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Does aneuploidy cause cancer? Beth AA Weaver and Don W Cleveland Aneuploidy has been recognized as a common characteristic of cancer cells for >100 years. Aneuploidy frequently results from errors of the mitotic checkpoint, the major cell cycle control mechanism that acts to prevent chromosome missegregation. The mitotic checkpoint is often compromised in human tumors, although not as a result of germline mutations in genes encoding checkpoint proteins. Less obviously, aneuploidy of whole chromosomes rapidly results from mutations in genes encoding several tumor suppressors and DNA mismatch repair proteins, suggesting cooperation between mechanisms of tumorigenesis that were previously thought to act independently. Cumulatively, the current evidence suggests that aneuploidy promotes tumorigenesis, at least at low frequency, but a definitive test has not yet been reported. Addresses Ludwig Institute for Cancer Research, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0670, USA Corresponding author: Cleveland, Don W ([email protected]) was already well recognized 100 years ago. The prevalence of aneuploidy in cancer cells, and its relatively low incidence in normal cells, led the German zoologist and cytologist Theodor Boveri to propose aneuploidy as a cause of tumorigenesis in 1902 [2] and 1914 [3]. Boveri observed that sea urchin embryos manipulated to undergo mitosis in the presence of multipolar spindles produced aneuploid progeny and suggested that tumors arise from normal cells that have become aneuploid as a result of passage through an aberrant mitosis. With the discovery of oncogenes and tumor suppressors in the 1970s and 1980s, aneuploidy-induced loss of heterozygosity (LOH) of tumor suppressor genes seemed to offer a simple, direct molecular mechanism for Boveri’s hypothesis. This has not, however, resulted in consensus. Some have argued aneuploidy to be irrelevant to tumor initiation [4], while others have argued it to be a completely benign side-effect of transformation [5], and an additional hypothesis suggests that aneuploidy contributes to tumor progression but not tumor initiation [6]. Current Opinion in Cell Biology 2006, 18:658–667 This review comes from a themed issue on Cell division, growth and death Edited by Bill Earnshaw and Yuri Lazebnik Available online 12th October 2006 0955-0674/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.ceb.2006.10.002 Introduction: aneuploidy correlates with tumorigenicity Aneuploidy, an aberrant chromosome number that deviates from a multiple of the haploid, is a remarkably common feature of human cancers (Table 1, compiled from the Mitelman database of cancer chromosomes [1]). Even haematological cancers, which typically maintain a stable, near-diploid chromosome number, have frequently gained or lost one or a few chromosomes (Table 1). Near-tetraploid karyotypes, which result from missegregation of single chromosomes before or after doubling of the genome (usually from failure of cytokinesis), are also observed in solid tumors, although not as commonly as near-diploid karyotypes (Table 1). Although methodological advances have permitted significantly more refined analysis of the chromosomal abnormalities in cancer cells in recent years, the abundance of aneuploid chromosome contents in tumor cells Current Opinion in Cell Biology 2006, 18:658–667 Genetic instability due to mutations in mismatch repair (MMR) enzymes has become well established as a causative mechanism for tumorigenesis. Biallelic mutations in MMR genes lead to expansion and contraction of short, repetitive sequences of DNA known as microsatellites, causing microsatellite instability (MIN). Germline mutations in one of five MMR genes, predominantly MSH2 and MLH1, are implicated in hereditary nonpolyposis colon cancer (HNPCC), and predispose individuals to a variety of cancers. HNPCC patients have an 80% lifetime risk of colorectal cancer and a 30–50% chance of endometrial cancer [7]. However, although MIN occurs in 90% of cancers in HNPCC patients, it is found in only 15% of sporadic cancers of the colon/rectum [7,8]. Aneuploidy represents a second, more common form of genetic abnormality found in human cancers. An important cause of aneuploidy is chromosomal instability (CIN), a form of genetic instability in which the gain or loss of entire chromosomes is elevated, producing an evolving, unstable karyotype. Although many aneuploid cells exhibit CIN, aneuploid karyotypes may also be stably maintained, as observed in some haematological cancers. On the basis of the cumulative evidence, it is likely that aneuploidy promotes tumorigenesis, at least at low frequency. However, a definitive empirical test of this hypothesis has not yet been reported, and alternative interpretations cannot be excluded. Here we summarize www.sciencedirect.com Does aneuploidy cause cancer? Weaver and Cleveland 659 Table 1 The majority of human cancers are near-diploid. Number of tumors that have not gained Number of aneuploid tumors Number of aneuploid tumors with a or lost chromosomes * with a near-diploid number of near- tetraploid number of chromosomes (68) chromosomes (69) Solid tumors Astrocytoma, grade III–IV Basal cell carcinoma Breast cancer Cervical cancer Colon adenocarcinoma Embryonal rhabdomyosarcoma Hepatoblastoma Leiomyosarcoma Lung cancer Malignant melanoma Neuroblastoma Osteosarcoma Ovarian cancer Prostate cancer Retinoblastoma Squamous cell carcinoma Teratoma Percent of solid tumors (n = 2780) Haematopoietic cancers Acute myeloid leukemia Adult T-cell lymphoma/leukemia B-prolymphocytic leukemia Burkitt lymphoma/leukemia Chronic myeloid leukemia Follicular lymphoma Hodgkins disease Multiple myeloma T-prolymphocytic leukemia Percent of haematopoietic cancers (n = 1973) Percent of solid and haematopoietic cancers (n = 4753) 10 23 31 4 1 9 17 7 36 30 28 6 5 16 10 12 3 8.9% 228 75 140 51 124 53 80 68 119 138 109 86 158 141 111 149 166 71.8% 62 4 29 29 19 12 3 34 45 31 58 59 37 43 1 39 31 19.3% 88 21 20 86 90 55 26 64 25 24.1% 207 224 72 75 110 228 129 217 111 69.6% 3 8 1 2 0 17 77 17 0 6.3% 15.2% 70.9% 13.9% * These cancer cells have 46 chromosomes containing translocations, inversions, deletions and/or additions but have not gained or lost entire chromosomes. the current evidence for and against a role for aneuploidy and CIN in tumorigenesis. The mitotic checkpoint: the major cell cycle checkpoint guarding against aneuploidy Beginning with the drawings of aberrant mitosis in cancer cells published by David van Hansemann in 1890 [9], defects during mitosis have been implicated as a major contributor to aneuploidy and CIN. The major cell cycle control mechanism that acts during mitosis is the mitotic checkpoint, also known as the spindle assembly checkpoint. The mitotic checkpoint prevents chromosome missegregation and aneuploidy by inhibiting the irreversible transition to anaphase until all of the replicated chromosomes have made productive attachments to spindle microtubules (Figure 1a, panel 2). Mitotic checkpoint proteins are recruited to the microtubule attachment sites (kinetochores) of unattached chromosomes, where they generate an at least partially diffusible signal that inhibits the anaphase promoting complex/cyclosome (APC/C). www.sciencedirect.com The APC/C is an E3 ubiquitin ligase that ubiquitinates substrates whose degradation is required for anaphase onset (securin) and mitotic exit (cyclin B). Destruction of securin after its ubiquitination frees its binding partner separase, while simultaneous loss of cyclin B-dependent Cdk1 kinase activity leads to dephosphorylation of separase. Both events activate separase, which then cleaves the cohesins that hold replicated chromosomes together and initiates anaphase (reviewed in [10,11]). Even a single unattached kinetochore can be sufficient to delay anaphase onset [12,13]. Treatment of cells with drugs producing depolymerization of spindle microtubules causes mitotic arrest as a result of activation of the mitotic checkpoint. >70% of many types of asynchronously cycling cells with an intact mitotic checkpoint accumulate in mitosis after 12–24 h treatment with microtubule poisons. Complete absence of the mitotic checkpoint leads to rapid cell-autonomous lethality due to massive chromosome missegregation Current Opinion in Cell Biology 2006, 18:658–667 660 Cell division, growth and death Figure 1 Mechanisms generating aneuploidy. (a) Wild type division producing identical diploid progeny in a hypothetical cell containing four chromosomes. (b–g) Mitotic errors that produce aneuploid progeny. For each part (a–g), from left to right, the first image depicts a diploid (a–f) or tetraploid (g) cell in interphase. The second panel represents metaphase, the stage of mitosis when all chromosomes have aligned in the middle of the mitotic spindle. Some errors (b,c,f) prevent full alignment of chromosomes in metaphase. The third image depicts chromosome segregation in anaphase (a–e,g). The last image represents the daughter cells that were produced, now in G1. The ploidy of the initial cells and their progeny is shown. [14,15]. However, an impaired mitotic checkpoint response, or more precisely an impaired ability to sustain mitotic checkpoint signaling, has been observed in many human tumor cell lines treated with microtubule poisons (Table 2), as evidenced by a decreased percentage of cells in mitosis and/or a decreased length of arrest. In 1998, Vogelstein and colleagues reported mutations in two mitotic checkpoint genes in a small subset of colorectal cancer cell lines [16]. This finding launched an extensive search for additional mutations in mitotic checkpoint genes. While mutations of several checkpoint proteins have been found in multiple cancer types, these mutations are not common (Table 3). This is not completely surprising, as a large number of gene products contribute to the mitotic checkpoint response (including, but not limited to, BUB1, BUBR1, BUB3, MAD1, MAD2, MPS1, MAPK, ROD, ZW10, Zwint, CENP-E Current Opinion in Cell Biology 2006, 18:658–667 and Aurora B) and mutation in any one could lead to weakening of the checkpoint. Additionally, mutations leading to complete inactivation of the mitotic checkpoint would be eliminated by cell death. In comparison to direct mutation, alterations in the level of expression of mitotic checkpoint genes appear to occur much more commonly (Table 3). Both decreases and, more surprisingly, increases in expression have been reported. Lower expression of checkpoint proteins would be predicted to lead to CIN and aneuploidy, at least in components whose accumulation was rate-limiting for checkpoint signaling at individual kinetochores. Consistent with this, mice that are heterozygous for the checkpoint proteins MAD2, BUBR1 and BUB3 exhibit an impaired mitotic checkpoint response and develop aneuploidy in vitro and in vivo [17–19]. The mechanism by which overexpression of individual checkpoint proteins www.sciencedirect.com Does aneuploidy cause cancer? Weaver and Cleveland 661 Table 2 Frequent impairment of the mitotic checkpoint in human cancers. Tumor type Frequency with impaired checkpoint Percentage frequency Notes Reference Adult T-cell leukemia Breast Breast Breast Colorectal Head and neck Hepatocellular carcinoma 6 7 1 7 3 6 5 100% 78% 100% 70% 100% 100% 62% Reduced expression of MAD1 in all, MAD2 in two No mutations in BUB1, BUBR1 or CDC20 [66] [67] [68] [67] [16] [69] [70] Hepatoma Lung Lung adenocarcinoma 6 of 11 4 of 9 1 of 2 55% 44% 50% Reduced expression of Mad2 in all No mutations in MAD2 or CDC20 Levels of MAD2 and BUB1 similar in both lines [71] [72] [73] Nasopharyngeal carcinoma 2 of 5 40% [74] Ovarian 3 of 7 43% No mutations in MAD2, MAD1 or CDC20. Reduced expression of MAD1 and MAD2 in both Reduced expression of MAD2 and MAD1 in all three Pancreatic Rhabdomyosarcoma Thyroid 3 of 3 1 of 1 4 of 8 100% 100% 50% MAD2 expression at wild-type levels Reduced BUBR1 expression in 3 of 4 of of of of of of of 6 9 1 10 3 6 8 can provoke aneuploidy is not so clear. Two possibilities are feasible. Overexpression of a single component, such as MAD2, could disrupt signaling by trapping limiting components in partial, non-productive signaling complexes. Alternatively, and perhaps more simply, increased levels of components that can directly bind to APC/C and/or its activator Cdc20 may provoke sustained arrest, as has been seen for Mad2 [20]. Escape from this type of arrest may occur without cytokinesis, which would produce tetraploid cells with two centrosomes that could produce aneuploid progeny in a subsequent multipolar mitosis (see below). Neither scenario has been demonstrated directly. Mechanisms generating aneuploidy Multiple defects occurring during mitosis can lead to the production of aneuploid cells. Mitotic checkpoint errors can give rise to near-diploid aneuploidy or to cell death, depending on the extent of the remaining checkpoint signal. Weakening of the mitotic checkpoint due to reduction in levels of one or more checkpoint components leads to near-diploid aneuploidy from nondisjunction errors, in which both copies of one or a few replicated chromosomes are deposited in the same daughter cell (Figure 1b). Conversely, complete inactivation of the mitotic checkpoint resulting from elimination of a key component such as MAD2 or BUBR1 leads to rampant aneuploidy and massive chromosome missegregation (Figure 1c). This mechanism is not expected to make a major contribution to tumorigenesis, as cells with an inactive checkpoint die within six divisions as a result of rampant aneuploidy [14]. Mitotic errors leading to aneuploidy can also occur despite intact mitotic checkpoint signaling. These include missegregation events that occur when the kinetochore of a single replicated chromosome becomes attached to microtubules from both spindle poles, a situation known as www.sciencedirect.com Reduced expression of Mad2 CIN correlated with lack of a mitotic checkpoint [75] [76] [68] [77] merotelic attachment. Since the chromosome is attached and under tension, no mitotic checkpoint signal is generated [21]. The inappropriate attachment is often resolved, but in some cases it produces a lagging chromosome with a stretched kinetochore [22] that either remains in the mitotic midzone, becoming excluded from both daughter cells during cytokinesis (Figure 1d), or is segregated into one daughter, where it may form a micronucleus. Segregation errors resulting from multipolar spindles also cannot be prevented by the mitotic checkpoint, because the chromosomes make productive attachments to two of the available poles. When three or more daughter cells are created by multiple cytokinetic furrows, aneuploid progeny are produced (Figure 1e). By contrast, cells containing monopolar spindles (resulting from failure of centrosome duplication or inhibition of the apparatus required for centrosome separation) have at least a few unattached chromosomes and will undergo sustained mitotic checkpoint-dependent arrest. Some of these cells die during the prolonged mitotic arrest and others undergo adaptation. Adaptation is a poorly understood (and frequently poorly defined) process that occurs when cells exit mitosis after long-term mitotic arrest without undergoing cytokinesis to produce one tetraploid G1 cell, despite the fact that the cell still contains unattached chromosomes and the mitotic checkpoint has not been satisfied (Figure 1f). Weakening of mitotic checkpoint signaling at individual kinetochores shortens the time a cell remains arrested before adapting and may contribute to the survival of cells treated with microtubule poisons. It is not yet known what determines whether cells will die or adapt after long-term mitotic arrest. Recently, a surprising proposal was made that nondisjunction produces tetraploid cells instead of the expected Current Opinion in Cell Biology 2006, 18:658–667 662 Cell division, growth and death Table 3 Genes preventing aneuploidy that are mutated and/or misregulated in human cancers. Gene Primary function Mutated in, frequency, reference Upregulated in, frequency, reference Downregulated in, frequency, reference BUB1 Mitotic checkpoint Colorectal, 2/19, [16] Colorectal, 1/31, [78] Colorectal, 1/1, [76] Leukemia (ATLL), 4/10, [79] Leukemia (T lymphoblastic), 2/2, [80] Lung, 1/60, [81] Lung, 1/88, [82] Thyroid, 1/27, [77] Barrett’s oesophagus (precancercous), 12/33, [48] Breast, 20/21, [83] Gastric, 36/43, [84] Gastric, 8/20, [84,85] Melanoma, 21/30, [86] Leukemia, (t-AML), not specified, [87] Oesophageal, 1/4, [48] Barrett’s oesophagus (precancerous), 9/33, [48] Colorectal, 10/110, [78] Gastric, 4/20, [85] Oesophageal, 1/4, [48] BUBR1 Mitotic checkpoint Colorectal, 2/19, [16] Lymphoma, 1/8, [79] MVAa, 5/8, [56] MVA, 6/6, [57] Breast 20/21, [83] Gastric, 29/43, [84] Lung, 8/8, [88] Colorectal, 10/116, [78] Thyroid, 3/8, [77] BUB3 Mitotic checkpoint Breast, 2/21, [83] Lung, 7/18, [89] MAD1 Mitotic checkpoint Breast, 18/21, [83] Gastric, 34/43, [84] Lung, 5/18, [89] Breast, 16/17, [83] Lung, 13/14, [90] MAD2 Mitotic checkpoint Breast, 1/22, [91] Breast, 1/1, [68] Gastric, 23/54, [92] Barrett’s oesophagus (precancerous), 8/33, [48] Bladder, not specified, [93] Breast, 3/13, [83] Breast, 15/21, [83] Colorectal, not specified, [94,95] Neuroblastoma, not specified, [93] Oesophageal, 1/4, [48] Barrett’s oesophagus (precancerous), 8/33, [48] Breast, 5/21, [68,83] Hepatocellular carcinoma, 5/10, [96] Hepatoma, 6/11, [71] Leukemia (ATL), 2/6 [66] Nasopharyngeal, 3/5, [74] Oesophageal, 2/4, [48] Ovarian, 3/7, [75] AdAPC b Tumor suppressor Colorectal, 76/115, [97] Duodenum, 16/19, [98] BRCA1 c Tumor suppressor BRCA2 d Tumor suppressor Msh2 e DNA mismatch repair Breast, 3/32, [103] Familial breast, 41/264, [104] Ovarian 1/12, [103] Ovarian, 15/103, [105] Ovarian, 39/649, [106] Breast, 2/70, [111] Familial breast, 60/264, [104] Ovarian, 0/55, [111] Ovarian, 21/649, [106] Colorectal, 1/509, [112] Colorectal with replication errors, 1/63, [112] Nasopharyngeal, 3/5, [74] Leukemia (ATL), 6/6, [66] Breast, 11/27, [99] Colorectal, 110/137, [100] Oesophageal, 4/35, [101] Oral, 15/50, [102] Breast, 39/48, [107] Breast, 51/162, [108] Colon, 5/5, [109] Ovarian, 54/76, [110] Pancreatic, 25/50, [107] Gallbladder, 35/46, [113] Urothelial, 17/17, [114] Leukemia (ATL), 11/11, [115] Melanoma, 45/106, [116] Skin (SCC), 2/125, [117] a Mosaic variegated aneuplody (MVA) is a rare condition associated with childhood cancers and growth retardation. bPatients with germline mutations in AdAPC have familial adenomatous polyposis (FAP) and develop colorectal cancer with almost 100% penetrance. FAP individuals also have an increased risk of duodenum (50–90% risk), thyroid, hepatoblastoma and adrenal cancers [7,118]. cHeterozygous germline mutations in BRCA1 occur in 20% of hereditary breast cancer patients. Individuals with germline mutations in BRCA1 have a 50–80% risk of developing breast cancer and a 40% risk of ovarian cancer [119,120]. dHeterozygous germline mutations in BRCA2 occur in 20% of hereditary breast cancer patients. Individuals with germline mutations in BRCA2 have a 45% lifetime risk of breast cancer and an 11% risk of ovarian cancer [120]. ePatients with germline mutations in Msh2 develop hereditary nonpolyposis colon cancer (HNPCC) syndrome and have increased risk of colorectal (80% lifetime risk), endometrial (30–50% risk), gastric, ovarian, urothelial, pancreatic and biliary cancers [7,118]. 2n + 1 and 2n 1 aneuploid progeny [23]. This idea was based on fluorescence in situ hybridization (FISH) data showing that cells that become binucleate after failing to complete cytokinesis have a higher incidence of missegregation of individual chromosomes into their two nuclei than do cells still in anaphase. This led to the conclusion Current Opinion in Cell Biology 2006, 18:658–667 that newly formed daughter cells are in some way able to sense nondisjunction events and cause cytokinetic furrow regression as a result. However, the correlation between nondisjunction and binucleation does not prove causality any more than does the correlation between aneuploidy and cancer. A direct test of this hypothesis posed in www.sciencedirect.com Does aneuploidy cause cancer? Weaver and Cleveland 663 primary cells that displayed elevated levels of nondisjunction (due to specific disruption of the gene encoding the mitotic motor CENP-E) showed no increase in binucleation [24]. Additionally, nondisjunction in mice and humans produces high levels of near-diploid aneuploidy, not tetraploidy [17,19,25]. Thus, nondisjunction produces near-diploid aneuploidy in most instances and is unlikely to serve as a major mechanism of tetraploidization. How does tetraploidy contribute to aneuploidy? Exit from mitosis without attempting cytokinesis (or with a failed cytokinesis) produces tetraploid cells containing two centrosomes. After replication, these centrosomes are capable of producing multipolar spindles with three or four poles, which would result in the production of aneuploid progeny during a subsequent mitosis, provided that the tetraploid cells were able to undergo successful cytokinesis (Figure 1g). However, a cell cycle checkpoint known as the tetraploidy checkpoint has been proposed to sense the presence of tetraploid cells and arrest them in G1 [26]. This mechanism would have the obvious advantage of preventing genomic instability caused by multipolar mitoses. However, recent work has raised concerns about the evidence for such a checkpoint. Initially, nontransformed rat embryonic fibroblasts were found to arrest in G1 after treatment with an actin inhibitor (cytochalasin) caused them to fail cytokinesis and become tetraploid. However, re-examination of the same cells indicated that tetraploid cells did proceed through the cell cycle if a lower concentration of cytochalasin was used [27]. Additionally, similar efforts with tetraploid primary human fibroblasts formed by drug treatment or cell fusion did not support the presence of a tetraploidy checkpoint [27,28]. Moreover, primary murine fibroblasts cycle despite being tetraploid, as recently observed with securin / separase / fibroblasts [29,30], and in numerous wild type examples [31,32,33]. Finally, tetraploid rat hepatocytes, HeLa cells and telomerase-immortalized human keratinocytes (N/ TERT-1 cells) have recently been filmed undergoing mitosis [23,34]. Thus, the balance of the evidence weighs heavily against the presence of a tetraploidy checkpoint as a general mechanism for blocking the proliferation of tetraploid cells. Mutations in tumor suppressors and DNA mismatch repair genes generate aneuploidy Germline mutations in the tumor suppressor gene adenomatous polyposis coli (AdAPC) cause familial adenomatous polyposis (FAP), a syndrome leading to the development of hundreds to thousands of colorectal polyps, resulting in colorectal cancer with almost complete penetrance. A large percentage of spontaneous colorectal tumors also contain mutations in AdAPC (Table 3). AdAPC plays a well-characterized role in down-regulating Wnt signaling by contributing to the degradation of b-catenin. Mutations in AdAPC result in stabilization of b-catenin, www.sciencedirect.com which leads to transcription of proliferation-associated genes, including c-myc and cyclin D1 [35]. However, it has recently been found that mutations in AdAPC also produce whole chromosomal aneuploidy in mouse embryonic stem cells [36,37]. Cells expressing truncated or mutant versions of AdAPC have a weakened mitotic checkpoint and unstable kinetochore-microtubule interactions during mitosis, which cooperate to produce lagging chromosomes in anaphase [36–38,39,40]. Thus, mutations in AdAPC may contribute to tumorigenesis via two distinct mechanisms, upregulation of Wnt signaling and generation of aneuploidy and CIN. Two other tumor suppressor genes, the breast cancer associated genes BRCA1 and BRCA2, have also recently been demonstrated to produce aneuploidy when mutated, in addition to their previously identified roles in DNA repair. Murine embryonic fibroblasts (MEFs) derived from mice expressing mutated forms of BRCA1 or BRCA2 contain highly aneuploid numbers of chromosomes. BRCA1 mutant cells exhibit lagging chromosomes and an apparently weakened mitotic checkpoint, which may be due to decreased expression of the essential mitotic checkpoint protein MAD2 [41]. BRCA2 mutant MEFs exhibit cytokinesis defects [42] and, consistent with this, contain supernumery centrosomes [43]. Thus, mutations in BRCA1 and BRCA2 appear to contribute to tumorigenesis through both the DNA damage and aneuploidy pathways. Similarly, mutations in the MMR gene Msh2 appear to cause both DNA damage and aneuploidy. As introduced above, mutations in Msh2 lead to MIN. MIN is thought to promote tumorigenesis when insertions or deletions of microsatellites occur in growth-regulatory genes. However, in addition to defects in MMR, Msh2 / primary MEFs develop rampant aneuploidy. Eighty percent of Msh2 / MEFs contained a non-diploid number of chromosomes at passage 2, as compared to 30% of wild type cells [31]. Thus, mutations in Msh2 may contribute to tumorigenesis through both the MIN and the CIN pathways. Conclusions: the evidence is equivocal on whether aneuploidy is a direct cause of cancer Aneuploidy is a remarkably common characteristic of tumor cells (Figure 1), which is a major reason why it has been proposed to initiate tumorigenesis. This proposal makes several predictions. First, aneuploidy should precede transformation. Indeed, aneuploidy is found in pre-cancerous lesions of the cervix [44,45], head and neck [46], colon [45,47], oesophagus [48] and bone marrow [49]. Aneuploidy has also been detected in premalignant breast [50] and skin [51] lesions in experimental animals. Second, aneuploidy should disrupt global transcription leading to upregulation of growth-promoting genes and downregulation of genes involved in growth control. Recent work indicates that aneuploidy due to the gain Current Opinion in Cell Biology 2006, 18:658–667 664 Cell division, growth and death of a single chromosome can indeed result in the misregulation of 100–200 genes. Strikingly, only 5–20% of misregulated genes were contained on the trisomic chromosome [52]. Third, transformation and tumorigenesis due to aneuploidy should require many generations to establish the complicated karyotypes contained in human tumor cells that permit patterns of gene misexpression supportive of uninhibited cell growth. This is consistent with the wellknown increase in cancer incidence with age. Although aneuploidy correlates with transformation, empirical tests of the hypothesis that aneuploidy drives tumorigenesis have been hampered by the difficulty of generating aneuploidy without causing other cellular defects, particularly DNA damage. Early attempts to test the effects of aneuploidy relied on drugs, many of which have subsequently been shown to be mutagenic. More recent attempts have used mice expressing reduced levels of mitotic checkpoint genes. Mice heterozygous for the mitotic checkpoint gene MAD2 are more susceptible to spontaneous, benign lung tumors after a long latency [19]. BUB3+/ mice are not predisposed to spontaneous tumors, but they may be more susceptible to carcinogen-induced tumors [17], as are mice expressing reduced levels of BUBR1 [53,54]. Interestingly, BUBR1 heterozygosity accelerates tumorigenesis in the large intestine and inhibits tumorigenesis in the small intestine in mice expressing a mutated allele of the AdAPC tumor suppressor gene [55]. Mutations in BUBR1 have also been found in families exhibiting mosaic variegated aneuploidy (MVA) [56,57], a rare condition associated with growth retardation and predisposition to various tumor types. However, all of these checkpoint proteins are expressed throughout the cell cycle and have been implicated in diverse cellular processes. BUBR1 functions in apoptosis [58–60], megakaryopoiesis [61], the DNA damage checkpoint [62], aging and fertility [18]. BUB3 acts as a transcriptional repressor [63] and MAD2 localizes to the nucleus and nuclear pores and participates in the DNA replication checkpoint [64]. All three contribute to gross chromosomal rearrangements [65]. Thus, interpretation of their tumor-prone phenotype is complicated by the fact that they participate in cellular functions other than chromosome segregation. Ultimately, a true test of the aneuploidy hypothesis will require a method to generate aneuploidy in the absence of other defects, a feat not yet reported. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1. Mitelman F, Johansson B, Mertens FE: Mitelman Database of Chromosome Aberrations in Cancer (2006). URL: http://cgap.nci.nih.gov/Chromosomes/Mitelman 2006. Current Opinion in Cell Biology 2006, 18:658–667 2. Boveri T: Ueber mehrpolige Mitosen als Mittel zur Analyse des Zellkerns. URL for English translation: http://8e.devbio.com/ article.php?ch=4&id=24. 3. Boveri T: Zur Frage der Entstehung maligner Tumoren. (The origin of malignant tumors.). Gustav Fischer, Jena 1914. 4. Hahn WC, Counter CM, Lundberg AS, Beijersbergen RL, Brooks MW, Weinberg RA: Creation of human tumour cells with defined genetic elements. Nature 1999, 400:464-468. 5. Marx J: Debate surges over the origins of genomic defects in cancer. Science 2002, 297:544-546. 6. Zimonjic D, Brooks MW, Popescu N, Weinberg RA, Hahn WC: Derivation of human tumor cells in vitro without widespread genomic instability. Cancer Res 2001, 61:8838-8844. 7. Strate LL, Syngal S: Hereditary colorectal cancer syndromes. Cancer Causes Control 2005, 16:201-213. 8. Rajagopalan H, Lengauer C: CIN-ful cancers. Cancer Chemother Pharmacol 2004, 54(Suppl 1):S65-S68. 9. von Hansemann D: Ueber asymmetrische Zelltheilung in Epithelkrebsen und deren biologische Bedeutung. Virchow’s Arch Path Anat. 1890, 119:299-326. 10. Kops GJ, Weaver BA, Cleveland DW: On the road to cancer: aneuploidy and the mitotic checkpoint. Nat Rev Cancer 2005, 5:773-785. 11. Taylor SS, Scott MI, Holland AJ: The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation. Chromosome Res 2004, 12:599-616. 12. Rieder CL, Cole RW, Khodjakov A, Sluder G: The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores. J Cell Biol 1995, 130:941-948. 13. Rieder CL, Schultz A, Cole R, Sluder G: Anaphase onset in vertebrate somatic cells is controlled by a checkpoint that monitors sister kinetochore attachment to the spindle. J Cell Biol 1994, 127:1301-1310. 14. Kops GJ, Foltz DR, Cleveland DW: Lethality to human cancer cells through massive chromosome loss by inhibition of the mitotic checkpoint. Proc Natl Acad Sci USA 2004, 101:8699-8704. See annotation to [15]. 15. Michel L, Diaz-Rodriguez E, Narayan G, Hernando E, Murty VV, Benezra R: Complete loss of the tumor suppressor MAD2 causes premature cyclin B degradation and mitotic failure in human somatic cells. Proc Natl Acad Sci USA 2004, 101:4459-4464. This paper and [14] demonstrate that complete loss of the mitotic checkpoint due to severe depletion of MAD2 or BUBR1 causes rapid cell death. 16. Cahill DP, Lengauer C, Yu J, Riggins GJ, Willson JK, Markowitz SD, Kinzler KW, Vogelstein B: Mutations of mitotic checkpoint genes in human cancers. Nature 1998, 392:300-303. 17. Babu JR, Jeganathan KB, Baker DJ, Wu X, Kang-Decker N, van Deursen JM: Rae1 is an essential mitotic checkpoint regulator that cooperates with Bub3 to prevent chromosome missegregation. 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