Abstract

In order for genomic stability, mitosis is a delicate event that must be executed accurately.In recent research, we have provided insight into how mitotic errors reshape cancer genomes by causing structural and numerical alterations in chromosomes, which lead to tumor initiation and progression.In this article we discuss the causes of mitotic errors in human tumors and their impact on fitness and transformation.New research reveals that chromosome missegregation can produce a pro-inflammatory environment that impacts tumor response to immunotherapy.


Keywords



To err is human

In our bodies, millions of cells divide each day to support growth and replace damaged or lost cells. .Aneuploidy occurs when there is too many or too few chromosomes in a daughter cell during mitosis.Trisomy 21, which is very rare in humans, is the exception to the rule of aneuploidies occurring as a result of mistakes in meiosis or during early embryonic development.In contrast, mitotic errors that result in aneuploidy later in life have been linked to tumorigenesis (Naylor and van Deursen 2016).

Aneuploidy is a very common feature of cancer, arising in almost 70% of solid human tumors (Duijf et al. 2013). In addition to alterations in chromosome number, tumor cells show frequent structural alterations in chromosomes that include deletions, amplifications, and translocations. Errors in mitosis are the major source of numerical changes in chromosome number observed in cancer and also have been recognized recently to be a contributing factor in the generation of chromosomal rearrangements (Bakhoum et al. 2014; Leibowitz et al. 2015). In this review, we discuss possible sources of mitotic errors and the effect of these mistakes on cell physiology and tumorigenesis. We then describe recent findings suggesting that errors in cell division are recognized by the immune system and that tumor cells with complex karyotypes may evolve mechanisms to counteract this recognition. We conclude with a discussion of how mistakes in cell division or their associated consequences can be targeted therapeutically to benefit patients with cancer.



Sources of mitotic errors

In the case of cancer genomes, this is occurring because of the genetic instability phenotype, each of which exhibits a unique mutational signature.Chromosome instability (CIN) refers to the process of acquiring genomic alterations that involve both gain and loss of chromosomes (Lengauer et al., 1997).Cellular heterogeneity is a characteristic of most aneuploid cancer cell lines and allows for adaptation to changing environments (van Jaarsveld and Kops, 2016).There is a need to recognize that CIN and aneuploidy are two distinct traits, which influence tumor evolution and behavior differently. .Thus, while CIN invariably results in aneuploidy, cells can still be aneuploid without exhibiting CIN.In the following sections, we examine the causes of CIN and how cell division errors lead to the evolution of malignant karyotypes in human cancer.


Spindle assembly checkpoint (SAC) defects

.In mitosis, chromosomes are attached to microtubules (MTs) at the mitotic spindle apparatus.Chromatin is assembled onto centromeric chromatin by a specialized protein structure called a kinetochores.When the replicative chromosomes bind their sister kinetochore MTs, biorientation occurs. . .In Anaphase, when that occurs, the sister chromatids are separated due to cleavage and inactivation of the cohesin complex held together by Securin (Fig. 1B).When Cyclin B is degraded, it inactivates cyclin-dependent kinase 1 (Cdk1), allowing the cell to exit mitosis and complete cell division.


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Chromosome segregation and sources of mitotic errors. (A) Unattached kinetochores activate an inhibitory SAC signal, which in turn blocks progression to anaphase. The target of the SAC is the APC/C, an E3 ubiquitin ligase that targets several proteins for degradation, including Cyclin B1 and Securin. When all kinetochores are correctly attached to MTs emerging from opposite poles of the cell (biorientation), the SAC is silenced, and APC/CCDC20 ubiquitinates and targets for degradation Cyclin B (to inactivate CDK1 and allow for mitotic exit) and Securin (to liberate the protease Separase and initiate the onset of anaphase). (B) Replicated sister chromatids are held together by the cohesin complex of proteins. Following silencing of the SAC, Securin is degraded, and the protease Separase is activated. Separase cleaves the cohesin complex to allow for sister chromatid separation and anaphase onset. (C) Extra centrosomes can generate a transient multipolar spindle, which, following centrosome clustering, leads to an increased rate of merotelic attachments, where one sister kinetochore is attached to MTs emerging from opposite poles. Merotelically attached chromosomes can lag in the middle of the spindle during anaphase and may subsequently be missegregated or incorporated into micronuclei. (D) After centrosome duplication, the two centrosomes are attached by a protein linker. This linker is disassembled prior to mitotic entry to allow the centrosomes to migrate apart and form opposite poles of the spindle. Delays in centrosome separation can lead to misattached chromosomes and/or abnormal spindle geometry that results in increased rates of chromosome missegregation. (E) Cleavage furrow regression leads to cytokinesis failure and the formation of a binucleate tetraploid cell with twice the normal centrosome content.


In mammals, inactivation of the SAC leads to dramatic chromosome segregation errors; thus, the SAC is essential for organismal development and the viability of most mammalian cells. However, while most cells require the SAC for continued growth, examples have emerged where the requirement for the SAC can be bypassed. For example, extending the time for chromosome alignment by lowering APC/C activity can render the SAC nonessential in human colorectal cancer cells (Wild et al. 2016; Sansregret et al. 2017), and some cells lacking the SAC component MAD2 can proliferate in vitro and in vivo if p53 is inactivated to allow tolerance to high levels of genome instability (Burds et al. 2005; Foijer et al. 2017).

The SAC is not an all-or-nothing response, but rather the strength of the signal depends on the number of unattached kinetochores (Collin et al. 2013). Thus, mutations that weaken the SAC can result in precocious anaphase onset before complete kinetochore attachment, which dramatically increases the probability of chromosome missegregation. Mouse models have shown that attenuating the SAC promotes aneuploidy and genome instability in vivo (Simon et al. 2015). Moreover, mutations in the SAC proteins TRIP13 and BUBR1 cause mosaic variegated aneuploidy (MVA), a rare disorder characterized by high levels of aneuploidy and an increased incidence of tumorigenesis (Hanks et al. 2004; Suijkerbuijk et al. 2010; Yost et al. 2017). Nevertheless, mutations in SAC genes are rare in human tumors, and cells with CIN do not generally enter anaphase precociously, indicating that SAC dysfunction is not a major contributor to the mitotic errors and karyotypic heterogeneity observed in human cancer cells (Holland and Cleveland 2012b).


Cohesion defects

At anaphase, chromosome separation occurs as a result of sister cohesion loss (Figure 1B).. .Although the implications of these mutations on chromosome segregation fidelity have not been examined.A cohesin defect can lead to dysregulated gene expression that drives cancer development since cohesin helps organize higher-order chromatin during interphase. .The STAG2-related mutations in the cohesin complex appear to exert their tumorigenic effects outside of mitosis.


Merotelic attachments

. .As a result, erroneous K–MT attachments must be converted to bidirectional attachments to allow faithful chromosome segregation. .

Most merotelically attached chromosomes segregate correctly during anaphase (Cimini et al. 2004). However, a proportion of chromosomes with these attachments are delayed in their segregation and end up lagging in the middle of the spindle (Fig. 1C). Lagging anaphase chromosomes are frequently observed in chromosomally unstable cancer cells. These tardy chromosomes can be missegregated to produce aneuploid daughter cells (Cimini et al. 2001; 2003). More frequently, however, lagging chromosomes are segregated to the correct daughter cell but fail to reach the main chromosome mass prior to nuclear envelope reassembly and are partitioned into a micronucleus (Thompson and Compton 2011). As described below, DNA trapped within micronuclei undergoes extensive DNA damage that can lead to chromosome rearrangements (Zhang et al. 2015). Merotelic attachments are thus likely to be a major source of genetic instability in human tumors; three main sources of these attachment errors—hyperstabilized K–MT interactions, centrosome amplification, and altered timing of centrosome separation—are discussed below.


K–MT stability

.In turn, a reduction in turnover of K–MT interactions could lead to the persistence of erroneous attachments and lead to an increase in chromosome segregation errors.Chromosomally unstable tumors demonstrate hyperstable interactions between MT and K (Bakhoum et al., 2009a).Moreover, reducing KMT attachment stability restored faithful chromosome segregation (Bakhoum et al. 2009b).This indicates that enhanced K-MT attachment stability may be a major driver of chromosome segregation errors.


Centrosome amplification

A further source of merotelic attachments arises from the acquisition of extra copies of the centrosome, known as centrosome amplification (Fig. 1C). Supernumerary centrosomes are a common feature of human cancers and can arise through several different pathways, including a cell division failure, cell fusion, and centrosome overduplication (Chan 2011; Nigg and Holland 2018). The presence of extra centrosomes leads to the formation of a multipolar mitotic spindle, which, if not corrected prior to anaphase, results in the segregation of chromosomes into more than two daughter cells. Live-cell imaging has revealed that the progeny of multipolar divisions are frequently inviable, since daughter cells are unlikely to inherit a full complement of chromosomes (Ganem et al. 2009). The best-characterized mechanism for dealing with this burden is the clustering of extra centrosomes to form a pseudobipolar spindle (Fig. 1C; Quintyne et al. 2005; Basto et al. 2008; Kwon et al. 2008; Leber et al. 2010). Efficient centrosome clustering is required for the survival of cancer cells with extra centrosomes and requires multiple factors, including the minus end-directed motor protein HSET/KIFC1 (Kwon et al. 2008). A recent study revealed that centrosome clustering in epithelial cells was inhibited by E-Cadherin, which increases cortical contractility and suppresses centrosome movement (Rhys et al. 2018). Loss of E-Cadherin is frequently observed in breast cancer cells with high levels of centrosome amplification, suggesting that cancer cells can select for genetic changes that enable efficient centrosome clustering. While the coalescence of centrosomes in a multipolar spindle provides a pathway to avoid lethal divisions, it also promotes the formation of merotelic K–MT attachments that lead to lagging anaphase chromosomes (Ganem et al. 2009; Silkworth et al. 2009). This provides an explanation for the association of centrosome amplification with CIN and aneuploidy.

Besides supernumerary centrosomes, additional mechanisms can contribute to multipolarity and/or aberrant spindle geometry in cancer cells. For example, multipolar spindles can form independently of centrosome amplification following a loss of spindle pole integrity (Maiato and Logarinho 2014). In addition, overexpression of Aurora A kinase or loss of its negative regulator, CHK2 kinase, increases MT assembly rates. This leads to transient alterations in spindle geometry that promote the generation of erroneous K–MT attachments and lagging anaphase chromosomes (Ertych et al. 2014). Since overexpression of Aurora A and loss of CHK2 occur frequently in human cancers, this may represent an important pathway influencing CIN in tumors.


Timing of centrosome separation

The improper timing of centrosome separation prior to cell division is emerging as an additional source of genetic instability (Nam et al. 2015). After centrosome duplication, the two centrosomes are connected by a protein linker, which is dissolved prior to entry into mitosis (Fig. 1D). Both delaying and accelerating centrosome separation elevate the frequency of chromosome misattachments to the mitotic spindle, leading to chromosome segregation errors (Silkworth et al. 2012; Zhang et al. 2012; Nam and van Deursen 2014; Kanakkanthara et al. 2016; van Ree et al. 2016). The deubiquitinase USP44 localizes to the centrosome, and loss of this protein results in incomplete centrosome separation and elevated frequencies of lagging chromosomes. Importantly, USP44 knockout mice are prone to aneuploidization and spontaneous tumor formation (Zhang et al. 2012). Defective centrosome separation can also occur as a result of misregulation of the EG5/KIF11 motor protein that drives centrosome separation. Overexpression of EG5 leads to chromosome missegregation and increased tumor incidence (Castillo et al. 2007). Moreover, in addition to negatively regulating PI(3)K signaling through its phosphatase activity, the tumor suppressor protein PTEN also functions to promote the centrosomal recruitment of EG5 and control timely centrosome separation (van Ree et al. 2016). Mice carrying a PTEN mutant that is defective in promoting EG5 loading onto the centrosomes but active in antagonizing PI(3)K signaling show increased aneuploidy and tumor susceptibility, suggesting that PTEN"s centrosomal role contributes to its tumor-suppressive function (van Ree et al. 2016). Defects in centrosome dynamics may therefore constitute a source of erroneous kinetochore attachments, which in turn drive CIN in human tumors.


Tetraploidy

A final source of mitotic errors is the proliferation of tetraploid cells, which contain twice as many chromosomes as normal cells.There are three ways that tetraploidy develops.Those that fail to separate their daughter cells following cell division (Fig. 1E) exhibit cytokinesis failure.Tetraploidization can also occur as a result of spontaneous fusion between cells, or as a result of viral infection (Duelli et al. 2005, 2007). .The failure of cytokinesis or the inability to complete the mitotic cycle is likely to be the most significant factor involved in the production of tetraploid cells in premalignant lesions.The reason for this may be due to chromatin retention in middle of the spindle, which in turn causes furrow regression (Steigemann et al. 2009).

. .Additionally, the extra chromosomes in tetraploid cells act as a buffer against deleterious mutations in essential and haploinsufficient genes, allowing cells to survive otherwise lethal genomic changes.A computational analysis of *5000 cancer genome sequences suggests that ∼37% of human cancers undergo a genome doubling event at some point during their evolution (Zack et al. 2013). .This suggests that genome doubling is an important step in the development of many genomically unstable tumors.


Mitotic errors lead to DNA damage

Mitotic errors have long been recognized to be a major source of whole-chromosomal aneuploidy, but recent evidence has also linked chromosome segregation errors to the generation of DNA damage that promotes structural alterations in chromosomes. Structural rearrangements alter the linear organization of chromosomes and are an established driver of tumorigenesis. Emerging evidence has suggested that lagging anaphase chromosomes, in addition to having a high risk of missegregation, are uniquely susceptible to the acquisition of DNA damage.

Chromosomes that lag in the middle of the spindle can be damaged if they fail to clear the spindle midzone prior to completion of cytokinesis (Fig. 2A). These chromosomes become trapped in the cleavage furrow, generating DNA double-strand breaks that are erroneously repaired to produce unbalanced translocations (Janssen et al. 2011). In addition to suffering direct DNA damage during cytokinesis, lagging chromosomes are often partitioned into micronuclei, where they acquire DNA damage in the following cell cycle. This arises in part because the nuclear envelope of micronuclei is unusually fragile and prone to spontaneous rupture, exposing the micronuclear DNA to potentially damaging cytoplasmic components (Hatch et al. 2013). Collapse of the micronuclear envelope during S phase leads to stalled replication and associated DNA damage (Crasta et al. 2012; Zhang et al. 2015). Moreover, micronuclei show delayed DNA replication kinetics, resulting in cells that enter into mitosis while replication of the micronuclear DNA is ongoing (Crasta et al. 2012). This leads to premature condensation and the fragmentation of the micronuclear chromosome (Crasta et al. 2012; Ly et al. 2017).


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Mitotic errors can generate DNA damage. (A) Lagging chromosomes in anaphase can acquire DNA damage directly by being trapped in the spindle midzone during cytokinesis. In addition, lagging chromosomes that are partitioned into micronuclei can acquire DNA damage in interphase of the subsequent cell cycle. Extensive damage leads to chromosome shattering, a phenomenon known as chromothripsis, which results in the production of highly localized chromosome rearrangements and/or the production of double-minute chromosomes. (B) Extensive shortening of telomeres (telomere crisis) can result in the end-to-end fusion of two telomeres and the generation of a dicentric chromosome. Dicentric chromosomes can attach to opposite sides of the cell and be pulled apart during mitosis, resulting in a chromatin bridge that connects the two daughter nuclei. The nuclear membrane surrounding the bridging DNA ruptures in interphase, and the exposed DNA can be subject to cleavage by a cytoplasmic nuclease to resolve the bridge. The DNA exposed to the cytoplasm may undergo chromothriptic-like chromosome rearrangements and/or hypermutation generated by APOBEC cytidine deaminases. (C) DNA entanglements between sister chromatids can form at underreplicated regions or as a result of persistent DNA catenation. If these linkages are not resolved by topoisomerases and helicases, they can form ultrafine DNA bridges that connect the segregating sister chromatids in anaphase. Ultrafine bridges can lead to cytokinesis failure, resulting in a binucleated cell, or be broken during anaphase, creating DNA damage and micronuclei.


Massive DNA damage occurring on chromosomes isolated within micronuclei can produce complex patterns of localized chromosome rearrangements that are highly reminiscent of those observed following a phenomenon known as “chromothripsis” (Fig. 2A; Zhang et al. 2015). Chromothripsis is characterized by the presence of extensive chromosomal rearrangements restricted to one or a few chromosomes (Stephens et al. 2011; Holland and Cleveland 2012a). These alterations have been observed in a broad array of tumor types and occur at a higher frequency in specific types of cancers, including those that arise from the blood and brain (Rode et al. 2016). Therefore, partitioning of chromosomes into micronuclei offers an attractive mechanistic explanation for how mitotic errors promote acquisition of highly localized DNA damage.

.There are often several circular chromosomes present together with oncogenes that drive tumor growth.Double minutes have been discovered in nearly half of tumors. Their random segregation during cell division leads to heterogeneous copy numbers of oncogenes, which in turn makes tumors more adaptable to changing environmental conditions (Turner et al. 2017).

.Diacentric chromosomes create chromatin bridges between the nuclei of the two daughter cells in early G1.. .As a consequence, more often than not, the daughter cells separate, and the nuclear membrane surrounding the bridged DNA ruptures, allowing a cytoplasmic nuclease to attack the exposed chromatin, leading to the resolution of the bridge (Maciejowski et al., 2015). .This pattern of hypermutation is also associated with "kataegis," possibly resulting from edits on exposed ssDNA bound to chromatin bridges by APOBEC cytidine deaminases (Fig. 2B).Mitotic errors are thought to produce focal rearrangements and clusters of hypermutation.

Finally, DNA damage can also arise from the inappropriate resolution of DNA ultrafine bridges (UFBs). UFBs are thin segments of naked DNA that connect the segregating sister chromatids at anaphase. They are formed as a result of topological links between sister chromatids that arise as a result of persistent DNA catenation or replication stress (Bizard and Hickson 2018). Replication stress has been proposed to contribute to genomic alterations and CIN as a result of attempts to segregate underreplicated regions of the genome (Burrell et al. 2013). These DNA entanglements often occur at defined chromosomal loci, such as common fragile sites or centromeric regions, and are usually resolved prior to mitosis. If left unresolved, UFBs can break during anaphase and form micronuclei or lead to a failure or abscission, resulting in the production of binucleated tetraploid cells (Fig. 2C). These events occur frequently in some cancer cells, indicating that aberrant resolution of UFBs can lead to mitotic DNA damage that contributes to numerical and structural alterations in the tumor karyotype (Chan et al. 2009; Naim and Rosselli 2009; Tiwari et al. 2018).


Mitotic errors can trigger activation of p53

.Mitotic errors can activate p53 in a complex and multifactorial manner.Following chromosome missegregation, p53 is activated, and, therefore, loss of p53 is often linked to aneuploidy in human cancers (Burds et al. 2005; Li et al. 2010; Thompson and Compton 2010).However, it remains unclear whether aneuploidy directly triggers the activation of p53. .When aneuploidy involves structural and genetic mutations, p53 is activated, which is likely to be caused by DNA damage acquired during or following mitotic errors.Alternatively, aneuploidies involving a small piece of the genome can be propagated in a p53-competent background (Santaguida et al. 2017; Soto et al. 2017).According to these findings, chromosome imbalances by themselves are not sufficient to activate p53, but missegregation or aneuploidy may.

Additional features of an erroneous mitosis could also contribute to p53 activation after division. For example, aneuploidy was proposed to increase the levels of reactive oxygen species that lead to activation of the ataxia telangiectasia-mutated (ATM) DNA damage signaling kinase and p53 (Fig. 3A; Li et al. 2010). Moreover, p53 stabilization following chromosome missegregation has been linked with the entrapment of chromatin in the cytokinetic furrow or the damage that accumulates in underreplicated DNA contained within micronuclei (Fig. 3A; Janssen et al. 2011; Crasta et al. 2012). A prolonged mitosis even in the absence of overt cell division errors can also trigger a p53-dependent cell cycle arrest in nontransformed cells (Uetake and Sluder 2010). Eliminating cells that delay in mitosis could serve as a quality control to prevent the proliferation of cells that have experienced stresses during mitosis (Lambrus and Holland 2017). Finally, recent work has proposed that differential phosphorylation of histones on lagging anaphase chromosomes is required to initiate p53 stabilization in the subsequent G1 (Hinchcliffe et al. 2016). However, how specific missegregated chromosomes could be marked by histone phosphorylation and how this feeds into p53 activation remain unclear.


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Mitotic errors can trigger activation of p53. (A) Lagging chromosomes that become damaged in the cleavage furrow or in micronuclei can elicit the canonical DNA damage repair pathway that activates p53. In addition, aneuploidy can also cause increased reactive oxidative species that lead to activation of DNA damage signaling. (B) Cytokinesis failure has been proposed to activate p53 through two distinct pathways. (1) Activation of the Hippo pathway. The Hippo pathway kinase LATS2 is activated in a tetraploid cell, leading to the phosphorylation and cytoplasmic sequestration of the transcription factor YAP. In addition, LATS2 binds and inactivates MDM2, a negative regulator of p53 stability. Inhibiting MDM2 allows for the increased accumulation of p53, which up-regulates p21 to elicit a growth arrest. (2) Activation of PIDDosome signaling. Tetraploid cells activate the PIDDosome, a multiprotein complex comprised of PIDD and RAIDD that in turn activates Caspase-2 (CASP2). Caspase 2 cleaves and inactivates MDM2, allowing p53 stabilization.


Given the potential harm that can arise as a result of the uncontrolled proliferation of tetraploid cells, it is perhaps unsurprising that mammalian cells have also evolved systems to limit the division of cells with increased ploidy. While some cells can proliferate following cytokinesis failure (Uetake and Sluder 2004; Wong and Stearns 2005), in many instances, tetraploid cells arrest in G1 due to the stabilization of p53 and up-regulation of the CDK inhibitor p21 (Andreassen et al. 2001; Kuffer et al. 2013). Two distinct pathways have been implicated in activating p53 to suppress the proliferation of tetraploid cells (Fig. 3B). In one pathway, cytokinesis failure activates the Hippo pathway kinase LATS2, which stabilizes p53 and inactivates the progrowth transcriptional regulator YAP (Ganem et al. 2014). In an alternative pathway, cytokinesis failure promotes the activation of the PIDDosome, a multiprotein complex that activates Caspase-2, leading to subsequent p53 stabilization (Fava et al. 2017). The presence of extra centrosomes in tetraploid cells has been suggested to be a key trigger for the activation of both LATS2 and the PIDDosome, but the extent to which these pathways act independently or collaborate to restrain the growth of tetraploid cells remains to be determined.

Since mitotic errors frequently trigger a cell cycle arrest in nontransformed cells, tumor cells with CIN are likely to acquire alterations that allow them to circumvent the anti-proliferative effects of p53 activation. Indeed, disruption of p53 is one of the most frequent events in tumorigenesis and allows cells to tolerate a broad range of insults, including CIN. Caspase 2 has been proposed to activate p53 following chromosome missegregation by cleaving MDM2, an E3 ubiquitin ligase that acts to target p53 for degradation by the proteasome. Consistently, loss of Caspase 2 is associated with an increased tolerance for karyotype imbalances (Dorstyn et al. 2012; Puccini et al. 2013; Dawar et al. 2017). In colorectal tumors, mutations in BCL9L confer tolerance to CIN by reducing Caspase 2 levels and preventing p53 stabilization (Lopez-Garcia et al. 2017). Finally, overexpression of cyclin D1 enables cells to circumvent a G1 arrest following genome doubling by sequestering p21 (Crockford et al. 2017). In summary, mitotic errors can directly or indirectly trigger activation of p53; thus, mechanisms that suppress or circumvent p53 activation are likely to be key contributors to the propagation of chromosomally unstable tumor cells.


Impact of mitotic errors on cell fitness

Given the detrimental effects of mitotic errors on genome stability, the question that naturally arises is how frequently these events occur in vivo. While mitotic errors are difficult to observe directly in tissues, several studies have measured the degree of aneuploidy in normal cells using fluorescence in situ hybridization (FISH), chromosome spreads, or spectral karyotyping. Surprisingly, initial estimates performed with FISH in healthy tissues suggested that 30%–50% of cells in the mammalian brain (Rehen et al. 2001; Pack et al. 2005; Yurov et al. 2007; Faggioli et al. 2012) and up to 50% of cells in the liver are aneuploid (Duncan et al. 2010, 2012). More recently, however, single-cell sequencing studies in these same tissues reported much lower levels of aneuploidy (McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2014; van den Bos et al. 2016). Since single-cell sequencing offers a more reliable technology for examining karyotypes at high resolution in an unbiased manner, these data indicate that cells with abnormal karyotypes are likely to be rare in healthy tissues (Bakker et al. 2015).

Somatic tissues exhibiting low levels of aneuploidy indicate either a low frequency of mitotic errors in vivo or that aneuploid cells are selectively eliminated.The proposition that aneuploid cells are selected against in vivo is likely to be accurate based on recent research. .Aneuploid cells are depleted from peripheral blood over time in experiments with chromosomally unstable BubR1H/H HSCs.A large number of the nonproliferating tissues from BubR1H/H mice were aneuploid, while regenerative tissues were largely euploid in nature (Pfau et al., 2016)..

Similar to the observations made in vivo, aneuploidy is generally detrimental to cell proliferation in vitro (Gordon et al. 2012; Santaguida and Amon 2015). This fitness defect arises as a result of changes in the copy number of genes located on the aneuploid chromosomes (Torres et al. 2007, 2010; Pavelka et al. 2010; Stingele et al. 2012; Dephoure et al. 2014). The loss or gain of an entire chromosome alters the production of hundreds, if not thousands, of proteins. While altering the copy number of specific genes can bring about strong phenotypic changes, most phenotypes associated with aneuploidy arise from the simultaneous alteration of many gene products that have little effect when modified individually (Torres et al. 2007; Pavelka et al. 2010; Oromendia et al. 2012; Bonney et al. 2015). Analysis of yeast or human cells with extra copies of an individual chromosome revealed that while the abundance of most proteins correlated with increased gene dosage, ∼20%–25% of the proteins encoded on the additional chromosomes were expressed at close to diploid levels (Stingele et al. 2012; Dephoure et al. 2014). Importantly, the majority of these proteins is components of macromolecular complexes. These data suggest that aneuploid cells counteract the production of partially assembled multisubunit complexes by degrading uncomplexed subunits.

The degradation of protein subunits produces an increased load on protein folding and degradation pathways of aneuploid cells, explaining why these cells exhibit traits indicative of protetoxic stress (Torres et al. 2007; Oromendia et al. 2012; Sheltzer et al. 2012; Stingele et al. 2012). Aneuploid cells are also prone to protein aggregation and up-regulate autophagy-mediated protein degradation (Santaguida et al. 2015). The stress produced from aneuploidy-induced protein imbalances results in an increased sensitivity to compounds that inhibit autophagy or interfere with protein folding or degradation (Tang et al. 2011). In addition, mutations that compromise the ubiquitin–proteasome pathway produce synthetic fitness defects in aneuploid cells (Dodgson et al. 2016a,b). These data suggest that, along with chromosome-specific effects elicited by dosage alteration of certain genes, aneuploid cells also share a set of associated stress phenotypes that is largely independent of the specific karyotypic alteration.

The association of aneuploidy with decreased cellular fitness is seemingly counterintuitive with the observation that aneuploidy is a nearly universal feature of human tumors. This “aneuploidy paradox” remains to be fully resolved, but several possible explanations have emerged. First, the effects of chromosome segregation errors may be revealed only under the appropriate evolutionary constraints. Under the stringent selective pressure in the tumor microenvironment, most karyotypic alterations are expected to reduce cell fitness and be selected against. However, in rare instances, karyotypes may emerge that provide a selective advantage in a specific environmental setting. Indeed, aneuploidy has been shown to offer a selective advantage to yeast and human cells under conditions of environmental stress (Pavelka et al. 2010; Chen et al. 2012; Rutledge et al. 2016). Moreover, some nontumor cell lines acquire specific aneuploidies in culture that increase cellular proliferation, showing that certain aneuploid karyotypes can be beneficial even in the absence of cellular transformation (Ben-David et al. 2014). An alternative hypothesis for the prevalence of aneuploidy in human tumors is that cancer cells acquire alterations that allow them to tolerate the adverse stresses associated with karyotype imbalances. For example, loss of the deubiquitinating enzyme UBP6 improves the proliferation rate of several aneuploid yeast strains (Torres et al. 2010). Together, these studies show that aneuploidy does not inevitably suppress cellular proliferation but can in fact be selected for under specific environmental conditions.


Aneuploidy can promote further genome instability

Mitosis is a dynamic and finely tuned event that is particularly sensitive to perturbations in gene expression arising from karyotype alterations. Consistently, analysis in yeast has shown that aneuploid cells are less genomically stable and show increased rates of chromosome missegregation, mitotic recombination, and defective DNA damage repair (Sheltzer et al. 2011; Zhu et al. 2012). In human cells, specific aneuploidies have also been shown to increase mitotic error frequency (Nicholson et al. 2015; Passerini et al. 2016). In addition, while human cell lines with defined trisomies show reduced cell growth in vitro and in xenograft tumor assays, spontaneous karyotype evolution occurs during prolonged growth and improves cellular fitness (Sheltzer et al. 2017). These data suggest that aneuploidy can promote further karyotype instability and facilitate the acquisition of growth-promoting alterations.

Recent work has begun to elucidate how karyotype alterations impair mitotic fidelity. Even the presence of a single extra chromosome can trigger genomic instability by reducing the abundance of key replication proteins and impairing DNA replication (Passerini et al. 2016). These replication defects lead to the acquisition of DNA damage that promotes chromosome rearrangements and an increased frequency of mitotic errors (Santaguida et al. 2017). In addition, aneuploidy can also generate karyotype-specific phenotypic changes that lead to mitotic defects. For example, trisomy of chromosome 13 results in a cytokinesis defect because of increased expression of a gene encoded on the aneuploid chromosome (Nicholson et al. 2015). However, CIN is clearly not a necessary outcome of aneuploidy, as cells from individuals with specific trisomies exhibit rates of chromosome missegregation similar to those of euploid cells in vitro, and single-cell sequencing of neurons from an individual with Down syndrome failed to reveal additional aneuploidies (Valind et al. 2013; van den Bos et al. 2016).


Mitotic errors and tumorigenesis

The notion that mitotic errors could contribute to tumorigenesis was first postulated >100 years ago by Boveri (1914). However, whether cell division errors promote tumorigenesis or arise as a byproduct of transformation has remained an area of active debate. The fact that genes that control chromosome segregation are rarely mutated in human cancers raises the possibility that inducing CIN is a passenger event in tumor development. Indeed, inactivation of several tumor suppressor genes has been shown to promote CIN and aneuploidy (Manning et al. 2010; van Ree et al. 2016). Nevertheless, the weight of evidence suggests that, at least under some circumstances, mitotic errors do contribute to tumorigenesis. First, patients with MVA exhibit high levels of aneuploidy and an increased predisposition to certain types of cancers (Garcia-Castillo et al. 2008). Second, mitotic errors and aneuploidization can be found early during tumor evolution, and the extent of chromosomal aberrations correlates with tumor grade and poor prognosis (Mugneret et al. 2003; van de Wetering et al. 2007; Walther et al. 2008; M"Kacher et al. 2010; Bakhoum et al. 2011). Finally, perhaps the most persuasive evidence to support a causative link between CIN and tumor development comes from the study of mouse models with increased rates of mitotic errors caused by reduced or elevated levels of SAC components. Many of these models with CIN exhibit an increased incidence of spontaneous tumors and/or elevation of chemically or genetically induced tumor formation (Dobles et al. 2000; Michel et al. 2001; Babu et al. 2003; Dai et al. 2004; Jeganathan et al. 2006, 2007; Iwanaga et al. 2007; Sotillo et al. 2007; Weaver et al. 2007; Baker et al. 2009; Li et al. 2009; van Ree et al. 2010; Foijer et al. 2014). For an extensive discussion of the spectrum of tumors formed in these animals, see the following reviews: Holland and Cleveland (2009), Simon et al. (2015), and Naylor and van Deursen (2016).

How mitotic defects can act to promote tumor development remains an area of intense study. As a first path to facilitate tumorigenesis, CIN drives a continually evolving karyotype that produces genetic diversity in the tumor cell population. In addition to population-level genetic variation, the dosage imbalances produced by aneuploidy have been shown to reduce the robustness of biological networks and increase cellular variability (Beach et al. 2017). Together, this genetic and nongenetic heterogeneity creates phenotypic diversity. While the vast majority of alterations to the karyotype is expected to be detrimental, a small fraction of those changes could be advantageous and selected for during tumor evolution. The chromosomal location and relative density of tumor suppressor genes and oncogenes have been proposed to play an important role in shaping the tissue-specific patterns of aneuploidy observed in different types of cancer (Davoli et al. 2013; Sack et al. 2018). This may explain why certain chromosomal alterations are observed recurrently in some tumor types, such as gain of chromosome 8, which is observed in 25% of chronic myeloid leukemia cases and 10%–15% of cases of acute myeloid leukemia (Paulsson and Johansson 2007).

A widely proposed mechanism by which mitotic errors facilitate tumor development is through the loss of a chromosome that contains the remaining wild-type copy of a tumor suppressor gene. Indeed, this has been shown to occur in chromosomally unstable mice that were heterozygous for p53 or carried a mutated APC allele (Baker et al. 2009). In addition to promoting primary tumor growth, recent work functionally linked CIN with metastasis by showing that chromosomally unstable tumor cell lines are more likely to spread and form new tumors when compared with the same cells in which CIN was suppressed (Bakhoum et al. 2018). Finally, the genetic instability produced by CIN could contribute to the evolution of resistance in response to targeted anti-cancer therapies. In mice, CIN driven by overexpression of the SAC protein MAD2 provides the evolutionary fuel to facilitate tumor recurrence following withdrawal of the KRAS oncogene (Sotillo et al. 2010). Genetically engineered mice that recapitulate the ongoing karyotype changes observed in the majority of human tumors are thus likely to represent powerful models for testing the efficacy of emerging clinical drug candidates.

The most extensive characterization of the role of mitotic errors in tumorigenesis has emerged from the development of mouse models that possess elevated or reduced levels of SAC proteins. These animals display ongoing CIN and increased aneuploidy in cells and tissues. While many of these models are tumor-prone, some exhibit high levels of aneuploidy without an increase in tumor predisposition, demonstrating that the degree of aneuploidy is not an accurate predictor of tumor susceptibility (Baker et al. 2006). One possible explanation for this observation is that some of the proteins that are manipulated have functions outside of mitosis that confound interpretations of the tumor phenotypes (Funk et al. 2016). For example, SAC proteins have been proposed to play roles in insulin signaling (Choi et al. 2016), transcriptional repression (Yoon et al. 2004), DNA replication and repair (Sugimoto et al. 2004; Dotiwala et al. 2010), and membrane trafficking (Wan et al. 2014). An alternative possibility is that the genes manipulated to induce chromosome missegregation lead to distinct types of mitotic errors that change the karyotype in different ways. For example, reduction in the levels/activity of the MT motor protein CENP-E leads to the initiation of anaphase with polar chromosomes, resulting in whole-chromosome gain and loss events (Weaver et al. 2003). On the other hand, overexpression of MAD2 produces lagging anaphase chromosomes that can be subjected to DNA double-strand breaks and serve as a source of chromosomal rearrangements (Sotillo et al. 2007). It will be interesting to determine to what extent the frequency of DNA breaks that result from mitotic errors correlates with the propensity for tumor development.

Although considerable effort has been focused on modeling mitotic errors using mice with altered levels of SAC components, SAC dysfunction does not appear to be a major driver of CIN in human tumors. Given the established role of centrosome amplification in promoting CIN and its widespread presence in aneuploid human tumors, recent attention has turned to generating mice in which extra centrosomes could be generated by overexpressing PLK4, the master regulator of centrosome biogenesis (Marthiens et al. 2013; Coelho et al. 2015; Kulukian et al. 2015; Vitre et al. 2015; Sercin et al. 2016; Levine et al. 2017). Modest overexpression of Plk4 produced chronic centrosome amplification and aneuploidy in multiple tissues and was sufficient to drive the formation of lymphomas and squamous cell carcinomas (Levine et al. 2017). Strikingly, these tumors exhibited high levels of aneuploidy, ongoing chromosome segregation errors, and defective p53 signaling. Furthermore, tumors that formed as a result of centrosome amplification exhibited complex karyotypes that mimicked those frequently found in human tumors. It will now be valuable to develop additional animal models that mimic other mitotic aberrations frequently observed in human tumor cells, such as hyperstabilized K–MT interactions.

Although mitotic errors have long been implicated in driving cancer, it is becoming clear that in some contexts, increasing chromosome segregation errors can act to suppress tumorigenesis. In several examples where tumors develop with low rates of CIN, further increasing CIN suppressed tumor incidence (Weaver et al. 2007; Baker et al. 2009; Silk et al. 2013). Indeed, combining low rates of chromosome missegregation from expression of a mutant APC allele with additional CIN from reduced levels of CENP-E resulted in increased cell death that suppressed tumor progression but not initiation (Zasadil et al. 2016). This suggests that low rates of chromosome missegregation can promote tumor development, while high levels of CIN lead to the loss of essential chromosomes and tumor suppression (Funk et al. 2016). This explains the seemingly paradoxical observation that low levels of CIN are associated with a poor outcome in estrogen receptor-negative breast cancer, while high levels of CIN correlate with improved long-term survival (Birkbak et al. 2011; Roylance et al. 2011; Jamal-Hanjani et al. 2015). Since excessive chromosome segregation errors are lethal, tumors may select for alterations that antagonize the effects of excessive CIN. Subtly increasing the duration of mitosis by reducing APC/C activity reduces chromosome segregation errors in cells with CIN (Sansregret et al. 2017). Therefore, increasing the duration of mitosis could be a strategy used by cancer cells to tune the level of CIN and counteract the long-term fitness defects caused by excessive chromosome segregation errors. Consistently, single-cell genome sequencing of human breast tumors revealed that aneuploidy occurs early during tumor evolution but remains relatively stable during tumor outgrowth (Wang et al. 2014). This suggests that once a critical point has been reached, increased genome stability can be selected for to aid tumor growth.

Taken together, the available data suggest that mitotic errors can have distinct impacts at different points during tumor development. Low rates of mitotic errors can be tumor-promoting, particularly in the context of inactivating pathways that suppress the growth of aneuploid or polyploid cells. Nevertheless, higher rates of chromosome segregation errors lead to loss of essential chromosomes and tumor suppression. Identifying genetic alterations that cooperate to facilitate the transformation of chromosomally unstable cells is an important area of future work.