The Cancer Biology of Whole-Chromosome Instability

REVIEW The cancer biology of whole-chromosome instability

PHG Duijf1,2 and R Benezra1

One form of chromosome instability (CIN), the recurrent missegregation of whole chromosomes during cell division (W-CIN), leads to aneuploidy. Although W-CIN is a hallmark of most cancers, mutations in genes involved in chromosome segregation are exceedingly rare. We discuss an oncogene-induced mitotic stress model that provides a mechanistic framework to explain this paradox. We also review the tumor-promoting and tumor-suppressing consequences of W-CIN. Importantly, we do this in the context of cancer as a complex systemic disease, rather than as a simple linearly progressing disorder that arises from a single abnormal cell population. Accordingly, we highlight the often neglected effects of W-CIN on key non-cell-autonomous entities, such as the immune system and the tumor microenvironment. Distinct tissue-speciﬁc susceptibilities to W-CIN-induced tumorigenesis and the clinical implications of W-CIN are also discussed.

Oncogene (2013) 32, 4727–4736; doi:10.1038/onc.2012.616; published online 14 January 2013 Keywords: whole-chromosome instability; aneuploidy; cancer biology; oncogene-induced mitotic stress; non-cell autonomous; tumor microenvironment

Cancer cells frequently missegregate their chromosomes during cope with W-CIN tumor cells? How does W-CIN affect metastasis? mitosis. This phenomenon, known as whole-chromosome instabil- Do the respective outcomes promote or suppress tumorigenesis? ity (W-CIN) leads to aneuploidy, a hallmark of most solid tumors. In this review, we will discuss these questions with an emphasis As extensively discussed elsewhere, tumor cells acquire W-CIN on the often overlooked non-cell-autonomous consequences of through three major mechanisms: mitotic checkpoint defects, W-CIN in cancer. centrosome overduplication or faulty sister chromatid cohesion (Figure 1, bottom).1–5 Briefly, the mitotic checkpoint ensures faithful chromosome segregation during mitosis by delaying ONCOGENE-INDUCED MITOTIC STRESS anaphase onset until all chromosomes have aligned on the To explain why most human tumors are aneuploid in the absence metaphase plate. A weakened mitotic checkpoint results in of mutations in W-CIN genes, the term ‘oncogene-induced mitotic premature chromosome segregation. More commonly, the stress’ was recently introduced.9 The basic idea is that commonly overexpression of checkpoint components leads to a prolonged activated oncogenes or inactivated tumor-suppressor genes 2 mitosis, which also leads to chromosome missegregation. As a directly or indirectly affect mitotic processes that control result of centrosome overduplication, tumor cells often acquire chromosome segregation (Figure 1). For example, overexpression three or more centrosomes per cell. Even though centrosome of the Ras oncogene, which mimics the mutational overactivation clustering typically results in a normal-appearing bipolar mitosis, of Ras that is frequently observed in human tumors,10 induces the formation of improper microtubule–kinetochore attachments centrosome overduplication, anaphase bridges and the formation 6 leads to frequent chromosome missegregation events. Finally, of multipolar mitotic spindles. The consequent chromosome cohesion defects cause premature separation of the sister missegregation leads to aneuploidy and the formation of 7,8 chromatids. micronuclei and binucleate cells.11 Similar mitotic defects are Here, we discuss the recently proposed oncogene-induced observed upon amplification of CDK4, which encodes cell cycle- mitotic stress model that provides a mechanistic framework for dependent kinase 4, and oncogenic mutations in a downstream the long-standing paradox that the majority of cancers are activator of the Ras pathway, B-Raf, both of which are also aneuploid, whereas mutations in genes involved in chromosome common in human cancers.12–14 segregation are extremely rare in tumors. We will also assess Defective signaling of the Rb and p53 tumor-suppressor pertinent immediate effects of W-CIN on the tumor cell. pathways also causes W-CIN through various mitotic abnormal- Importantly, however, we call special attention to the conse- ities, including centrosome overduplication, chromosome breaks quences of W-CIN in the context of cancer as a complex systemic and defects in centromere structure, chromosome condensation disease, rather than as one in which W-CIN merely contributes to and cohesion.15–18 Rb and p53 loss have been shown to result in tumor progression in a simple linear, cell-autonomous fashion. the upregulation of the E2F target Mad2, a key component of the Does the cell type in which W-CIN arises influence its tumorigenic mitotic checkpoint that, when overexpressed, causes W-CIN and effects? What are the consequences of W-CIN for interactions with tumorigenesis in vivo.19–21 Mad2 overexpression-induced W-CIN the tumor microenvironment? How does the immune system may be due to microtubule stabilization at the kinetochore that

1Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA and 2The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, Queensland, Australia. Correspondence: Dr PHG Duijf, The University of Queensland Diamantina Institute, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane, Queensland 4102, Australia or Dr R Benezra, Department of Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 241, New York, NY 10065, USA. E-mail: [email protected] or [email protected] Received 28 September 2012; revised 12 November 2012; accepted 15 November 2012; published online 14 January 2013 The cancer biology of W-CIN PHG Duijf and R Benezra 4728

Figure 1. Oncogene-induced mitotic stress. Most human tumors are aneuploid, but mutations in genes involved in chromosome segregation are rare. Key oncogenic and tumor suppressor pathways (top) control the gene expression and/or activity of an array of proteins involved in faithful chromosome segregation during mitosis (bottom). Aberrations in these pathways impose a mitotic stress that leads to W-CIN, thus explaining why most tumors are aneuploid. Superscript—and þ indicate common inactivating and activating aberrations in human tumors, respectively. Proteins in red font are direct E2F transcriptional targets;134–136 those in blue font are p53 transcriptional targets.137,138 Targets of both are shown in green.

leads to improper attachments and lagging chromosomes.22 In W-CIN-induced tumor formation is higher in blood vessels and one study, the function of p53 to maintain genomic stability was skin (Figure 2b). Similarly, although the absolute increase in elegantly dissociated from its apoptosis-inducing properties. W-CIN-induced tumorigenesis is highest for carcinomas, followed Homozygous p53R172P knock-in mice carry alleles that encode a by lymphomas and sarcomas, respectively, in terms of fold mutant form of p53 that maintains the ability to induce p21 increase, this order is reversed (Figure 2c). Thus, W-CIN is a potent expression, yet is unable to trigger apoptosis.23 These animals driver of tumorigenesis as it not only exacerbates tumor incidence develop tumors with stable, diploid genomes. However, in this in inherently more susceptible tissues but also augments context, p21 loss, which results in the activation of E2F and Mad2 tumorigenesis in tissues that normally have a very low propensity overexpression, leads to the formation of highly aneuploid tumors to form tumors. with shorter latencies, indicating that p21 induction is a critical W-CIN has also been shown to cause a switch in the observed means by which p53 prevents genomic instability.24 Remarkably, tumor spectrum in mouse tumor models. For instance, p53 À / À by reducing the protein levels of Mad2, alleviation of at least part mice primarily develop thymic lymphomas. However, concomitant of the stress on the mitotic checkpoint in this setting, as well as in reduction of the protein level of the mitotic checkpoint enzyme a Rb-defective mammary tumor model, is sufficient to rescue Separase, which on its own does not cause tumorigenesis, leads to W-CIN.21 increased W-CIN and the development of a wider variety of aneuploid malignancies, including ones that originate from the spleen and bone marrow.25 Interestingly, in the p53R172P/R172P SUSCEPTIBILITY TO W-CIN-INDUCED TUMORIGENESIS IS mutant knock-in mouse model that develops euploid tumors, TISSUE-SPECIFIC mainly sarcomas,23 p21 loss predominantly leads to the formation Factors that might determine to what extent W-CIN causes cellular of aneuploid lymphomas24 and rescue of the aneuploidy by Mad2 transformation include its origin, or cell type, and the stromal normalization in the latter model also reverses the tumor environment or tissue that surrounds it. A number of studies using spectrum to that observed in p53R172P/R172P animals.21 Strikingly, mouse models have shown that W-CIN may cause or accelerate these examples are consistent with W-CIN inducing a greater tumorigenesis more readily in some organs than in others. To increase in lymphomagenesis than in sarcomagenesis in W-CIN assess which tumor sites are most commonly affected by W-CIN, mouse models involving a single genetic defect (Figure 2c). we compiled data from 14 separate mouse models that have Together, these observations provide strong evidence for the previously been published. We limited our analysis to single-gene existence of tissue-specific differences in the susceptibility to models that spontaneously develop solid aneuploid tumors and, W-CIN-induced tumorigenesis. for each tumor site, only included data when a minimum of 2 Finally, some organs may have an increased propensity to studies with a total of at least 30 animals were available. The become aneuploid because of certain inherent properties of their overall increases in tumor incidence in W-CIN mouse models cells. The liver is a particularly notorious example in this regard. A compared with non-W-CIN control animals were calculated per tendency to become polyploid has been observed in hepatocytes tumor site and tumor type (Figure 2a). This analysis indicates that of a wide variety of mammalian species.26 In fact, these cells W-CIN increases tumor development at all organ sites and in all frequently increase and reduce their ploidy status through failed cell types studied (Figures 2b and c). W-CIN induces the most cytokinesis and multipolar mitosis, respectively. The dynamic significant absolute increase in tumorigenesis in lung, liver and nature of these processes has been shown to inevitably lead to lymphoid organs. However, the percentage (or fold) increase in the acquisition of numerical chromosome abnormalities.27,28 This

Oncogene (2013) 4727 – 4736 & 2013 Macmillan Publishers Limited The cancer biology of W-CIN PHG Duijf and R Benezra 4729

Figure 2. Whole-chromosome instability-induced tumorigenesis affects various tumor sites and cell types disproportionately in mouse models. To analyze the extent to which whole-chromosome instability (W-CIN) may affect tumor development in different organs and cell types differentially, we combined data from fourteen previous studies. The studies included heterozygous knockout animals for Aurora A139 (reference a), Cdh138 (m), Cenpe28 (b), Mad1140 (c), Mad2141 (l), Plk1142 (d) and Plk4143 (e); knockout, hypomorphic and transgenic alleles for Bub157,144,145 (f-h); transgenic overexpression models for Hec1146 (i), Mad220 (j) and UbcH10147 (k); and a heterozygous mutant Cdc20 knock-in model88 (n). These studies were selected because: (1) the experimental animals had but a single genetic defect that leads to W-CIN (i.e., they had no additional commonly used driver mutations in other oncogenes or tumor suppressor genes), (2) the genetic defect affected the animals globally (i.e., all organs and tissues) and (3) tumors arose spontaneously (i.e., were not induced by carcinogens). (a) Using the respective reports, numbers of animals in each category were added and the indicated percentages were calculated. (b, c) W-CIN-induced tumorigenesis affects different organs (b) and cell types (c) disproportionately. As compared with non-W-CIN animals, W-CIN increases the tumor incidence in all of the indicated organs and cell types. However, some of these differences are not statistically significant (*Po0.05; **Po0.01; ***Po0.0001; ns, not significant; one-sided Fisher’s exact test). In terms of absolute increases, liver, lung and lymphoid organs are significantly more susceptible to W-CIN-induced tumor formation than blood vessel endothelium or skin. Similarly, the absolute increase in W-CIN-induced development of carcinomas is larger than those of lymphomas and sarcomas (two-sided Fisher’s exact tests).

may explain why the liver is particularly amenable to W-CIN- induced tumorigenesis in mice (Figure 2b) and why liver tumors are among the ones with the highest aneuploidy rates in humans.29

TUMOR-PROMOTING EFFECTS OF W-CIN Cell-autonomous tumor-promoting effects The genesis of tumors is a process that involves a stepwise acquisition of multiple malignant features. Hanahan and Wein- berg30 described eight hallmarks of cancer to better understand the course of tumor development conceptually (Figure 3, bottom). In addition, two characteristics have been proposed to facilitate the acquisition of these features: genomic instability and Figure 3. Cell-intrinsic and -extrinsic factors affect the acquisition— inﬂammation by innate immune cells.30 Indeed, genomic or loss—of cancer-promoting properties. Genomic instability, instability, which comprises both chromosome instability (CIN) comprising CIN and MIN, is the primary cell-autonomous driver of and microsatellite instability (MIN),31 is the only cell-autonomous tumor progression as it instigates genetic and genomic abnormalities (gains, losses, duplications, deletions, rearrangements, DNA feature that provides the evolving tumor cell with a mechanism damage, mutations) that contribute to the acquisition of the eight that fosters unremitted genomic and mutational plasticity leading hallmarks of cancer (bottom).30 Non-cell-autonomous factors from to survival, proliferation and dissemination (Figure 3 and the tumor micro-environment and the immune system can similarly 32 Table 1). In essence, therefore, W-CIN drives tumor progression support or restrain tumor development. Tumor-suppressive effects by accelerating the gain of oncogenic and the loss of tumor- include the loss or reversion of the acquisition of malignant features, suppressor loci, thus facilitating the acquisition of each of the as well as the induction of cell cycle arrest, apoptosis, necroptosis, eight hallmarks of cancer. Consistently, W-CIN accelerates tumor senescence or autophagy. See also main text for details. formation and increases tumor burden and malignancy in a wide range of in vivo settings.2,4,21 In addition to this direct tumor-promoting effect, whole- facilitate tumor progression (Figure 3). In mouse models of W-CIN, chromosome missegregation has been demonstrated in a variety loss of Pttg, which encodes securin, a target of the mitotic of contexts to trigger other forms of genomic instability—as well checkpoint, leads to increased levels of DNA damage.37 Moreover, as epigenetic changes and oxidative stress33–36—that further W-CIN is accompanied by structural chromosomal aberrations in

& 2013 Macmillan Publishers Limited Oncogene (2013) 4727 – 4736 The cancer biology of W-CIN PHG Duijf and R Benezra 4730 tumors that develop in animals overexpressing the mitotic unanswered question. Nonetheless, recent cancer genome checkpoint protein Mad2, as well as in primary cells sequencing efforts suggest that it is characteristic for all forms heterozygous for Cdh1 or overexpressing aurora B.20,38,39 Thus, of advanced human cancer.51–55 W-CIN induces an avalanche of further genomic damage, It remains unclear how some of the above in vivo and in vitro instability and mutations, a trait that has been referred to as the studies relate to several previous observations in other mouse mutator phenotype (Figure 3).40 models of mitotic checkpoint regulators. Mice hypomorphic for Several recent studies provide additional evidence to support Bub1 or haploinsufficient for Bub3/Rae1 exhibit W-CIN and develop the idea that W-CIN and aneuploidy provoke a mutator aneuploid tumors but do not show any signs of DNA damage or phenotype. In both fission and baker’s yeast, the presence of an structural chromosomal abnormalities.56,57 Also, if aneuploidy, or extra chromosome causes various forms of genomic instability, the presence of an extra chromosome, by itself facilitates genomic including increased rates of DNA damage, mutations, whole- instability, including continued chromosome missegregation,41 chromosome loss and mitotic recombination.41 Interestingly, the Down syndrome would be expected to be accompanied by authors find that the genomic instability in these aneuploid yeast increased aneuploidy and elevated solid cancer susceptibility, but cells is due to excess protein and not to excess DNA. In yeast and the opposite is true.58–60 Conversely, Klinefelter patients, who carry primary mouse cells, this phenomenon had previously already one or more additional X chromosomes per cell, are at an increased been shown to result in proteotoxic stress and changes in cellular risk of developing cancer—although that may be because of the metabolism.42,43 This can lead to oxidative stress,36,44 which could inherent hormonal deregulation.61,62 Perhaps the degree of other explain the observed DNA damage. In mammalian cells, W-CIN— genomic damage—or protection from it—that is triggered by rather than aneuploidy per se—also causes other types of genomic aneuploidy is dependent on which specific chromosome(s) or loci instability. W-CIN cells often have a prolonged metaphase. This are gained or lost. Another, not necessarily mutually exclusive, culminates in telomere dysfunction and DNA damage at possibility is that the gain of smaller chromosomes, such as chromosome ends.45 In addition, lagging chromosomes, which chromosome 21, has less detrimental effects because smaller are usually caused by improper microtubule–kinetochore attach- chromosomes encode fewer proteins, thus causing less stress ments in W-CIN cells, often become trapped in the cleavage because of increased protein production.41,42,63 Consistent with this, furrow during cytokinesis, the final stage of cell division. This in both mice and humans, trisomies for larger autosomes cause results in DNA double-strand breaks that are incorrectly repaired either embryonic or perinatal lethality, such as in Patau syndrome by non-homologous end joining, thus producing daughter cells (trisomy 13) and Edwards syndrome (trisomy 18).43,64 Future studies that suffer structural chromosome aberrations, including unba- might shed more light on the above paradox. lanced translocations.46 Inaccurate (merotelic) microtubule– kinetochore attachments have also been reported to result in pericentromeric DNA breaks in mutant mouse cells with mitotic Non-cell-autonomous tumor-promoting effects spindle defects.47 However, in human tumor cells, such aberrant W-CIN can also, directly and indirectly, accelerate tumor progres- attachments often cause hyperstabilization of microtubule– sion by affecting non-cell-autonomous mechanisms: evasion of kinetochore interactions, resulting in elevated chromosome anti-tumor immunity, triggering inflammation and remodeling of missegregation rates.22 Finally, lagging chromosomes can the tumor microenvironment (Figure 3 and Table 1).65 Elevated be encapsulated in micronuclei that form independently of the chromosome missegregation rates, as well as the consequences of primary nucleus in tissue culture models. Following extensive DNA the ensuing mutator phenotype, increase the likelihood that damage, DNA breaks and chromosomal rearrangements inside tumor cells will be able to evade eradication by the immune the micronucleus, the structurally altered chromosome can system.31,66 This may occur in a variety of ways. First, the adaptive segregate into the primary nucleus of daughter cells.48 In this immune system may no longer recognize the W-CIN cancer cell. manner, W-CIN could directly cause chromothripsis, the shredding This can be due either to the loss or mutation of tumor-specific and structural rearrangement of genomic fragments within antigens or to the acquisition of defects in antigen processing or chromosomes.49,50 Whether this phenomenon will be observed presentation.67,68 Second, the genomically unstable tumor cells in mouse models of chromosome missegregation is an important may become insensitive to destruction by cytotoxic cells of the

Table 1. Tumor-promoting and tumor-suppressive effects of W-CIN

Tumor-promoting effects of W-CIN Tumor-suppressive effects of W-CIN

Cell-autonomous Cell-autonomous – Gain of oncogenes – Loss of oncogenes – Loss of tumor suppressor genes – Gain of tumor suppressor genes – Mutations, epigenetic changes, DNA damage, telomere dysfunction, – Mutations, epigenetic changes, DNA damage, telomere structural chromosomal rearrangements dysfunction, structural chromosomal rearrangements – Formation of binucleate cells – Formation of binucleate cells – Proteotoxic stress – Excessive levels of W-CIN

Non-cell-autonomous Non-cell-autonomous – Evasion of anti-tumor immunity (for example, by altering tumor-specific – Triggering auto-destruction by cytotoxic immune cells (for antigens, acquiring defects in antigen presentation, inducing an example, via tumor-specific antigens) immunosuppressive microenvironment) – Protect the premetastatic site from colonization – Triggering inflammation – Remodeling of the tumor microenvironment (for example, induction of angiogenesis)

Oncogene (2013) 4727 – 4736 & 2013 Macmillan Publishers Limited The cancer biology of W-CIN PHG Duijf and R Benezra 4731 innate immune system.69 Finally, W-CIN cancer cells may create an facilitate the acquisition of tumor-promoting features, it is equally immunosuppressive microenvironment.65 The complex tumor- likely to facilitate the loss of such characteristics and the gain of suppressive (see also below) and tumor-promoting interactions tumor-suppressive properties (Figure 3 and Table 1).32 Outcomes between W-CIN tumors and the cells of the immune system can of the latter may include apoptosis, necroptosis (that is, lead to the in vivo selection and survival of specific cancer cells programmed necrosis82), senescence and autophagy (that is, the that can permanently evade destruction by the immune system. controlled lysosomal degradation of damaged or non-functional This phenomenon is known as cancer immunoediting.70,71 Thus, molecules or organelles within the cell83,84). Consistently, as W-CIN might facilitate cancer immunoediting in tumor cells.69 compared with control cells, a modestly increased fraction of Additionally, W-CIN may inadvertently lead to the tumor cell’s primary cells that exhibit W-CIN undergo cell death and/or enter secreting one or more of the many factors that recruit inflammatory senescence (see also below).20,37,56,57,85 immune cells or promote tissue remodeling, including tumor W-CIN and aneuploidy have also been shown to slow the angiogenesis. For instance, elevated expression of the pro- proliferation rate of cells. This has been observed in aneuploid inflammatory factors cyclooxygenase 2 (Cox-2) and interleukin-6 mouse embryonic fibroblasts that are trisomic and harbor (IL-6) has been observed in the colon mucosa of a heterozygous-null Robertsonian fusion chromosomes.43 Additionally, with very few mouse model for Sgo1, which encodes the chromosome cohesion exceptions,28,86 primary mouse embryonic fibroblasts derived factor Shugoshin-172 (Figure 1). Cox-2 deficiency protects against from genetic W-CIN models grow more slowly than wild-type mutant adenomatous polyposis coli-induced colon carcinogen- control fibroblasts20,38,85–88 and form colonies less efficiently.20,28 esis,73,74 and IL-6 is essential for colitis-associated colon cancer One explanation for this may lie in the fact that cells with extra development in the mouse.65 This suggests that these factors could copies of chromosomes suffer substantial proteotoxic stress play an important role in the Sgo1 þ / À phenotype. It is not clear yet because of increased protein production.42,43,63 Another may be whether elevated expression of Cox-2 and IL-6 is a direct result of that, in an attempt to accurately segregate sister chromatids the W-CIN and whether it is confined to this particular W-CIN tumor during mitosis, W-CIN cells prolong their metaphase. In addition to model. We note, however, that prolonged DNA damage signaling, a slowing the cell cycle and the production of aneuploid progeny, consequence of W-CIN, leads to the secretion of inflammatory such extended mitotic arrest may result in the formation of cytokines,75 andinonemousemodel,W-CINhasbeenassociated binucleate cells, which proliferate more slowly or not at all.20,89–91 with cancer progression through predisposition to chronic Alternatively, DNA damage, another consequence of W-CIN, may inflammation.76 Finally, W-CIN can induce necrosis or apoptosis, be responsible. W-CIN can cause DNA damage in vitro and either by virtue of random genetic changes or as a result of in vivo20,46–48 and excessive amounts of DNA damage have been excessive W-CIN or genomic instability (see below), and while these shown to induce apoptosis.92 Moreover, DNA damage triggers a forms of cell death may protect against tumor progression, they also response that, in early stages of tumorigenesis, delays tumor trigger an inflammatory response that promotes tumorigenesis.65 progression or prevents tumorigenesis altogether.93 Collectively then, these observations suggest that W-CIN may The rate at which cells missegregate their chromosomes also accelerate tumor progression in vivo by creating an inflammatory affects whether W-CIN is beneficial or detrimental for tumor environment. initiation and progression. Low rates of W-CIN are advantageous Inflammation and tissue changes in the tumor milieu, including as they enable the steady acquisition of malignant features. the recruitment of tumor-associated macrophages and neutrophils, Excessive W-CIN, however, results in a form of cell death during or facilitate tumor progression in a multitude of ways. They include the immediately after mitosis that has been termed mitotic immune cells’ production of growth factors, cytokines, chemokines, catastrophe.94,95 This phenomenon may explain why cells and matrix metalloproteinases, prostaglandins and angiogenic factors, animals that are completely deficient in key regulators of which stimulate survival, proliferation, invasion, angiogenesis, chromosome segregation are not viable, whereas heterozygous migration, extravasation and metastasis of malignant cells and knockout cells and mice often are, but exhibit W-CIN, resulting in weaken the anti-tumor adaptive immune response.65,77 Moreover, aneuploid tumor formation.2,4 As mentioned above, W-CIN cells inflammation is associated with cytokine release and has been attempt to faithfully segregate chromosomes during mitosis by shown to induce tumor-promoting epigenetic changes, structural prolonging metaphase. Increasing evidence indicates that when chromosomal abnormalities and, through the generation of reactive this arrest lasts too long, as is imperative in the case of excessive oxygen and nitrogen species, DNA damage, all of which in turn W-CIN, cells die in mitosis.96,97 Alternatively, mitotic slippage leads 78–80 promote further genomic aberrations (Figure 3). Specific to the cell’s entering the next G1 stage of the cell cycle, after which dampening of these inflammatory responses by genetic or drug it may permanently arrest or undergo apoptosis.89,95–98 intervention will be required to determine the contribution to Several lines of evidence suggest that the p53 pathway is W-CIN-induced disease. critically important for sensing excessive amounts of W-CIN, and It is important to note that W-CIN models created by global loss- the resulting genomic damage, and inducing cell cycle arrest, or gain-of-function also likely change the chromosome stability in senescence or cell death. A p53-dependent checkpoint that cells of the tumor microenvironment. These changes could affect arrests cells in G1 following mitotic slippage limits the proliferation the ability of these cells to support tumor growth in a positive or of aneuploid cells.45,89–91,98–101 An effective tumor-suppressive negative way. Here then, conditional inactivation of the genes response to DNA damage and telomere dysfunction critically relies controlling W-CIN in specific cell types will be required. Lung on p53 function.102,103 P53 prevents aneuploidy-induced tumor cell-specific overexpression of Mad2 does have a significant transformation in vivo by inducing cell death,36 and in C57BL/6 impact on oncogenic Kras-induced tumorigenesis81 independent mice, p21 loss only leads to the development of aneuploid tumors of any W-CIN effects on the microenvironment, but this does not when p53 cannot induce apoptosis.24 Finally, only one study has rule out compounding effects if Mad2 overexpression in the tumor thus far reported the recovery of viable cells with extreme levels of cell is combined with that in the tumor microenvironment. W-CIN and this required p53 loss.104

Non-cell-autonomous tumor-suppressive effects TUMOR-SUPPRESSIVE EFFECTS OF W-CIN How W-CIN may be tumor suppressive—or, as outlined above, Cell-autonomous tumor-suppressive effects tumor promoting—as a result of inducing non-cell-autonomous The random nature of chromosome missegregation and its effects has thus far been poorly acknowledged. The mechanisms consequences inherently implies that as much as W-CIN can and consequences of genomic instability are often studied on the

& 2013 Macmillan Publishers Limited Oncogene (2013) 4727 – 4736 The cancer biology of W-CIN PHG Duijf and R Benezra 4732 molecular and cell biological levels. However, it is important to tumor microenvironment and possibly inhibiting tumor growth. In realize that tumors, particularly larger ones, are not merely a future studies, it will be important to examine the consequences homogeneous mass of malignant cells. Not only do they often of genetically induced W-CIN in a specific cell type or organ in the consist of different clones of cancer cells that have acquired context of a wild-type tumor microenvironment and immune shared as well as distinct genetic changes that allow them to system. proliferate,105 they also contain a variety of normal cell types, including fibroblasts, endothelial cells and pericytes of the tumor vasculature, as well as various types of immune cells.30,106 CD4 þ CLINICAL IMPLICATIONS OF W-CIN T cells, CD8 þ T cells, natural killer T cells, natural killer cells, tumor- The ability to missegregate chromosomes equips cancer cells with associated macrophages, dendritic cells and neutrophils can act to a mechanism that fuels tumor progression. Consistently, aneu- promote tumor progression under certain conditions, but all these ploidy, the immediate consequence of W-CIN, has been associated cell types, as well as mast cells, Tie-2-expressing monocytes, with poor prognostic factors for a variety of cancers (Table 2).29,113 mesenchymal stem cells, fibroblasts and pericytes, have also been These include correlations with higher tumor grade, increases in found to possess anti-tumor activity.65,107–109 W-CIN, and the tumor recurrence and decreases in metastasis-free and overall consequent other forms of genomic damage, can lead to the patient survival. In line with this, primary tumors that are tumor cell’s producing signals that stimulate their own eradication aneuploid have an increased likelihood to metastasize and by cytotoxic leukocytes or inhibit their growth facilitation by other metastatic tumors often exhibit more chromosomal aberrations cell types. One such signal could be the production of novel than the primary tumors. For example, the brain metastases of immunogenic proteins due to, for instance, mutations or colorectal carcinomas have more structural and numerical translocations. Such proteins are also known as tumor-specific chromosomal abnormalities than the matched primary antigens and are actively sought after in order to develop cancer tumors.114 W-CIN increases the risk for lymph node metastasis of immunotherapies.110 squamous cell carcinomas115 and aneuploidy is associated with It is conceivable (but not yet shown) that W-CIN can also trigger poorer overall survival in breast, ovarian, lung and brain cancers non-cell-autonomous antimetastatic effects. For example, during (Table 2).29,113,116–118 tumorigenesis, cancer cells acquire the ability to entrain neutro- Importantly, one study has shown that even though W-CIN is phils to actively kill tumor cells at the premetastatic site and more frequent in metastasized pulmonary tumors, its presence in thereby inhibit seeding and colonization by these cells.111 In the primary tumor is not necessarily a reliable prognostic marker principle, this type of change could be enhanced by W-CIN in the for metastatic potential.119 Additionally, other studies have tumor cell. In addition, in the tumor cell, W-CIN may result in demonstrated that, before a clinically manifest metastasis, early changes in the expression levels of certain factors that are critical disseminated breast, esophageal and prostate carcinoma cells in for metastasis—such as the microRNA miR-126—that would then the lymph nodes or bone marrow exhibit significantly fewer in turn protect the metastatic site from the recruitment of whole-chromosome and segmental chromosomal aberrations endothelial cells, angiogenesis and colonization.112 Ultimately, than the matched primary tumors.120–123 Together, these results however, as the tumor continues to evolve, such tumor- suggest that at a time when primary tumors have adapted to use suppressive barriers may be overcome, after which metastasis aneuploidy to fuel tumor growth, there may be selective pressure does occur. against the most aneuploid cells in the population in early Finally, in one mouse model, W-CIN induced by the loss of one metastatic events. Once the metastatic niche is established, then copy of Cenpe, which encodes a kinetochore motor protein rapid tumor evolution may once again allow the expansion of the involved in chromosome segregation, has been shown to reduce more aneuploid and aggressive cell populations. the incidence of tumorigenesis in some settings.28 However, in Regardless of the order of genetic and genomic events, another this study, as well as in all other single-gene W-CIN models that malignant clinical consequence of W-CIN is enhanced resistance have thus far been created, the induction of aneuploidy is global, to cancer treatment.124 W-CIN has been associated with multidrug that is, in all cells and tissues of the animal, thereby affecting the resistance125 and, in the context of breast and ovarian cancer, it

Table 2. Elevated aneuploidy rates correlate with increased malignancy and metastasis in human cancers

Cancer type Primary observations

Astrocytoma Early-stage tumors have lower aneuploidy rates than late-stage tumors.29 Basal cell carcinoma Aneuploidy increases the likelihood of tumor recurrence.148 Breast cancer A W-CIN signature/score correlates with higher tumor grade, poorer survival and shorter times to recurrence and metastasis in patients with most breast cancer subtypes.113,116,117 Cervical carcinoma Aneuploidy status is a better prognostic predictor than lymph node metastasis status.149 Colorectal carcinoma Brain metastases have more chromosomal aberrations and whole-chromosome gains and losses than the primary tumors.114,150 Gastrointestinal stromal Increased AURKA expression and aneuploidy rates are associated with an elevated metastasis rate.151 tumors (GIST) Glioma A W-CIN signature correlates with poorer patient survival.113 Lung adenocarcinoma Aneuploidy and a W-CIN signature correlate with poorer patient prognosis.113,152 Lymphoma A W-CIN signature correlates with poorer patient survival.113 Medulloblastoma A W-CIN signature correlates with poorer patient survival.113 Melanoma Aneuploidy correlates with poorer patient survival.153 Ovarian carcinoma Aneuploidy correlates with higher tumor grade and increased risk of recurrence. Whole-chromosome aneuploidy correlates with poorer patient survival.29,118 Squamous cell carcinoma Increased aneuploidy rates reduce disease-free and overall survival and increase the risk of lymph node metastasis.115,154 Thyroid carcinoma Metastases are more often aneuploid than primary tumors.155

Oncogene (2013) 4727 – 4736 & 2013 Macmillan Publishers Limited The cancer biology of W-CIN PHG Duijf and R Benezra 4733 has been shown to confer resistance to taxanes.126 Taxanes are aneuploid cells is proportional to the transcript and protein levels microtubule-stabilizing drugs that are commonly used in of the gene32 predicts that this may become a feasible option. chemotherapy and are thought to induce cell death by causing a prolonged mitotic arrest.96,97,127 Finally, W-CIN is responsible for tumor recurrence after oncogene withdrawal, which may reflect CONCLUSIONS AND PERSPECTIVES 81 resistance to targeted anticancer therapies. In this regard, the As discussed above, W-CIN has tumor-promoting and tumor- ongoing chromosomal changes may lead to the tumor cells’ suppressive effects (Table 1). The question of what determines the becoming insensitive to therapy, for example, due to loss of the fate of a cell that missegregates its chromosomes may be target or the acquisition of other pro-proliferative signals that embedded in the temporal order of events. Oncogene (or permit the re-growth of the tumor even when target pathway tumor-suppressor loss)-induced mitotic stress is likely an early inhibition is effective. event that exacts a temporary fitness cost before the expansion of more aneuploid and more aggressive stress-resistant progeny. Enough missegregation and mutator events likely need to occur THERAPEUTIC TARGETING OF W-CIN TUMOR CELLS to efficiently drive tumor progression. Early dissemination events Because W-CIN is an important driver of tumor progression, an and the population of the early metastatic niche may counter- approach that specifically targets W-CIN tumor cells is likely to be select initially for the cells in the population with fewer effective against most forms of cancer. Several such strategies chromosomal imbalances until these cells can once again tolerate have been proposed. As mentioned above, an excessively high the stress of growing in a hostile tumor microenvironment. Thus, level of W-CIN results in cell death. The possibility of increasing the the evolutionary process of tumorigenesis involves overcoming levels of W-CIN in tumor cells in order to eradicate them has multiple critical tumor-suppressive barriers that are put in place in therefore been explored in vitro. Indeed, a combination of a normal cell and W-CIN is one mechanism that facilitates the reducing the expression level of the mitotic kinases Mps1 or breaking of these barriers. Importantly, in this review, we BubR1 and treatment with the taxane paclitaxel induces cell 128 underscored that non-cell-autonomous factors also have a critical death. However, at present, the development of a therapeutic influence on this W-CIN-driven selection process. Similar to the strategy based on this principle remains both challenging and macroevolution of species in which key genetic changes that drive precarious. adaptation occur over thousands or millions of years, this Another strategy involves the specific inhibition of centrosome microevolutionary process may be very slow, as evidenced by clustering. Tumor cells have often overduplicated their centro- the fact that it may take decades until an incipient tumor cell has somes such that they possess more than the normal number of developed into full-blown metastatic disease. two centrosomes per cell (Figure 1). Centrosome clustering allows The dynamic nature of W-CIN has several undesirable clinical cells to undergo a bipolar division, but this frequently leads to consequences, such as the increased likelihood of tumor aneuploid daughter cells secondary to merotelic chromosome 6 recurrence and the acquisition of resistance to cancer therapies. attachments and lagging chromosomes. The use of various It has also significantly hampered the development of aneuploidy- drugs, such as griseofulvin and EM011, and small-molecule specific regimens. However, over the past decades, a number of inhibitors or small interfering RNAs against various mitotic promising strategies have emerged. The further improvement of proteins, including HSET and HEC1, have shown various degrees these strategies and the design of novel ones may in the not too of specificity in inhibiting the proliferation of cancer cells with 129–132 distant future lead to the development of more effective cancer overduplicated centrosomes. The latter study has in fact treatment options. demonstrated some in vivo efficacy.132 In addition to these approaches that aim to target W-CIN cells per se, other strategies have focused on targeting the resulting CONFLICT OF INTEREST aneuploid cells. The presence of an additional chromosome in disomic yeast or in trisomic mouse embryonic fibroblasts, The authors declare no conflict of interest. generated by Robertsonian fusion chromosomes, has been shown to cause aberrations in cellular metabolism and protein folding and turnover.42,43 Importantly, three drugs that induce energy ACKNOWLEDGEMENTS stress or inhibit autophagy or protein folding have been shown to We thank Peter J Cook, Zvi Granot, Rozario I Thomas, Daniel Marks and Rocıó Sotillo specifically inhibit proliferation of the aforementioned trisomic for critical reading of the manuscript. PHGD is supported by a Young Investigator mouse embryonic fibroblasts and aneuploid human cells.133 It Award from Alex’s Lemonade Stand Foundation and RB by the Breast Cancer Research Foundation. remains unclear, however, whether these agents will be effective in specifically killing aneuploid cells that have lost chromosomes, a phenomenon that is in fact more common in human tumors.29 REFERENCES Finally, aneuploid cells may be targeted specifically by virtue of a common gain or loss of a whole chromosome, a chromosome 1 Holland AJ, Cleveland DW. Boveri revisited: chromosomal instability, aneuploidy arm or a combination thereof. Chromosome 7 is frequently gained and tumorigenesis. Nat Rev Mol Cell Biol 2009; 10: 478–487. 2 Schvartzman JM, Sotillo R, Benezra R. Mitotic chromosomal instability and can- in solid tumors, particularly in carcinomas, and chromosome 22 is cer: mouse modelling of the human disease. Nat Rev Cancer 2010; 10: 102–115. often lost, for example, in nearly half of ovarian adenocarcino- 3 Thompson SL, Bakhoum SF, Compton DA. Mechanisms of chromosomal 29 mas. Similarly, an approach that combines targeting the gain of instability. Curr Biol 2010; 20: R285–R295. chromosome 7 and the loss of chromosome 10 is anticipated to 4 Ricke RM, Van Ree JH, Van Deursen JM. Whole chromosome instability and be effective for up to 75% of glioblastomas and astrocytomas.29,53 cancer: a complex relationship. Trends Genet 2008; 24: 457–466. The small arms of chromosomes 3 and 8 are frequently lost and 5 Gordon DJ, Resio B, Pellman D. Causes and consequences of aneuploidy in gained in tumors, respectively.52,53 The gains and/or losses of cancer. Nat Rev Genet 2012; 13: 189–203. these whole chromosomes or chromosome arms, or deletions or 6 Duensing A, Duensing S. Centrosomes, polyploidy and cancer. Adv Exp Med Biol amplification of smaller chromosome segments, may be targeted 2010; 676: 93–103. 7 Oliveira RA, Nasmyth K. Getting through anaphase: splitting the sisters and using the respective increased or decreased expression of one or beyond. Biochem Soc Trans 2010; 38: 1639–1644. multiple genes on the involved chromosomes. Genes encoding 8 Solomon DA, Kim T, Diaz-Martinez LA, Fair J, Elkahloun AG, Harris BT et al. cell membrane proteins would represent the most promising Mutational inactivation of STAG2 causes aneuploidy in human cancer. Science candidates. The observation that the copy number of genes in 2011; 333: 1039–1043.

& 2013 Macmillan Publishers Limited Oncogene (2013) 4727 – 4736 The cancer biology of W-CIN PHG Duijf and R Benezra 4734 9 Malumbres M. Oncogene-induced mitotic stress: p53 and pRb get mad too. 38 Garcia-Higuera I, Manchado E, Dubus P, Canamero M, Mendez J, Moreno S et al. Cancer Cell 2011; 19: 691–692. Genomic stability and tumour suppression by the APC/C cofactor Cdh1. Nat Cell 10 Sherr CJ, McCormick F. The RB and p53 pathways in cancer. Cancer Cell 2002; 2: Biol 2008; 10: 802–811. 103–112. 39 Nguyen HG, Makitalo M, Yang D, Chinnappan St D, Hilaire C, Ravid K. 11 Saavedra HI, Fukasawa K, Conn CW, Stambrook PJ. MAPK mediates RAS-induced Deregulated Aurora-B induced tetraploidy promotes tumorigenesis. FASEB J chromosome instability. J Biol Chem 1999; 274: 38083–38090. 2009; 23: 2741–2748. 12 Nelsen CJ, Kuriyama R, Hirsch B, Negron VC, Lingle WL, Goggin MM et al. Short 40 Loeb LA. A mutator phenotype in cancer. Cancer Res 2001; 61: 3230–3239. term cyclin D1 overexpression induces centrosome amplification, mitotic spindle 41 Sheltzer JM, Blank HM, Pfau SJ, Tange Y, George BM, Humpton TJ et al. abnormalities, and aneuploidy. J Biol Chem 2005; 280: 768–776. Aneuploidy drives genomic instability in yeast. Science 2011; 333: 1026–1030. 13 Kamata T, Hussain J, Giblett S, Hayward R, Marais R, Pritchard C. BRAF inacti- 42 Torres EM, Sokolsky T, Tucker CM, Chan LY, Boselli M, Dunham MJ et al. Effects of vation drives aneuploidy by deregulating CRAF. Cancer Res 2010; 70: 8475–8486. aneuploidy on cellular physiology and cell division in haploid yeast. Science 14 Cui Y, Borysova MK, Johnson JO, Guadagno TM. Oncogenic B-Raf(V600E) induces 2007; 317: 916–924. spindle abnormalities, supernumerary centrosomes, and aneuploidy in human 43 Williams BR, Prabhu VR, Hunter KE, Glazier CM, Whittaker CA, Housman DE et al. melanocytic cells. Cancer Res 2010; 70: 675–684. Aneuploidy affects proliferation and spontaneous immortalization in mamma- 15 Coschi CH, Martens AL, Ritchie K, Francis SM, Chakrabarti S, Berube NG et al. lian cells. Science 2008; 322: 703–709. Mitotic chromosome condensation mediated by the retinoblastoma protein is 44 Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria in cancer cells: what is so tumor-suppressive. Genes Dev 2010; 24: 1351–1363. special about them? Trends Cell Biol 2008; 18: 165–173. 16 Van Harn T, Foijer F, Van Vugt M, Banerjee R, Yang F, Oostra A et al. Loss of Rb 45 Hayashi MT, Cesare AJ, Fitzpatrick JA, Lazzerini-Denchi E, Karlseder J. A telomere- proteins causes genomic instability in the absence of mitogenic signaling. Genes dependent DNA damage checkpoint induced by prolonged mitotic arrest. Nat Dev 2010; 24: 1377–1388. Struct Mol Biol 2012; 19: 387–394. 17 Manning AL, Longworth MS, Dyson NJ. Loss of pRB causes centromere dys- 46 Janssen A, Van der Burg M, Szuhai K, Kops GJ, Medema RH. Chromosome function and chromosomal instability. Genes Dev 2010; 24: 1364–1376. segregation errors as a cause of DNA damage and structural chromosome 18 Fukasawa K, Choi T, Kuriyama R, Rulong S, Vande Woude GF. Abnormal cen- aberrations. Science 2011; 333: 1895–1898. trosome amplification in the absence of p53. Science 1996; 271: 1744–1747. 47 Guerrero AA, Gamero MC, Trachana V, Futterer A, Pacios-Bras C, Diaz-Concha NP 19 Hernando E, Nahle Z, Juan G, Diaz-Rodriguez E, Alaminos M, Hemann M et al. Rb et al. Centromere-localized breaks indicate the generation of DNA damage by inactivation promotes genomic instability by uncoupling cell cycle progression the mitotic spindle. Proc Natl Acad Sci USA 2010; 107: 4159–4164. from mitotic control. Nature 2004; 430: 797–802. 48 Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y et al. DNA 20 Sotillo R, Hernando E, Diaz-Rodriguez E, Teruya-Feldstein J, Cordon-Cardo C, breaks and chromosome pulverization from errors in mitosis. Nature 2012; 482: Lowe SW et al. Mad2 overexpression promotes aneuploidy and tumorigenesis in 53–58. mice. Cancer Cell 2007; 11: 9–23. 49 Stephens PJ, Greenman CD, Fu B, Yang F, Bignell GR, Mudie LJ et al. Massive 21 Schvartzman JM, Duijf PH, Sotillo R, Coker C, Benezra R. Mad2 is a critical genomic rearrangement acquired in a single catastrophic event during cancer mediator of the chromosome instability observed upon Rb and p53 pathway development. Cell 2011; 144: 27–40. inhibition. Cancer Cell 2011; 19: 701–714. 50 Liu P, Erez A, Nagamani SC, Dhar SU, Kolodziejska KE, Dharmadhikari AV et al. 22 Kabeche L, Compton DA. Checkpoint-Independent Stabilization of Kinetochore- Chromosome catastrophes involve replication mechanisms generating complex Microtubule Attachments by Mad2 in Human Cells. Curr Biol 2012; 22: 638–644. genomic rearrangements. Cell 2011; 146: 889–903. 23 Liu G, Parant JM, Lang G, Chau P, Chavez-Reyes A, El-Naggar AK et al. Chro- 51 Molenaar JJ, Koster J, Zwijnenburg DA, Van Sluis P, Valentijn LJ, Van der Ploeg I mosome stability, in the absence of apoptosis, is critical for suppression of et al. Sequencing of neuroblastoma identifies chromothripsis and defects in tumorigenesis in Trp53 mutant mice. Nat Genet 2004; 36: 63–68. neuritogenesis genes. Nature 2012; 483: 589–593. 24 Barboza JA, Liu G, Ju Z, El-Naggar AK, Lozano G. p21 delays tumor onset 52 Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, Donovan J et al. The by preservation of chromosomal stability. Proc Natl Acad Sci USA 2006; 103: landscape of somatic copy-number alteration across human cancers. Nature 19842–19847. 2010; 463: 899–905. 25 Mukherjee M, Ge G, Zhang N, Huang E, Nakamura LV, Minor M et al. Separase 53 TCGARN. Integrated genomic analyses of ovarian carcinoma. Nature 2011; 474: loss of function cooperates with the loss of p53 in the initiation and progression 609–615. of T- and B-cell lymphoma, leukemia and aneuploidy in mice. PLoS One 2011; 6: 54 Curtis C, Shah SP, Chin SF, Turashvili G, Rueda OM, Dunning MJ et al. The e22167. genomic and transcriptomic architecture of 2,000 breast tumours reveals novel 26 Celton-Morizur S, Desdouets C. Polyploidization of liver cells. Adv Exp Med Biol subgroups. Nature 2012; 486: 346–352. 2010; 676: 123–135. 55 Puente XS, Pinyol M, Quesada V, Conde L, Ordonez GR, Villamor N et al. Whole- 27 Duncan AW, Taylor MH, Hickey RD, Hanlon Newell AE, Lenzi ML, Olson SB et al. genome sequencing identifies recurrent mutations in chronic lymphocytic leu- The ploidy conveyor of mature hepatocytes as a source of genetic variation. kaemia. Nature 2011; 475: 101–105. Nature 2010; 467: 707–710. 56 Baker DJ, Jeganathan KB, Malureanu L, Perez-Terzic C, Terzic A, Van Deursen JM. 28 Weaver BA, Silk AD, Montagna C, Verdier-Pinard P, Cleveland DW. Early aging-associated phenotypes in Bub3/Rae1 haploinsufficient mice. JCell Aneuploidy acts both oncogenically and as a tumor suppressor. Cancer Cell 2007; Biol 2006; 172: 529–540. 11: 25–36. 57 Schliekelman M, Cowley DO, O’Quinn R, Oliver TG, Lu L, Salmon ED et al. 29 Duijf PH, Schultz N, Benezra R. Cancer cells preferentially lose small chromo- Impaired Bub1 function in vivo compromises tension-dependent checkpoint somes. Int J Cancer (e-pub ahead of print 5 November 2012; doi:10.1002/ function leading to aneuploidy and tumorigenesis. Cancer Res 2009; 69: 45–54. ijc.27924). 58 Hasle H, Clemmensen IH, Mikkelsen M. Risks of leukaemia and solid tumours in 30 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; individuals with Down’s syndrome. Lancet 2000; 355: 165–169. 144: 646–674. 59 Satge D, Sasco AJ, Carlsen NL, Stiller CA, Rubie H, Hero B et al. A lack of 31 Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. neuroblastoma in Down syndrome: a study from 11 European countries. Cancer Nature 1998; 396: 643–649. Res 1998; 58: 448–452. 32 Pavelka N, Rancati G, Zhu J, Bradford WD, Saraf A, Florens L et al. Aneuploidy 60 Yang Q, Rasmussen SA, Friedman JM. Mortality associated with Down’s confers quantitative proteome changes and phenotypic variation in budding syndrome in the USA from 1983 to 1997: a population-based study. Lancet 2002; yeast. Nature 2010; 468: 321–325. 359: 1019–1025. 33 Rodriguez-Paredes M, Esteller M. Cancer epigenetics reaches mainstream 61 Swerdlow AJ, Schoemaker MJ, Higgins CD, Wright AF, Jacobs PA. Cancer inci- oncology. Nat Med 2011; 17: 330–339. dence and mortality in men with Klinefelter syndrome: a cohort study. J Natl 34 Jones PA, Baylin SB. The epigenomics of cancer. Cell 2007; 128: 683–692. Cancer Inst 2005; 97: 1204–1210. 35 Visconti R, Grieco D. New insights on oxidative stress in cancer. Curr Opin Drug 62 Hasle H, Mellemgaard A, Nielsen J, Hansen J. Cancer incidence in men with Discov Devel 2009; 12: 240–245. Klinefelter syndrome. Br J Cancer 1995; 71: 416–420. 36 Li M, Fang X, Baker DJ, Guo L, Gao X, Wei Z et al. The ATM-p53 pathway 63 Torres EM, Dephoure N, Panneerselvam A, Tucker CM, Whittaker CA, suppresses aneuploidy-induced tumorigenesis. Proc Natl Acad Sci USA 2010; 107: Gygi SP et al. Identification of aneuploidy-tolerating mutations. Cell 2010; 143: 14188–14193. 71–83. 37 Wang Z, Yu R, Melmed S. Mice lacking pituitary tumor transforming gene show 64 Lakovschek IC, Streubel B, Ulm B. Natural outcome of trisomy 13, trisomy 18, and testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aber- triploidy after prenatal diagnosis. Am J Med Genet A 2011; 155A: 2626–2633. rant cell cycle progression, and premature centromere division. Mol Endocrinol 65 Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2001; 15: 1870–1879. 2010; 140: 883–899.

Oncogene (2013) 4727 – 4736 & 2013 Macmillan Publishers Limited The cancer biology of W-CIN PHG Duijf and R Benezra 4735 66 Dunn GP, Bruce AT, Ikeda H, Old LJ, Schreiber RD. Cancer immunoediting: from 93 Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K et al. DNA damage immunosurveillance to tumor escape. Nat Immunol 2002; 3: 991–998. response as a candidate anti-cancer barrier in early human tumorigenesis. 67 Chang CC, Ogino T, Mullins DW, Oliver JL, Yamshchikov GV, Bandoh N et al. Nature 2005; 434: 864–870. Defective human leukocyte antigen class I-associated antigen presentation 94 Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through a tragedy: mitotic caused by a novel beta2-microglobulin loss-of-function in melanoma cells. J Biol catastrophe. Cell Death Differ 2008; 15: 1153–1162. Chem 2006; 281: 18763–18773. 95 Manchado E, Guillamot M, Malumbres M. Killing cells by targeting mitosis. Cell 68 Sanda MG, Restifo NP, Walsh JC, Kawakami Y, Nelson WG, Pardoll DM et al. Death Differ 2012; 19: 369–377. Molecular characterization of defective antigen processing in human prostate 96 Gascoigne KE, Taylor SS. Cancer cells display profound intra- and interline cancer. J Natl Cancer Inst 1995; 87: 280–285. variation following prolonged exposure to antimitotic drugs. Cancer Cell 2008; 69 Khong HT, Restifo NP. Natural selection of tumor variants in the generation of 14: 111–122. ‘tumor escape’ phenotypes. Nat Immunol 2002; 3: 999–1005. 97 Terrano DT, Upreti M, Chambers TC. Cyclin-dependent kinase 1-mediated Bcl-xL/ 70 Schreiber RD, Old LJ, Smyth MJ. Cancer immunoediting: integrating immunity’s Bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and roles in cancer suppression and promotion. Science 2011; 331: 1565–1570. apoptosis. Mol Cell Biol 2010; 30: 640–656. 71 DuPage M, Mazumdar C, Schmidt LM, Cheung AF, Jacks T. Expression of 98 Thompson SL, Compton DA. Proliferation of aneuploid human cells is limited by tumour-specific antigens underlies cancer immunoediting. Nature 2012; 482: a p53-dependent mechanism. J Cell Biol 2010; 188: 369–381. 405–409. 99 Mikule K, Delaval B, Kaldis P, Jurcyzk A, Hergert P, Doxsey S. Loss of 72 Yamada HY, Yao Y, Wang X, Zhang Y, Huang Y, Dai W et al. Haploinsufficiency of centrosome integrity induces p38-p53-p21-dependent G1-S arrest. Nat Cell Biol SGO1 results in deregulated centrosome dynamics, enhanced chromosomal 2007; 9: 160–170. instability and colon tumorigenesis. Cell Cycle 2012; 11: 479–488. 100 Lanni JS, Jacks T. Characterization of the p53-dependent postmitotic checkpoint 73 Oshima M, Dinchuk JE, Kargman SL, Oshima H, Hancock B, Kwong E et al. following spindle disruption. Mol Cell Biol 1998; 18: 1055–1064. Suppression of intestinal polyposis in Apc delta716 knockout mice by inhibition 101 Minn AJ, Boise LH, Thompson CB. Expression of Bcl-xL and loss of p53 can of cyclooxygenase 2 (COX-2). Cell 1996; 87: 803–809. cooperate to overcome a cell cycle checkpoint induced by mitotic spindle 74 Chulada PC, Thompson MB, Mahler JF, Doyle CM, Gaul BW, Lee C et al. Genetic damage. Genes Dev 1996; 10: 2621–2631. disruption of Ptgs-1, as well as Ptgs-2, reduces intestinal tumorigenesis in Min 102 Reinhardt HC, Schumacher B. The p53 network: cellular and mice. Cancer Res 2000; 60: 4705–4708. systemic DNA damage responses in aging and cancer. Trends Genet 2012; 28: 75 Rodier F, Coppe JP, Patil CK, Hoeijmakers WA, Munoz DP, Raza SR et al. Persistent 128–136. DNA damage signalling triggers senescence-associated inflammatory cytokine 103 Deng Y, Chan SS, Chang S. Telomere dysfunction and tumour suppression: the secretion. Nat Cell Biol 2009; 11: 973–979. senescence connection. Nat Rev Cancer 2008; 8: 450–458. 76 Zheng L, Dai H, Zhou M, Li M, Singh P, Qiu J et al. Fen1 mutations result in 104 Burds AA, Lutum AS, Sorger PK. Generating chromosome instability through the autoimmunity, chronic inflammation and cancers. Nat Med 2007; 13: 812–819. simultaneous deletion of Mad2 and p53. Proc Natl Acad Sci USA 2005; 102: 77 Joyce JA, Pollard JW. Microenvironmental regulation of metastasis. Nat Rev 11296–11301. Cancer 2009; 9: 239–252. 105 Greaves M, Maley CC. Clonal evolution in cancer. Nature 2012; 481: 306–313. 78 Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A. Cancer-related inflam- 106 Hanahan D, Coussens LM. Accessories to the crime: functions of cells recruited to mation, the seventh hallmark of cancer: links to genetic instability. Carcinogen- the tumor microenvironment. Cancer Cell 2012; 21: 309–322. esis 2009; 30: 1073–1081. 107 Ostrand-Rosenberg S. Immune surveillance: a balance between protumor and 79 Lonkar P, Dedon PC. Reactive species and DNA damage in chronic inflammation: antitumor immunity. Curr Opin Genet Dev 2008; 18: 11–18. reconciling chemical mechanisms and biological fates. Int J Cancer 2011; 128: 108 Mason SD, Joyce JA. Proteolytic networks in cancer. Trends Cell Biol 2011; 21: 1999–2009. 228–237. 80 Aivaliotis IL, Pateras IS, Papaioannou M, Glytsou C, Kontzoglou K, Johnson EO 109 Lewis CE, De Palma M, Naldini L. Tie2-expressing monocytes and tumor et al. How do cytokines trigger genomic instability? J Biomed Biotechnol 2012; angiogenesis: regulation by hypoxia and angiopoietin-2. Cancer Res 2007; 67: 2012: 536761. 8429–8432. 81 Sotillo R, Schvartzman JM, Socci ND, Benezra R. Mad2-induced chromosome 110 Seremet T, Brasseur F, Coulie PG. Tumor-specific antigens and immunologic instability leads to lung tumour relapse after oncogene withdrawal. Nature 2010; adjuvants in cancer immunotherapy. Cancer J 2011; 17: 325–330. 464: 436–440. 111 Granot Z, Henke E, Comen EA, King TA, Norton L, Benezra R. Tumor 82 Galluzzi L, Vanden Berghe T, Vanlangenakker N, Buettner S, Eisenberg T, entrained neutrophils inhibit seeding in the premetastatic lung. Cancer Cell 2011; Vandenabeele P et al. Programmed necrosis from molecules to health and 20: 300–314. disease. Int Rev Cell Mol Biol 2011; 289:1–35. 112 Png KJ, Halberg N, Yoshida M, Tavazoie SF. A microRNA regulon that mediates 83 Kimmelman AC. The dynamic nature of autophagy in cancer. Genes Dev 2011; endothelial recruitment and metastasis by cancer cells. Nature 2012; 481: 25: 1999–2010. 190–194. 84 White E. Deconvoluting the context-dependent role for autophagy in cancer. Nat 113 Carter SL, Eklund AC, Kohane IS, Harris LN, Szallasi Z. A signature of chromo- Rev Cancer 2012; 12: 401–410. somal instability inferred from gene expression profiles predicts clinical outcome 85 Baker DJ, Jeganathan KB, Cameron JD, Thompson M, Juneja S, Kopecka A et al. in multiple human cancers. Nat Genet 2006; 38: 1043–1048. BubR1 insufficiency causes early onset of aging-associated phenotypes and 114 Gutenberg A, Gerdes JS, Jung K, Sander B, Gunawan B, Bock HC et al. High infertility in mice. Nat Genet 2004; 36: 744–749. chromosomal instability in brain metastases of colorectal carcinoma. Cancer 86 Babu JR, Jeganathan KB, Baker DJ, Wu X, Kang-Decker N, Van Deursen JM. Rae1 Genet Cytogenet 2010; 198:47–51. is an essential mitotic checkpoint regulator that cooperates with Bub3 to pre- 115 Bhattacharya A, Roy R, Snijders AM, Hamilton G, Paquette J, Tokuyasu T et al. vent chromosome missegregation. J Cell Biol 2003; 160: 341–353. Two distinct routes to oral cancer differing in genome instability and risk for 87 Chesnokova V, Kovacs K, Castro AV, Zonis S, Melmed S. Pituitary hypoplasia in cervical node metastasis. Clin Cancer Res 2011; 17: 7024–7034. Pttg À / À mice is protective for Rb þ / À pituitary tumorigenesis. Mol Endocrinol 116 Kreike B, Halfwerk H, Armstrong N, Bult P, Foekens JA, Veltkamp SC et al. Local 2005; 19: 2371–2379. recurrence after breast-conserving therapy in relation to gene expression pat- 88 Li M, Fang X, Wei Z, York JP, Zhang P. Loss of spindle assembly checkpoint- terns in a large series of patients. Clin Cancer Res 2009; 15: 4181–4190. mediated inhibition of Cdc20 promotes tumorigenesis in mice. J Cell Biol 2009; 117 Smid M, Hoes M, Sieuwerts AM, Sleijfer S, Zhang Y, Wang Y et al. Patterns and 185: 983–994. incidence of chromosomal instability and their prognostic relevance in breast 89 Andreassen PR, Lohez OD, Lacroix FB, Margolis RL. Tetraploid state induces p53- cancer subtypes. Breast Cancer Res Treat 2011; 128: 23–30. dependent arrest of nontransformed mammalian cells in G1. Mol Biol Cell 2001; 118 Skirnisdottir I, Sorbe B, Karlsson M, Seidal T. Prognostic importance of DNA 12: 1315–1328. ploidy and p53 in early stages of epithelial ovarian carcinoma. Int J Oncol 2001; 90 Rieder CL, Maiato H. Stuck in division or passing through: what 19: 1295–1302. happens when cells cannot satisfy the spindle assembly checkpoint. Dev Cell 119 Warth A, Herpel E, Krysa S, Hoffmann H, Schnabel PA, Schirmacher P et al. 2004; 7: 637–651. Chromosomal instability is more frequent in metastasized than in non-metas- 91 Zhang D, Shimizu T, Araki N, Hirota T, Yoshie M, Ogawa K et al. Aurora A tasized pulmonary carcinoids but is not a reliable predictor of metastatic overexpression induces cellular senescence in mammary gland hyperplastic potential. Exp Mol Med 2009; 41: 349–353. tumors developed in p53-deficient mice. Oncogene 2008; 27: 4305–4314. 120 Schardt JA, Meyer M, Hartmann CH, Schubert F, Schmidt-Kittler O, Fuhrmann C 92 Bitomsky N, Hofmann TG. Apoptosis and autophagy: Regulation of et al. Genomic analysis of single cytokeratin-positive cells from bone apoptosis by DNA damage signalling - roles of p53, p73 and HIPK2. FEBS J 2009; marrow reveals early mutational events in breast cancer. Cancer Cell 2005; 8: 276: 6074–6083. 227–239.

& 2013 Macmillan Publishers Limited Oncogene (2013) 4727 – 4736 The cancer biology of W-CIN PHG Duijf and R Benezra 4736 121 Schmidt-Kittler O, Ragg T, Daskalakis A, Granzow M, Ahr A, Blankenstein TJ et al. 139 Lu LY, Wood JL, Ye L, Minter-Dykhouse K, Saunders TL, Yu X et al. Aurora A is From latent disseminated cells to overt metastasis: genetic analysis of systemic essential for early embryonic development and tumor suppression. J Biol Chem breast cancer progression. Proc Natl Acad Sci USA 2003; 100: 7737–7742. 2008; 283: 31785–31790. 122 Stoecklein NH, Hosch SB, Bezler M, Stern F, Hartmann CH, Vay C et al. Direct 140 Iwanaga Y, Chi YH, Miyazato A, Sheleg S, Haller K, Peloponese Jr JM et al. genetic analysis of single disseminated cancer cells for prediction of outcome Heterozygous deletion of mitotic arrest-deficient protein 1 (MAD1) increases the and therapy selection in esophageal cancer. Cancer Cell 2008; 13: 441–453. incidence of tumors in mice. Cancer Res 2007; 67: 160–166. 123 Weckermann D, Polzer B, Ragg T, Blana A, Schlimok G, Arnholdt H et al. Perio- 141 Michel LS, Liberal V, Chatterjee A, Kirchwegger R, Pasche B, Gerald W et al. MAD2 perative activation of disseminated tumor cells in bone marrow of patients with haplo-insufficiency causes premature anaphase and chromosome instability in prostate cancer. J Clin Oncol 2009; 27: 1549–1556. mammalian cells. Nature 2001; 409: 355–359. 124 McClelland SE, Burrell RA, Swanton C. Chromosomal instability: a composite phe- 142 Lu LY, Wood JL, Minter-Dykhouse K, Ye L, Saunders TL, Yu X et al. Polo-like kinase notype that influences sensitivity to chemotherapy. Cell Cycle 2009; 8: 3262–3266. 1 is essential for early embryonic development and tumor suppression. Mol Cell 125 Duesberg P, Stindl R, Hehlmann R. Explaining the high mutation rates of cancer Biol 2008; 28: 6870–6876. cells to drug and multidrug resistance by chromosome reassortments that are 143 Ko MA, Rosario CO, Hudson JW, Kulkarni S, Pollett A, Dennis JW et al. Plk4 catalyzed by aneuploidy. Proc Natl Acad Sci USA 2000; 97: 14295–14300. haploinsufficiency causes mitotic infidelity and carcinogenesis. Nat Genet 2005; 126 Swanton C, Nicke B, Schuett M, Eklund AC, Ng C, Li Q et al. Chromosomal 37: 883–888. instability determines taxane response. Proc Natl Acad Sci USA 2009; 106: 144 Jeganathan K, Malureanu L, Baker DJ, Abraham SC, Van Deursen JM. Bub1 8671–8676. mediates cell death in response to chromosome missegregation and acts to 127 Bedard PL, Di Leo A, Piccart-Gebhart MJ. Taxanes: optimizing adjuvant che- suppress spontaneous tumorigenesis. J Cell Biol 2007; 179: 255–267. motherapy for early-stage breast cancer. Nat Rev Clin Oncol 2010; 7: 22–36. 145 Ricke RM, Jeganathan KB, Van Deursen JM. Bub1 overexpression induces 128 Janssen A, Kops GJ, Medema RH. Elevating the frequency of chromosome mis- aneuploidy and tumor formation through Aurora B kinase hyperactivation. JCell segregation as a strategy to kill tumor cells. Proc Natl Acad Sci USA 2009; 106: Biol 2011; 193: 1049–1064. 19108–19113. 146 Diaz-Rodriguez E, Sotillo R, Schvartzman JM, Benezra R. Hec1 overexpression 129 Rebacz B, Larsen TO, Clausen MH, Ronnest MH, Loffler H, Ho AD et al. Identifi- hyperactivates the mitotic checkpoint and induces tumor formation in vivo. Proc cation of griseofulvin as an inhibitor of centrosomal clustering in a phenotype- Natl Acad Sci USA 2008; 105: 16719–16724. based screen. Cancer Res 2007; 67: 6342–6350. 147 Van Ree JH, Jeganathan KB, Malureanu L, Van Deursen JM. Overexpression of the 130 Karna P, Rida PC, Pannu V, Gupta KK, Dalton WB, Joshi H et al. A novel micro- E2 ubiquitin-conjugating enzyme UbcH10 causes chromosome missegregation tubule-modulating noscapinoid triggers apoptosis by inducing spindle multi- and tumor formation. J Cell Biol 2010; 188: 83–100. polarity via centrosome amplification and declustering. Cell Death Differ 2011; 148 Janisson-Dargaud D, Durlach A, Lorenzato M, Grange F, Bernard P, Birembaut P. 18: 632–644. Aneuploidy, but not Ki-67 or EGFR expression, is associated with recurrences in 131 Kwon M, Godinho SA, Chandhok NS, Ganem NJ, Azioune A, Thery M et al. basal cell carcinoma. J Cutan Pathol 2008; 35: 916–921. Mechanisms to suppress multipolar divisions in cancer cells with extra centro- 149 Susini T, Olivieri S, Molino C, Amunni G, Rapi S, Taddei G et al. DNA ploidy is somes. Genes Dev 2008; 22: 2189–2203. stronger than lymph node metastasis as prognostic factor in cervical carcinoma: 132 Wu G, Qiu XL, Zhou L, Zhu J, Chamberlin R, Lau J et al. Small molecule targeting 10-year results of a prospective study. Int J Gynecol Cancer 2011; 21: 678–684. the Hec1/Nek2 mitotic pathway suppresses tumor cell growth in culture and in 150 Walther A, Houlston R, Tomlinson I. Association between chromo- animal. Cancer Res 2008; 68: 8393–8399. somal instability and prognosis in colorectal cancer: a meta-analysis. Gut 2008; 133 Tang YC, Williams BR, Siegel JJ, Amon A. Identification of aneuploidy-selective 57: 941–950. antiproliferation compounds. Cell 2011; 144: 499–512. 151 Lagarde P, Perot G, Kauffmann A, Brulard C, Dapremont V, Hostein I et al. Mitotic 134 Ishida S, Huang E, Zuzan H, Spang R, Leone G, West M et al. Role for E2F in checkpoints and chromosome instability are strong predictors of clinical control of both DNA replication and mitotic functions as revealed from DNA outcome in gastrointestinal stromal tumors. Clin Cancer Res 2012; 18: 826–838. microarray analysis. Mol Cell Biol 2001; 21: 4684–4699. 152 Choi CM, Seo KW, Jang SJ, Oh YM, Shim TS, Kim WS et al. Chromosomal 135 Polager S, Kalma Y, Berkovich E, Ginsberg D. E2Fs up-regulate expression of instability is a risk factor for poor prognosis of adenocarcinoma of the lung: genes involved in DNA replication, DNA repair and mitosis. Oncogene 2002; 21: Fluorescence in situ hybridization analysis of paraffin-embedded tissue from 437–446. Korean patients. Lung Cancer 2009; 64: 66–70. 136 Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young RA et al. E2F integrates 153 Ehlers JP, Worley L, Onken MD, Harbour JW. Integrative genomic analysis of cell cycle progression with DNA repair, replication, and G(2)/M checkpoints. aneuploidy in uveal melanoma. Clin Cancer Res 2008; 14: 115–122. Genes Dev 2002; 16: 245–256. 154 Sato H, Uzawa N, Takahashi K, Myo K, Ohyama Y, Amagasa T. Prognostic utility of 137 Zhou Y, Mehta KR, Choi AP, Scolavino S, Zhang X. DNA damage-induced inhi- chromosomal instability detected by fluorescence in situ hybridization in fine- bition of securin expression is mediated by p53. J Biol Chem 2003; 278: 462–470. needle aspirates from oral squamous cell carcinomas. BMC Cancer 2010; 10: 182. 138 Oikawa T, Okuda M, Ma Z, Goorha R, Tsujimoto H, Inokuma H et al. Transcrip- 155 Pinto AE, Silva GL, Pereira T, Cabrera RA, Santos JR, Leite V. S-phase fraction and tional control of BubR1 by p53 and suppression of centrosome amplification by ploidy as predictive markers in primary disease and recurrence of papillary BubR1. Mol Cell Biol 2005; 25: 4046–4061. thyroid carcinoma. Clin Endocrinol (Oxf) 2012; 77: 302–309.

Oncogene (2013) 4727 – 4736 & 2013 Macmillan Publishers Limited