to a second round of mitosis. The authors propose a model in which DNA damage that is incurred during a prolonged mitotic arrest leads to caspase 9-mediated cell death, but that the timing of cell death depends on whether cells remain in mitosis or slip through it following degradation of cyclin B1. These two thresholds, activation of caspase 9 and degradation of cyclin B1, are thought to be responsible for the observed intra- and inter-line variations in drug response65. Given the pro-tumorigenic effects of mitotic checkpoint overactivation, it is possible that the use of microtubule drugs that overactivate the mitotic checkpoint might occasionally result in tumour progression after an initial response. In line with these findings, a recent study has shown that preventing mitotic slippage by downregulating CDC20 may increase the sensitivity of tumour cells to microtubule-targeting agents97, providing an alternative therapeutic window.

Table 2.

Drugs that target mitosis*

The fact that cells with CIN have evolved to survive repeated rounds of mitotic arrests suggests that it might be preferable to inhibit the mitotic checkpoint or the centrosome abnormalities previously described. The MPS1 and AURKA kinases show some promise in this regard, and the cell lethality of mitotic checkpoint inhibition supports this approach although a means of specifically targeting tumour cells is not yet apparent. Targeted drug delivery or perhaps the hypersensitivity of tumour cells addicted to an overactive checkpoint might provide the therapeutic window required for drug efficacy.

In addition, centrosome clustering seems to be a survival mechanism used by cells that might otherwise carry out an abnormal and lethal multipolar mitosis81. Centrosome clustering therefore becomes an attractive target that is remarkably specific to cancer cells. Indeed, the normally non-essential kinesin motor protein KIFC1 is required for the viability of extra centrosome-containing cells81. It remains unclear, however, how efficient this targeting approach will be given the high rate of escape of cancer cells already observed in cell line analyses.

Future perspectives

The role of CIN in cancer remains filled with questions and some contradictions between the observations that arise from different laboratories and different model organisms. Although it now seems likely that CIN provides the evolutionary fuel to initiate and propagate the transformed state in several solid tumours, the oncogenic pathways that are activated or the tumour suppressor pathways compromised have yet to be elucidated. In addition, although the role of CIN in inhibiting tumour formation in humans is a possibility, the nature of this tumour suppressive role remains poorly defined. We stress that the observations made in model organisms must be viewed in relation to human cancer. The coming years will most likely see new answers to questions, such as when does mitotic CIN arise in human tumours and in which tumour types does it have an important role in growth, progression and/or metastasis? Can cells that have mitotic CIN be targeted efficiently from a therapeutic standpoint? On the 200th anniversary of the birth of Charles Darwin, it is striking that an analysis of evolution and natural selection — in this case in the form of CIN and cancer progression — is at the forefront of our battle against this devastating disease.

At a glance.

• Chromosomal instability (CIN), the inability to correctly segregate sister chromatids during mitosis, provides the evolutionary fuel to initiate and propagate the transformed state of multiple forms of cancer.
• The mitotic checkpoint is seldom lost or weakened in human tumours.
• Mitotic checkpoint overactivation is a more frequent observation in human tumours and is sufficient to generate CIN in vivo and in vitro. Mitotic checkpoint overactivation results in a prolonged mitosis, abnormal stabilization of cyclin B1 and securin, and an increased incidence of merotelic attachments and lagging chromosomes.
• Many of the key regulators of the mitotic checkpoint are downstream targets of the Rb tumour suppressor pathway and are therefore upregulated in most human tumours.
• The consequences of CIN are manifold and context-dependent. Although CIN can initiate tumour formation in many mouse models, under some conditions it decreases cellular fitness, providing a potential tumour suppressor effect. This effect is nevertheless often overcome, giving rise to the karyotypic complexity observed in advanced tumours.
• Mitotic checkpoint overactivation could prove effective as a novel therapeutic target as mitotic checkpoint loss is incompatible with cellular viability.

Acknowledgments

We apologize to those authors whose work is not cited because of space limitations. We would like to thank A. Unni and W. Forrester for helpful discussions of the manuscript. J.M.S. is supported by a Breast Cancer Research Program Predoctoral Traineeship Award from the Department of Defense (Congressionally Directed Medical Research Program). R.S. was supported by the Charles H. Revson Foundation. R.B. is supported by the National Institutes of Health.

Glossary

Chromosome instability

The inability to maintain a correct chromosome complement after cell division.

Aneuploidy

An abnormal chromosome number.

Mitotic checkpoint

A cell cycle checkpoint that arrests cell division at metaphase until all sister kinetochores are attached to microtubules from opposite spindle poles.

Breakage–fusion–bridge cycles

A process of amplification in which two centromeres of a dicentric chromosome are pulled to opposite poles during mitosis. If the chromosome breaks then the double-stranded breaks persist in the following S-phase and can contribute to translocations or form new dicentric chromosomes that continue the process of instability.

Whole chromosome instability

This describes CIN in terms of abnormal numbers of chromosomes.

Segmental chromosome instability

This describes CIN in terms of structural abnormalities, such as translocations, inversions, interstitial deletions and amplifications.

Kinetochore

The protein complex that assembles around centromeric chromosome regions and is the source of the signal that activates the mitotic checkpoint and the site of spindle fibre attachment.

Spindle pole

The site of origin of microtubule fibres in mitosis. In most cells this site is delineated by the presence of centrosomes that act as microtubule organizing centers.

Cohesin

A protein complex composed of structural maintenance of chromosomes 1A (SMC1A), SMC3, sister chromatid cohesion 1 (SSC1, also known as RAD21) and SSC3 (also known as stromal antigen 1), the function of which is to topologically link sister chromatids before metaphase.

Anaphase promoting complex/cyclosome

A large E3 ubiquitin ligase complex that degrades cyclin B1 and securin once the mitotic checkpoint is satisfied.

Spindle poison

A compound that affects microtubule function and therefore mitotic spindle formation by stabilizing (such as taxanes) or depolymerizing (such as vinka alkaloids or nocodazole) microtubules.

Transformation

A mechanistically defined process in which a primary cell acquires the ability to grow indefinitely in vitro (immortalization), form colonies in soft agar (anchorage-independent growth) and form tumour xenografts when implanted intradermally in nude mice.

Microcephaly

An abnormally small head circumference, which usually results from abnormal brain development.

Mosaic aneuploidy

A tissue in which groups of cells contain chromosome complements that differ from those of neighbouring cells.

Merotelic attachment

When a single kinetochore is attached to microtubules from two spindle poles rather than to one pole.

Mitotic slippage

The process by which a cell arrested in mitosis proceeds through anaphase despite an active mitotic checkpoint.

Monastrol

A small molecule inhibitor of the plus-end directed KIF11 kinesin motor, the function of which is required for chromosome segregation in mitosis.

Nocodazole

A chemical inhibitor of microtubule polymerization often used to activate the mitotic checkpoint and therefore arrest cells in the G2/M phase of the cell cycle.

Lagging chromosome

In anaphase, pairs of sister chromatids that remain at the metaphase plate, often as a consequence of merotelic attachment, can be the source of aneuploidy in the resulting daughter cells. This is distinct from a single chromatid that fails to segregate upon disjunction from its sister chromatid.

Chromosome bridge

In anaphase, a chromosome that bridges the two separating daughter nuclei as a result of abnormal attachments.

Robertsonian translocation

A non-reciprocal chromosomal translocation in which two distinct acrocentric chromosomes become fused and share a single centromere.

DMBA-induced tumour

An induced tumour model in mice in which the carcinogen DMBA (7,12-dimethylbenz(a) anthracene) is applied to the skin of 5–7 day old pups. This results in the appearance of skin and lung tumours once animals reach adulthood.

Array-comparative genomic hybridization

A genomic DNA hybridization technique that allows high-resolution analysis of copy number changes between two populations (such as normal versus tumour DNA).

Non-cell-autonomous effect

A phenotypic effect seen in a field of cells that are mediated by cells that are not part of that field, such as the clearance of tumour cells by bone marrow cells or cells of the tumour microenvironment.

Footnotes

Competing interests statement

The authors declare no competing financial interests.

DATABASES

Entrez Gene: http://www.ncbi.nlm.nih.gov/gene

Apc | ATM | Cdkn2aARF | CENPA | Cenpe | FBXO5 | Fzr1 | KIFC1 | MLH1 | MSH2 | NDC80 | Plk4 | Rae1 | RB1 | Rbl1 | Rbl2

OMIM: http://www.ncbi.nlm.nih.gov/omim

ataxia–telangiectasia | Li–Fraumeni | mosaic variegated aneuploidy

UniProtKB: http://www.uniprot.org

AURKA | BUB1 | BUB3 | BUBR1 | CDC20 | CDK1 | cyclin A | cyclin B1 | KIF11 | MAD1 | MAD2 | MPS1 | NEK2 | p53 | RANBP2 | SCC1 | securin | separase | UBCH10

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