Genetic Instability: Tipping the Balance

REVIEW Genetic instability: tipping the balance

A Janssen1,2 and RH Medema1

Tumor cells typically contain a genome that is highly divergent from the genome of normal, non-transformed cells. This genetic divergence is caused by a number of distinct changes that the tumor cell acquires during its transformation from a normal cell into a tumorigenic counterpart. Changes to the genome include mutations, deletions, insertions, and also gross chromosomal aberrations, such as chromosome translocations and whole chromosome gains or losses. This genetic disorder of the tumor cell has complicated the identification of crucial driver mutations that cause cancer. Moreover, the large genetic divergence between different tumors causes them to behave very differently, and makes it difficult to predict response to therapy. In addition, tumor cells are genetically unstable and frequently acquire new mutations and/or gross chromosomal aberrations as they divide. This is beneficial for the overall capacity of a tumor to adapt to changes in its environment, but newly acquired genetic alterations can also compromise the genetic dominance of the tumor cell and thus affect tumor cell viability. Here, we review the mechanisms that can cause gross chromosomal aberrations, and discuss how these affect tumor cell viability.

Oncogene (2013) 32, 4459–4470; doi:10.1038/onc.2012.576; published online 17 December 2012 Keywords: chromosome instability; cancer; mitosis; DNA damage; translocations; aneuploidy

STRUCTURAL CHROMOSOMAL INSTABILITY Breakage Syndrome, Bloom’s syndrome, ataxia telangiectasia or Translocations, one of the most prominent types of structural mutations in BRCA1 and 2, display an increased susceptibility to 18 chromosomal changes, have been found in many cancer types1 form structural chromosomal changes. Cells from these patients (Figure 1). In hematological malignancies, several translocations accumulate translocations due to mutations in DNA repair have been identified that contribute to specific gene fusions, which proteins, such as NBS1, BLM helicase, ATM kinase or the BRCA1 are thought to be drivers in the process of tumorigenesis.2 The first and BRCA2 proteins. Moreover, it was shown long ago that several identified translocation in human cancer was the Philadelphia DSB-inducing agents, such as ionizing irradiation, UV-light and 3 chemical mutagens, can also result in the formation of chromosome, which results in the formation of a fusion between 19 the BCR and Abl genes, and is causative in the development of chromosomal aberrations. chronic myeloid leukemia.4 Translocations found in cancer can be The structural instability of cancer cells is not merely caused by balanced, creating two reciprocal chromosomal fusions that are inherited genetic defects or damage induced by exogenous homogeneously present in all tumor cells.5 However, more often, agents. Cancer cells can also acquire new translocations through the breakage-fusion-bridge (BFB) cycle, a process first described in cancer cells within a tumor display mosaic structural changes, 20,21 indicating that chromosomes continue to rearrange at a high rate in maize meiosis. In the BFB cycle, chromosomes that are broken 6–11 by DSBs first fuse with other broken chromosomal parts, for the established tumor. Generally, these chromosome structure 22 instabilities are thought to promote tumorigenesis by providing example, through fusion at dysfunctional telomeres. These telomere fusion events result in dicentric (two centromeres) continuous genetic diversification within the tumor that facilitates chromosomes, often found in tumors.22,23 The presence of two adaptation to environmental changes, for example through loss of centromeres on these aberrantly shaped chromosomes can result certain tumor suppressors or gain of specific oncogenes.7,12 in improper microtubule (MT) attachments in mitosis, such that Moreover, this continued genetic diversification could help the the two centromeres on a single chromatid become attached to tumor in acquiring drug resistance, cope with increased hypoxia, or opposite spindle poles. These attachments can induce chromatin escape challenges by the immune system. In line with this, an bridging in telophase, resulting in breakage of the fused increased occurrence of structural chromosomal aberrations 20,24 1,13,14 chromosome during cytokinesis, starting another BFB cycle. correlates with increased tumor grade. Such repetitive BFB events increase the number of aberrant Structural chromosomal instabilities arise through mis- or chromosomes in the offspring.20,21 BFB has indeed been unrepaired DNA double-stranded breaks (DSBs). Homologous correlated with an increase in intratumor heterogeneity and recombination and non-homologous end joining are thought to might therefore be an important factor in structural instability.25 be the two main repair pathways contributing to the formation of structural aberrations.15–17 Especially the error-prone non- homologous end joining pathway, which can ligate any two broken DNA ends together, is held responsible for the formation NUMERICAL CHROMOSOMAL INSTABILITY of structural aberrations.15 Indeed, individuals with genetic defects Another striking hallmark of cancer cells is the presence of an that affect repair of DSBs, such as patients with Nijmegen abnormal chromosome number8,26,27 (Figure 1), termed

1Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands and 2Department of Medical Oncology and Cancer Genomics Center, University Medical Center Utrecht, Utrecht, The Netherlands. Correspondence: Dr R Medema, Netherlands Cancer Institute Division of Cell Biology, Plesmanlaan 121, Amsterdam, Holland 1066 CX, The Netherlands. E-mail: [email protected] Received 24 September 2012; revised 23 October 2012; accepted 24 October 2012; published online 17 December 2012 Genetic instability in tumors A Janssen and RH Medema 4460

Figure 1. Typical karyotype of a cancer cell. Karyotype of a human osteosarcoma cell line (U2OS) revealing a variety of numerical and structural chromosomal abnormalities.

aneuploidy, a state in which cells do not contain an exact multiple of the haploid DNA content. On average, 25 percent of the genome of a cancer cell is affected by numerical changes of either whole chromosomes or complete chromosomal arms.6 The aneuploid karyotype can be stably propagated in a population of (tumor) cells,6,28,29 but more often chromosome numbers continuously change between cancer cells and their offspring.30 This continuous change in chromosome number is termed whole chromosomal instability (CIN)30.31 and is correlated 32–38 Figure 2. Various mitotic defects can lead to CIN. Absence of mitotic with tumor grade, metastasis and poor prognosis. Moreover, checkpoint signaling allows anaphase initiation in the presence of CIN has been associated with resistance to chemotherapeutics, unattached kinetochores, whereas cohesion loss or merotelic such as the widely used MT-stabilizing agent Paclitaxel.39–41 attachments induce chromosomal segregation errors by incorrect Various hypotheses have been postulated on how CIN and kinetochore orientation or the induction of lagging chromatids aneuploidy could contribute to tumorigenesis.42–46 The general respectively. idea is that whole chromosome gains and losses during cell division can, as suggested above for structural changes, provide a mode for cancer cells to adapt to their environment and and causes embryonic lethality in mouse,51–58 likely because it continuously divide.47,48 results in continuous chromosome missegregation. Thus, subsequent cell divisions of a viable aneuploid karyotype results in improper propagation of the genetic material, resulting in high CAUSES OF NUMERICAL CIN rates of cell death in the respective daughter cells. In contrast, Since the initial discovery of CIN in a variety of colon cancer cell partial checkpoint dysfunction results in a low frequency of lines in 1997,30 many researchers have investigated the underlying chromosome segregation errors, a low rate of cell death and the cause of this striking phenotype, found to be present in many potential to generate a new viable karyotype that is relatively other tumor types as well. Several mechanisms have been stable. Partial mitotic checkpoint activity could be responsible for CIN in tumor cells by allowing mitotic exit in the presence of one proposed and tested using a variety of cancer cell lines and 51,58–60 mouse models. It seems highly unlikely that one single or more unattached kinetochores (Figure 2). However, CIN mechanism can be held responsible for the CIN observed in the cancer cell lines that were initially thought to have a severely different types of tumors. Below we outline the different cellular impaired mitotic checkpoint due to mutations in the mitotic checkpoint kinase Bub1,61 were subsequently shown to have a causes that have been postulated and the genetic defects that 62 could underlie these phenotypes. very sturdy mitotic checkpoint. Nonetheless, altered expression levels and mutations in mitotic checkpoint genes have been identified in human cancer that compromise checkpoint Causes of numerical CIN: Mitotic checkpoint defects function.43,63–76 In one specific syndrome, mosaic-variegated The mitotic checkpoint has evolved to safeguard genetic stability, aneuploidy, which is linked to cancer predisposition at a very a function it performs by monitoring the attachment status of young age, mono- and biallelic mutations in the BUB1B gene have chromosomes to the mitotic spindle. During mitosis, sister been identified.63 BUB1B encodes for the mitotic checkpoint chromatid pairs need to bi-orient on the mitotic spindle, meaning protein BubR1 and these mutations can lead to the complete that sister chromatids within a pair are attached to opposite absence of BubR1 mRNA or single amino-acid substitutions in the spindle poles. Improper attachment of as much as a single sister BubR1 protein product.77 In all cases, these mutations lead to chromatid is sufficient to sustain the mitotic checkpoint and lower BubR1 protein levels and decreased mitotic checkpoint prevent the onset of anaphase. Biorientation is achieved through activity,64,77,78 linking the cancer predisposition of mosaic- binding of the MT attachment site of each sister chromatid, called variegated aneuploidy patients to defects in the mitotic kinetochore, to MTs coming from one spindle pole. Once all checkpoint. kinetochores in a cell are properly attached to the spindle, the The occurrence of mutations in mitotic checkpoint genes in mitotic checkpoint is satisfied and anaphase onset is no longer human cancers remains extremely rare.79–82 Surprisingly, many inhibited.49 Defects in mitotic checkpoint signaling directly lead to CIN tumors actually display enhanced levels of mitotic checkpoint chromosome missegregations by allowing mitotic exit in the proteins.33,83–89 One explanation for this increase in expression of presence of unattached kinetochores (Figure 2). Indeed, the mitotic checkpoint genes lies in the dysfunction of the Rb hypothesis that has been investigated the most, postulates that pathway, resulting in overexpression of the mitotic checkpoint mitotic checkpoint defects could underlie CIN.50 Complete mitotic protein MAD2.90 Indeed, deregulation of the Rb pathway can checkpoint loss is lethal to all dividing cell types studied thus far result in CIN in several organisms.91,92 Notably, CIN induced in

Oncogene (2013) 4459 – 4470 & 2013 Macmillan Publishers Limited Genetic instability in tumors A Janssen and RH Medema 4461 Rb-negative cell lines was directly dependent on increased MAD2 causes of merotelic attachments have been hypothesized, of levels.90 In line with this, MAD2 overexpression in mice results in which the most signiﬁcant ones are outlined below. aneuploidy and spontaneous tumor formation93 (See Table 1) and CIN in Rb-negative tumors depends on increased MAD2 levels.94 Causes of merotelic attachments: aberrant spindle morphology. In summary, decreased expression levels of checkpoint proteins One way in which merotelic attachments can arise is through as well as mutations in mitotic checkpoint genes have been abnormal spindle assembly in mitosis. Initial clues on this came observed in tumor tissues, but these are relatively rare. Instead, from the observation that multipolar spindle assembly in mere overexpression of mitotic checkpoint proteins, in particular kangaroo cells often led to kinetochore-binding to two spindle MAD2, seems to be the more prevalent cause in the induction of poles,110 producing merotelic attachments and lagging CIN in various tumor types. chromosomes.111 Thus, proper spindle polarity is important for the establishment of proper chromosome attachments. In Causes of numerical CIN: cohesion loss mammalian cells, spindle polarity is largely dictated by the centrosomes. The two centrosomes that are normally present Duplicated sister chromatids are held together until anaphase by separate before mitosis, and subsequently organize the spindle the cohesin complex. Maintenance of cohesion between the two poles. Drugs that perturb centrosome separation also increase the sister centromeres is essential for chromosome biorientation and number of merotelic attachments and induce chromosome hence for proper chromosome segregation.95–97 Loss of cohesion segregation errors.107 before anaphase results in premature sister chromatid separation, Interestingly, multipolar spindles are often observed in CIN which eventually results in chromosome segregation errors and tumors,112–115 which indicates that aberrant spindle morphology aneuploidy (Figure 2). Various genes encoding proteins involved might be responsible for merotelic attachments and CIN induction in cohesion establishment or maintenance have been found to be in vivo as well. Multipolar spindles are often the result of mutated in aneuploid tumors.96,98 In addition, a recent report centrosome overduplication, a common trait of cancer cells.116 observed frequent deletion of part of the X chromosome in Persistence of multipolar spindles in anaphase results in severe genetically unstable tumor samples.99 This locus encodes for SA2, chromosome missegregations and the severely aneuploid which is a subunit of the cohesin complex. Interestingly, targeted daughter cells often die in the next G1 phase.117 However, inactivation of this locus, termed STAG2, resulted in CIN in tumor cells that harbor multiple centrosomes can progress otherwise chromosome-stable near-diploid cells. Moreover, through a transient multipolar state, after which the reconstitution of STAG2 in CIN tumor cells harboring a deletion supernumerary centrosomes cluster during mitosis and form a of the endogenous locus, reverted the CIN phenotype and seemingly normal bipolar spindle118,119 (Figure 3). The transient enhanced chromosomal stability in these lines.99 multipolar state that results from having too many centrosomes is Defective sister chromatid cohesion as a result of somatic very prone to attach single kinetochores to multiple spindle mutations may represent a major cause of CIN in human cancers. poles.110,111,118,119 These multipolar attachments are converted to In line with this, overexpression of Separase, the protease that merotelic attachments upon centrosome clustering and can cleaves cohesin upon mitotic exit, has been observed in breast produce chromosome segregation errors118,119 (Figure 3). cancer samples and transient overexpression of Separase can also Supernumerary centrosomes can arise through a number of produce aneuploidy in human cells.100 Besides its role in sister different mechanisms, such as centrosome overduplication, chromatid cohesion, cohesin has also been implicated in DNA cytokinesis failure or virus-induced cell fusion, with the last two replication, repair of DNA damage and protection of telomeres.101 also resulting in increased ploidy. Interestingly, tetraploidy is also Therefore, various other mechanisms might underlie the frequently observed in tumor cells and has been shown to aneuploidy observed in cells that carry mutations in one of the promote tumorigenesis in mice120 as well as transformation cohesin subunits102 and further research is needed to investigate in vitro and in vivo.121 It is currently unclear if the tumorigenic the contribution of cohesion defects in the establishment of CIN. nature of a tetraploid cell is due to the fact that it contains an extra copy of the genome or because it contains twice the amount of Causes of numerical CIN: Merotelic attachments centrosomes. The extra copy of the genome could set the stage Although CIN cell lines do not necessarily possess a weakened for subsequent chromosome loss to produce the genetic mitotic checkpoint, many CIN cell lines do have an increased imbalance that drives tumorigenesis. Alternatively, the extra occurrence of lagging chromosomes due to unresolved merotelic centrosomes could be the critical driving force to produce that attachments to the mitotic spindle103,104 (Figure 2). Merotelic genetic imbalance by enhancing the chance of creating merotelic attachments link a single kinetochore to two spindle poles, instead attachments. of one. If this merotelic attachment persists, the respective sister Very recently, a novel and remarkable mechanism to produce chromatid will end up in between the two packs of DNA during tetraploid cells was described. The process was named ‘entosis’: anaphase, producing a lagging chromosome105,106 (Figure 2). the invasion of a cell into another cell. Entosis has been described Most merotelic attachments are resolved by the Aurora B-depen- as a non-apoptotic cell death program of cells that have been dent error-correction machinery before anaphase.12,107–109 detached from the extracellular matrix.122 Internalization of these However, merotelic attachments are not actively being sensed detached cells into host cells most often results in cell death. by the mitotic checkpoint and therefore do not delay mitotic exit Entosis has been observed in tumors, and its occurrence correlates to allow for correction. Such merotelic attachments can therefore with tumor grade.122–124 Cells taken up by entosis are usually persist into anaphase and cause chromosome missegregations destroyed before the host divides, but if an intact invading cell and aneuploidy.103,106 Although not all lagging chromosomes ends up in the host cell’s cleavage plane, it can inhibit cytokinesis. necessarily lead to aneuploidy in the respective daughter cells,12 This produces a tetraploid cell that will build a multipolar spindle merotelic attachments are very prominent in CIN cells and in the next mitosis, a combination that is likely to produce therefore thought to be a major cause of aneuploidy. aneuploid daughter cells.124,125 This illustrates that one single As indicated in Figure 2, loss of cohesion between two sister cytokinesis failure could initiate chromosome segregation errors in chromatids could increase the occurrence of merotelic attach- the tumor cells’ offspring, ultimately leading to CIN. ments. In the absence of cohesion, kinetochores become more In addition to the abovementioned models for the induction of prone to bind MTs coming from two poles due to the absence of multiple centrosomes and subsequent aberrant spindle formation, the physical barrier normally created by the presence of another loss of the DNA damage checkpoint kinase Chk2 has also been sister chromatid. Many other cellular, genetic and molecular implicated in the induction of abnormal spindles through

& 2013 Macmillan Publishers Limited Oncogene (2013) 4459 – 4470 Genetic instability in tumors A Janssen and RH Medema 4462

Figure 3. Schematic representation of underlying causes of merotelic attachments. Centrosome coalescence creates merotelic attachments by allowing clustering of centrosomes, which had already established MT interactions with kinetochores. Impaired kinetochore MT dynamics inhibits the release of erroneous attachments, whereas the presence of multiple centromeres increases the chance of creating faulty attachments.

deregulation of MT dynamics.126 Chk2 is thought to be important when compared with that of tumor cells.131 Since MAD2 for proper spindle formation through phosphorylation of Brca1 on overexpression results in chromosome segregation errors and Serine 988. The fact that deregulated Chk2 functioning causes tumorigenesis in mice,90,93 aberrant Aurora B localization and numerical CIN through abnormal spindle formation is intriguing, activation could very well explain the CIN observed in MAD2- as the role of Chk2 in the maintenance of genetic stability has overexpressing tumors. mainly been ascribed to its function in DNA damage checkpoint One other interesting possibility that could explain the signaling.127 increased stability of kinetochore-MTs observed in CIN cells,128 is inactivation of the tumor-suppressor gene adenomatous polyposis coli (APC), which is frequently mutated in colon carcinomas.132 Causes of merotelic attachments: increased stability of kinetochore There is ample evidence that mutation of APC is an initiating microtubule attachments. The proper control of kinetochore–MT event in colon tumorigenesis, and that the effect of APC stability is another crucial factor that determines the ﬁdelity of inactivation on tumorigenesis is mediated through the Wnt chromosome segregation (Figure 3). Increasing the stability of MTs pathway.133 Nonetheless, APC also affects MT dynamics and APC bound to kinetochores through depletion of MCAK or Kif2b, two loss and CIN are found to coincide in early cancer lesions.134 Also, kinetochore-localized MT depolymerases, results in increased APC loss-of-function has been shown to directly result in genetic occurrence of merotelic attachments and severe chromosome 135,136 128 instability. This latter effect is thought to, at least partially, be missegregations in human tumor cells. A follow-up study in dependent on the function of APC in kinetochore–MT stability.137–139 which kinetochore-MT turnover was compared between an APC localizes to centrosomes, kinetochores and MT plus-ends,134 untransformed cell line and several CIN lines, revealed that and depletion of APC results in reduced inter-kinetochore tension, kinetochore MTs are more stable in the CIN lines. This increased which is thought to be due to decreased kinetochore–MT kinetochore MT stability could impede error correction and result 137,138 129 dynamics. Although a very interesting hypothesis, future in misattachments that can persist until anaphase. Importantly, research will have to assess if APC loss produces CIN through increasing kinetochore-MT turnover in these CIN cell lines by altered MT dynamics or through activation of Wnt signaling, as the overexpressing Kif2b or MCAK could partially revert the CIN 129 activation of Wnt target genes could also result in the induction of phenotype. CIN.140,141 More recently an unexpected link was reported between MAD2 overexpression, often found in CIN tumors, and increased kinetochore-MT stability. It was shown that MAD2 over- Causes of merotelic attachments: multiple attachment sites. The expression causes a reduction in Aurora B levels and activity at presence of extra centromeres on chromosomes could be another the centromeres, indicating that MAD2 overexpression inhibits cause of lagging chromosomes (Figure 3). As discussed above, error correction, providing a plausible explanation for the aberrantly structured chromosomes, such as ring chromosomes or increased segregation errors seen in these cells.130 The authors fusion of multiple chromosomes have a high chance of ending up showed that the effect on Aurora B localization is independent of in the cleavage furrow during telophase. These chromosomes the checkpoint function of MAD2, but the exact mechanism often have multiple centromeres and kinetochores, creating responsible for the loss of Aurora B remains to be elucidated. The multiple MT attachment sites that complicate chromosome effect on Aurora B localization and activity is in line with another biorientation. The multiple kinetochores present on a single study, which suggested that the Aurora B-dependent error chromatid can attach to opposite poles, effectively creating correction machinery works more efﬁciently in healthy cells ‘merotelic’ attachments142–144 (Figure 3). Upon exit from mitosis,

Oncogene (2013) 4459 – 4470 & 2013 Macmillan Publishers Limited Genetic instability in tumors A Janssen and RH Medema 4463 this bipolarly attached chromatid would lag behind, causing Besides DNA damaging events that occur in the S phase following whole-chromosome gain or loss,20,24,142,144 or initiate a BFB cycle an aberrant mitosis, it has also been shown that lagging as discussed above. These data indicate that structural chromosomes can be damaged during telophase.155 Following chromosomal aberrations and numerical CIN are closely linked both artificially induced chromosome segregation errors and and show that DNA damage due to, for example, replication spontaneous missegregations in CIN cancer cells, chromatin was defects,145,146 telomere defects24,102,144,147 or DNA-repair found to be damaged in telophase and the next G1 phase.155 The defects148 could, besides structural CIN, eventually result in occurrence of these DSBs could be (partially) rescued by inhibiting numerical CIN as well. cytokinesis, suggesting that cleavage furrow ingression causes Another cause of multiple centromere formation could be the damage to the lagging chromosomes (Figure 4). random incorporation of CENP-A at heterochromatic sites, other How ingression of the furrow could impose sufficient force to than the core centromeric region. CENP-A is a Histone H3-like damage the lagging chromatin is unclear. It could be that the protein, which specifically integrates at centromeric nucleosomes mechanical force exerted through contraction of the actin–myosin and is required for the proper formation of centromeres and ring can break the lagging chromatid. In this respect it will be kinetochores.149 It has been shown in Drosophila (cells) that interesting to see whether the furrow is actually in direct contact overexpression of CENP-A can directly result in the localization of with the lagging chromatin and whether this physical contact CENP-A into noncentromeric regions.142 This leads to ectopic coincides with the observed DNA damage. kinetochore formation, resulting in an increase in erroneous MT An interesting hypothesis is that fragile sites present on lagging attachments upon mitotic entry and ultimately severe chromosomes could render them more prone to breakage when chromosome segregation errors. Enhanced levels of CENP-A and present in the cleavage furrow. It has been shown that fragile sites its targeting factor HJURP have been found in both breast and induced by replication defects are protected from further colon cancer.150,151 However, in human cells, contradictory data destabilization and breaking in G1 through the recruitment of have been obtained on the effects of ectopic localization of CENP- 53BP1.162,163 Hypothetically, cleavage furrow ingression in the A. It has been suggested that CENP-A presence is not sufficient to presence of these underreplicated hotspots could promote drive complete de novo kinetochore formation,152 whereas other breakage of lagging chromosomes before 53BP1 has studies have shown it can.153 Thus, it is unclear if mere accumulated to protect these loci. Another cause of overexpression of CENP-A is sufficient for the induction of CIN missegregation-induced DNA damage could be the presence of through creation of multiple attachment sites. an increased, centromere-localized force imposed by MTs coming from the two spindle poles. This has also been suggested by an earlier study, in which the authors observed centromere-localized NUMERICAL CIN AND DNA DAMAGE DNA damage foci in interphase CIN cells.164 To address this point, Both structural and numerical chromosomal changes arise in the future studies should determine whether inhibition of this majority of cancer cells.6,8 As described above, it has been known centromere-localized force can reduce the damage. for several decades that structural chromosomal aberrations can Nevertheless, these recent findings154–156 reveal new induce chromosome segregation errors upon cell division.20 mechanisms by which CIN and aneuploidy promote genomic However, it has only recently been revealed that the opposite instability in cancer cells (Figure 4). The reciprocal relationship also occurs: chromosome segregation errors can induce structural between numerical and structural chromosomal changes compli- chromosomal changes through generation of DSBs.154–156 cates analysis of the direct effects of either structural chromoso- Duesberg et al.157,158 initially observed that the severity of mal changes or numerical CIN on tumor formation and reveals aneuploidy coincides with an increase in structural chromosomal additional ways in which heterogeneity arises in tumors. changes, and several CIN model systems displayed structural chromosomal aberrations as well.8,33,93,120,159 However, the underlying mechanisms for this correlation remained unknown. CHROMOSOME SEGREGATION ERRORS AND TUMORIGENESIS By generating 13 aneuploid yeast strains that each harbor an extra Boveri165 was the first to postulate that chromosomal aberrations single chromosome in addition to the haploid content, it was could be a causal factor in the occurrence of cancer after von recently shown that aneuploidy can directly induce genomic Hansemann had observed the presence of chromosomal instability156 (Figure 4). Aneuploidy of a single chromosome in abnormalities and mitotic errors in cancer cells.166 At present, some cases resulted in chromosome missegregations, whereas in the impact of structural changes on cancer progression is quite others, the abnormal chromosome number induced a high well understood,2 whereas the effects of numerical changes mutation rate. This increased genomic instability is thought to on tumorigenesis remain highly debated.46,167 Numerical be due to both an increase in DSBs and defects in recombinational chromosomal instability has been observed in early neoplastic repair.156 The underlying cause of the increase in DSBs remains lesions168 and is thought to be able to cause transformation.169–171 largely unknown, but could be due to aneuploidy-induced In addition, aneuploidy, one of the common consequences of CIN, imbalances in protein stoichiometry.160,161 Indeed, diploid yeast can drive evolution by introducing phenotypic variation in several strains carrying a single extra chromosome did not display an budding yeast strains.172,173 In line with a role for CIN in increase in genomic instability.156 In addition to defects induced tumorigenesis, CIN has been associated with poor prognosis and by aneuploidy per se, it was also recently shown that single resistance to chemotherapeutics in human patients.32–41 Below, we chromosome missegregation events, the underlying cause of summarize some of the main findings on the consequences of CIN aneuploidy induction, can also directly lead to DNA damage154,155 on tumor cells and tumor formation. For a more comprehensive (Figure 4). summary of the most recent advances in our understanding of the Micronuclei, small nuclear compartments that are surrounded consequences of CIN, we refer to a set of recently published reviews, by a nuclear envelope, in general, contain one or two chromo- which specifically summarize this aspect of CIN.46,174,175 somes that missegregated in the preceding mitosis.12,24 These Several CIN mouse models have been generated with deletions micronuclei undergo replication defects in the next S and G2 or hypomorphic alleles of genes important for mitotic fidelity phase, due to incomplete recruitment of DNA replication factors, (Table 1). Homozygous deletion of genes required for faithful such as the DNA helicase components MCM2 and MCM3 and the mitotic progression leads to embryonic lethality.53,57,58,79,171,176–179 initiation factor Cdt1.154 These replication defects result in Haploinsufficiency of mitotic genes, however, is tolerated in all persistent DNA damage in micronuclei, providing a link between cases and is therefore used extensively to investigate in the effects chromosome segregation errors and genomic instability (Figure 4). of CIN in tumorigenesis. Following recent findings that mitotic

& 2013 Macmillan Publishers Limited Oncogene (2013) 4459 – 4470 Genetic instability in tumors A Janssen and RH Medema 4464

Figure 4. Schematic representation of the proposed links between chromosomal instability (CIN), aneuploidy and genomic instability. The presence of an extra chromosome enhances protein synthesis, which can (in)directly lead to chromosome segregation errors, recombination problems and defects in DNA repair. Chromosome segregation errors, associated with CIN, could lead to DNA damage through the formation of replication-defective micronuclei (MN) or cleavage furrow-induced chromatin breakage.

genes are more frequently upregulated than downregulated in spontaneous tumor formation does not always correlate with the CIN tumors, various mouse models have been generated that level of aneuploidy found in MEFs. For example, BubR1H/H MEFs57 overexpress genes involved in mitotic progression, such as the have similar levels of aneuploidy as MEFs of Bub1 À /H and Bub1H/H mitotic checkpoint genes Bub1180 and MAD293 and the E2 enzyme animals,178 but BubR1H/H animals do not display an increased UbcH10181 (Table 1). Below we discuss the insights that these tumor incidence, whereas Bub1 hypomorphic animals do. These various models have generated with respect to the contribution of discrepancies could be due to additional roles of these checkpoint CIN in tumor formation. proteins in other cellular processes. Indeed, BubR1 hypomorphic animals also develop severe ageing-associated phenotypes, indicating that BubR1 has additive functions in maintaining cell Tumor-promoting role of numerical CIN homeostasis, possibly through regulation of p16 and p19 protein Haploinsufficiency, hypomorphy or overexpression of genes levels.57,184 One striking similarity in the spectrum of involved in mitotic progression can result in CIN. Although spontaneously developing tumors in the various CIN models is aneuploidy levels vary between different mouse models,167 CIN the formation of lung tumors. This resemblance indicates that, for generally seems to be able to enhance tumorigenesis. Treatment unknown reasons, CIN specifically enhances tumorigenesis in lung with carcinogens, such as DMBA, leads to increased tumor epithelial cells. incidence in 6 out of 10 CIN models tested (see Table 1) and One single chromosome missegregation event results in the crossing some of the CIN models with tumor-prone mouse loss or gain of one complete chromosome and thereby affects a models, such as Em-myc93,180 or APCmin/ þ 182,183 mice, results in large number of genes. The fact that CIN tumor cells continuously enhanced or accelerated tumor formation (Table 1). Many CIN change their chromosome composition indicates that CIN causes mouse models also display an increased susceptibility to extensive phenotypic changes, as signified by the increased spontaneous tumor formation in lung, liver and lymph nodes, overall transcriptomic and proteomic activity in aneuploid yeast albeit at a relatively old age (Table 1). Interestingly, the extent of strains.160 These dramatic changes in genetic make-up make it

Oncogene (2013) 4459 – 4470 & 2013 Macmillan Publishers Limited Genetic instability in tumors A Janssen and RH Medema 4465 Table 1. Mouse models of numerical CIN

Mouse model Spontaneous (organ) Carcinogen induced Crossed with Tumorigenesis in References tumors (organ) compound mice (organ)

MAD2 þ / À Yes (lung) Â p53 þ / À Increase 51,193 p53 þ / À /MAD1 þ / À Increase (lung, spleen, breast) MAD1 þ / À Yes (lung) Increase, vincristine p53 þ / À Normal 58,193 (lung, liver) p53 þ / À /MAD2 þ / À Increase (lung, spleen, breast) Bub3 þ / À No Normal, DMBA p53 þ / À Normal 55,60,177 Rb þ / À Normal Rae1 þ / À Increase, DMBA (lung) BubR1 þ / À No Increase, APCmin/ þ Increase (colon) 56,183 azoxymethane (lung, Decrease (small intestine) colon) BubR1 þ /H No ÂÂÂ 57 BubR1H/H No Increase, DMBA (lung) ÂÂ 57 Bub1 þ / À No Increase, DMBA (lung) p53 À / À Normal 178,182,188 APCmin/ þ Increase (colon) Bub1H/H Yes (liver, lung, lymph Â p53 þ / À Increase (thymus) 178,182,188,194 nodes) p53 À / À Increase (thymus) Bub1 À /H Yes (liver, lung, lymph Â p53 þ / À Increase (thymus) 178,182,188 nodes) p53 À / À Increase (thymus) APCmin/ þ Increase (colon) Rb þ / À Increase (pituitary) PTEN þ / À Decrease (prostate lesions) MAD2 ox Yes (liver, lung, lymph Â Em-myc Increase (lymph nodes) 93,195 nodes, intestine) KRASG12D Increased relapse (lung) Bub1T85/T264 Yes (liver, lymph Â Em-myc Increase (lymph nodes) 180 nodes and skin) CENP-E þ / À Yes (lung, colon, Decrease, DMBA p19 þ / À Decrease (lymph nodes) 171 spleen) (lung) Decrease (liver) Cdh1 þ / À Yes (breast, lung) ÂÂÂ 196 Hec1 ox Yes (lung, liver) ÂÂÂ 197 Cdc20 þ / À /H/H/ À /H No Normal, DMBA ÂÂ 198 Cdc20AAA/ þ Yes (lymph nodes, Â p53 À / À Increase (thymus) 188,199 liver) ATM À / À Increase (thymus) UbcH10T1/T2 Yes (lung, liver, blood, Increase, DMBA (lung) ÂÂ 181 skin) Pttg1 À / À No Â Rb þ / À Decrease (pituitary) 200,201 SA1 þ / À Yes (liver, pancreas, Decrease, 3-methyl- p53 À / À Normal 102 blood) colanthrene (fibrous PTEN þ / À Normal tissue) Decrease, diethyl-nitrosamine (liver) Incenp þ / À No ÂÂÂ 202 Survivin þ / À No ÂÂÂ 202 difficult to determine how CIN could be exactly contributing to in turn could allow the induction of other transforming changes, tumorigenesis. which ultimately lead to tumorigenesis. One, rather simple, hypothesis is that CIN is able to promote Alternatively, CIN and aneuploidy could promote tumorigen- tumorigenesis by inducing loss of certain tumor-suppressor genes esis through the creation of a mutator phenotype in cells.42,187 or gain of oncogenes. In line with this, crossing a CIN mouse Aneuploidy could affect levels of DNA repair genes, and thereby model carrying a hypomorphic Bub1 allele with tumor-prone promote genomic instability. In line with this hypothesis, mouse models heterozygous for p53 or APCmin resulted in loss of aneuploidy was initially found to correlate with genomic heterozygosity of these tumor-suppressor genes and enhanced instability in cancer cells158 and has recently been shown to tumor formation.182 CIN could indeed be quite effective in induce genomic instability in budding yeast.156 Moreover, CIN inducing loss of heterozygosity, as it will immediately induce could itself be a mutator by inducing DNA damage and loss of multiple genes. structural chromosomal changes through micronuclei However, since aneuploidy can lead to loss or gain of multiple formation154 or cleavage furrow ingression in the presence of essential genes, it is very likely to be detrimental to cells.52,54,160,161 lagging chromosomes.155 Interestingly, crossing a CIN mouse It is therefore thought that coinciding mutations would have to model with a mouse model deficient for ATM resulted in arise in other pathways to render the cell insensitive to changes in accelerated tumor formation,188 consistent with the notion that chromosome number.46 Examples of such changes are loss of CIN-induced tumor formation involves the induction of DNA function of p53185 or mutations that promote proteasomal damage. degradation in order to circumvent the increases in protein In summary, CIN and aneuploidy could together create a state production obtained by gain of chromosomes.186 These mutations in which cancer cells not only continuously lose and gain whole

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