Chromosomal instability,tolerance of mitotic errors and multidrug resistance are promoted by tetraploidization in human cells

Up to 80% of human cancers, in particular solid tumors, contain cells with abnormal chromosomal numbers, or aneuploidy, which is often linked with marked chromosomal instability. Whereas in some tumors the aneuploidy occurs by missegregation of one or a few chromosomes, aneuploidy can also arise during proliferation of inherently unstable tetraploid cells generated by whole genome doubling from diploid cells. Recent findings from cancer genome sequencing projects suggest that nearly 40% of tumors underwent whole genome doubling at some point of tumorigenesis, yet its contribution to cancer phenotypes and benefits for malignant growth remain unclear. Here, we investigated the consequences of a whole genome doubling in both cancerous and non-transformed p53 positive human cells. SNP array analysis and multicolor karyotyping revealed that induced whole-genome doubling led to variable aneuploidy. We found that chromosomal instability (CIN) is a frequent, but not a default outcome of whole genome doubling. The CIN phenotypes were accompanied by increased tolerance to mitotic errors that was mediated by suppression of the p53 signaling. Additionally, the expression of pro-apoptotic factors, such as iASPP and cIAP2, was downregulated. Furthermore, we found that whole genome doubling promotes resistance to a broad spectrum of chemotherapeutic drugs and stimulates anchorage-independent growth even in non-transformed p53-positive human cells. Taken together, whole genome doubling provides multifaceted benefits for malignant growth. Our findings provide new insight why genome-doubling promotes tumorigenesis and correlates with poor survival in cancer.     

Chromosome instability and tumor lethality suppression in carcinogenesis

The maintenance and survival of each organism depends on its genome integrity. Alterations of essential genes, or aberrant chromosome number and structure lead to cell death. Paradoxically, cancer cells, especially in solid tumors, contain somatic gene mutations and are chromosome instability (CIN), suggesting a mechanism that cancer cells have acquired to suppress the lethal mutations and/or CIN. Herein we will discuss a tumor lethality suppression concept based on the studies of yeast genetic interactions and transgenic mice. During the early stages of the multistep process of tumorigenesis, incipient cancer cells probably have adopted genetic and epigenetic alterations to tolerate the lethal mutations of other genes that ensue, and to a larger extent CIN. In turn, CIN mediated massive gain and loss of genes provides a wider buffer for further genetic reshuffling, resulting in cancer cell heterogeneity, drug resistance and evasion of oncogene addiction, thus CIN may be both the effector and inducer of tumorigenesis. Accordingly, interfering with tumor lethality suppression could lead to cancer cell death or growth defects. Further validation of the tumor lethality suppression concept would help to elucidate the role of CIN in tumorigenesis, the relationship between CIN and somatic gene mutations, and would impact the design of anticancer drug development.     

Chromosome segregation and genomic stability

The acquisition of genomic instability is a crucial step in the development of human cancer. Genomic instability has multiple causes of which chromosomal instability (CIN) and microsatellite instability (MIN) have received the most attention. Whereas the connection between a MIN phenotype and cancer is now proven, the argument that CIN causes cancer remains circumstantial. Nonetheless, the ubiquity of aneuploidy in human cancers, particularly solid tumors, suggests a fundamental link between errors in chromosome segregation and tumorigenesis. Current research in the field is focused on elucidating the molecular basis of CIN, including the possible roles of defects in the spindle checkpoint and other regulators of mitosis.     

A balancing act: focus on aneuploidy

Aneuploidy has emerged as a major health concern in cancer and fertility. This issue of EMBO reports features four reviews that discuss aneuploidy and its consequences from different viewpoints, and are contextualized in this editorial.EMBO reports (2012) 13, 472; doi:10.1038/embor.2012.66Faithful chromosome segregation is crucial for the viability of cells and organisms, as evidenced by the fact that in humans only one autosomic trisomy—and no autosomic monosomies—allow survival into adulthood. Cells therefore use sophisticated mechanisms to ensure that each daughter receives an intact copy of the genome during cell division. Eukaryotic chromosomes have a specialized region known as the centromere, which recruits a complex proteinaceus structure—the kinetochore—that binds spindle microtubules to enable the separation of chromosomes during mitosis. The mitotic checkpoint and the machinery that controls kinetochore–microtubule attachment ensure correct chromosome segregation. However, several processes can lead to aneuploidy—the deviation from a haploid chromosomal number—such as defects in mitotic checkpoint proteins or sister chromatid cohesion, incorrect or hyperstabilized chromosome-spindle attachments, centrosome amplification or defects in cytokinesis.Aneuploidy is a major health concern. It is the leading cause of mental retardation and spontaneous miscarriage, and the current trend towards advanced maternal age has increased the frequency of trisomic fetuses by 71% in the past ten years [1]. Furthermore, most solid tumours and about 50% of haematopoietic cancers are aneuploid. During the past few years, the cell-cycle, cancer and fertility fields have therefore made a substantial effort to understand the causes and consequences of aneuploidy.To bring together knowledge from different viewpoints and highlight recent advances in this exciting field, this issue of EMBO reports features four reviews on aneuploidy. An article by Rolf Jessberger analyses the process of oocyte meiosis and how it becomes less accurate with age, and reviews by Holland & Cleveland, Pfau & Amon and Swanton & colleagues focus on aneuploidy in the context of cancer.An overarching theme is the importance of intact sister chromatid cohesion to ensure the fidelity of chromosome segregation. In mammalian oocytes—which remain arrested in meiosis for up to four decades in humans—cohesin is loaded onto chromosomes during development and is probably not turned over for the life of the oocyte. Progressive loss of cohesin or ‘exhaustion'' seems responsible for the dramatic increase in aneuploid eggs with age. Similarly, defects in cohesion proteins are frequently found in various types of cancer.As will become apparent in the three cancer-related reviews, it is important to distinguish between aneuploidy and chromosomal instability (CIN)—a high rate of gain or loss of chromosomes. CIN leads to aneuploidy, but stable aneuploidy can occur without CIN, which is associated with a good prognosis in cancer and occurs in normal brain and liver tissue. An outstanding question is how and whether aneuploidy and CIN predispose to tumorigenesis. Technological advances have allowed the characterization of CIN status of a variety of cancers, underscoring the prevalence of aneuploidy. However, whether aneuploidy is a driving cause of tumour formation remains unclear. Despite the extensive association of aneuploidy with tumours in vivo, extensive data from yeast, mouse and human cell culture indicate that abnormal chromosome content provides a growth disadvantage in vitro, and the presence of CIN in some tumours correlates with good prognosis: this is the so-called ‘aneuploidy paradox''.In this review series, the Cleveland, Amon and Swanton groups provide their own particular views on this paradox. CIN could endow tumour cells with extreme evolvability that is beneficial in vivo, but would be a growth disadvantage under the constant, rich conditions of cell culture. On the other hand, aneuploidy could interfere with cell proliferation—as seen in vitro—and would be selected against; further mutations or chromosomal alterations would allow cells to overcome this restriction and reveal their full tumorigenic potential. According to this view, CIN would allow cells to overcome the negative effects of aneuploidy and promote tumorigenesis below a certain threshold. However, as Swanton and colleagues discuss, the nonlinear relationship between the extent of CIN and cancer prognosis suggests that, beyond this threshold, CIN would become unfavourable owing to the accumulation of deleterious genomic alterations.An increase in genomic material is generally accompanied by an increase in the expression of proteins encoded there, leading to altered metabolic properties, imbalances in the cell proteome and proteotoxic stress due to an overloading of protein degradation pathways. These effects imply that therapeutically targetable pathways would be common in a variety of aneuploid tumour cells. Initial proof-of-principle screens show promise in this regard and, as discussed in these reviews, have led to potential drug candidates.Swanton and colleagues provide a much needed—but rare—translational perspective into the issue of aneuploidy and CIN. Their review highlights the prognostic value of CIN assessment in human tumours, evaluates the methods used to analyse CIN and provides insights into how it could be therapeutically targeted.We hope this selection of comprehensive reviews will contribute to a better understanding of the complexities of aneuploidy and its causes. The possibility of targeting this imbalanced state in cancer therapy and harnessing our increasing knowledge to alleviate fertility problems are exciting prospects. We look forward to future developments in this fast-moving field.     

Chromosomal instability and aneuploidy in cancer: from yeast to man

Aneuploidy is frequently associated with disease and developmental abnormalities. It is also a key characteristic of cancer. Several model systems have been developed to study the role of chromosomal instability and aneuploidy in tumorigenesis. The results are surprisingly complex, with the conditions sometimes promoting and sometimes inhibiting tumour formation. Here, we review the effects of aneuploidy and chromosomal instability in cells and model systems of cancer, propose a model that could explain these complex findings and discuss how the aneuploid condition could be exploited in cancer therapy.     

Aneuploidy and tumorigenesis

Aneuploidy is a prominent phenotype of cancer. It refers to deviations from the normal number of chromosomes in a cell, as a result of whole-chromosome loss or gain. In most cases, aneuploidy is caused by mitotic errors due to defects in the mechanisms that have evolved to ensure faithful chromosome segregation, such as the spindle assembly checkpoint (SAC). The observation that SAC-deficient mice are tumor prone demonstrates that this checkpoint is important in suppressing tumor formation and suggests that aneuploidy can induce tumorigenesis. In this review, we will summarize our current knowledge about the cause of aneuploidy and discuss the cellular response to aneuploidy in the context of tumorigenesis.     

Proliferation of aneuploid human cells is limited by a p53-dependent mechanism

Most solid tumors are aneuploid, and it has been proposed that aneuploidy is the consequence of an elevated rate of chromosome missegregation in a process called chromosomal instability (CIN). However, the relationship of aneuploidy and CIN is unclear because the proliferation of cultured diploid cells is compromised by chromosome missegregation. The mechanism for this intolerance of nondiploid genomes is unknown. In this study, we show that in otherwise diploid human cells, chromosome missegregation causes a cell cycle delay with nuclear accumulation of the tumor suppressor p53 and the cyclin kinase inhibitor p21. Deletion of the p53 gene permits the accumulation of nondiploid cells such that CIN generates cells with aneuploid genomes that resemble many human tumors. Thus, the p53 pathway plays an important role in limiting the propagation of aneuploid human cells in culture to preserve the diploid karyotype of the population. These data fit with the concordance of aneuploidy and disruption of the p53 pathway in many tumors, but the presence of aneuploid cells in some normal human and mouse tissues indicates that there are known exceptions to the involvement of p53 in aneuploid cells and that tissue context may be important in how cells respond to aneuploidy.     

CIN and Aneuploidy: Different Concepts,Different Consequences

Chromosomal instability (CIN) and aneuploidy are similar concepts but not synonymous. CIN is the process that leads to chromosome copy number alterations, and aneuploidy is the result. While CIN and resulting aneuploidy often cause growth defects, they are also selected for in cancer cells. Although such contradicting fates may seem paradoxical at first, they can be better understood when CIN and aneuploidy are assessed separately, taking into account the in vitro or in vivo context, the rate of CIN, and severity of the aneuploid karyotype. As CIN can only be measured in living cells, which proves to be technically challenging in vivo, aneuploidy is more frequently quantified. However, CIN rates might be more predictive for tumor outcome than assessing aneuploidy rates alone. In reviewing the literature, we therefore conclude that there is an urgent need for new models in which we can monitor chromosome mis‐segregation and its consequences in vivo. Also see the video abstract here: https://youtu.be/fL3LxZduchg     

CINister thoughts

Chromosome instability (CIN) is the process that leads to aneuploidy, a known hallmark of human tumours for over a century. Nowadays, it is believed that CIN promotes tumorigenesis by shuffling the genome into a malignant order through translocations, amplifications, deletions (structural CIN), and gains and losses of whole chromosomes (numerical CIN or nCIN). The present review focuses on the causes and consequences of nCIN. Several roads can lead to nCIN, including a compromised spindle assembly checkpoint, cohesion defects, p53 deficiency and flawed microtubule-kinetochore attachments. Whereas the link between nCIN and tumorigenesis is becoming more evident, indications have emerged recently that nCIN can suppress tumour formation as well. To understand these paradoxical findings, novel reagents and more sophisticated mouse models are needed. This will provide us with a better understanding of nCIN and eventually with therapies that exploit this characteristic of human tumours.     

Chromosome instability and kinetochore dysfunction

Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Thus, defect in kinetochore function is a candidate source for CIN and the generation of aneuploidy. Recently, a number of kinetochore components have been shown to be mutated and/or aberrantly expressed in human cancers, which suggests an important role of kinetochore for CIN and carcinogenesis. In this article, we will discuss about how kinetochore dysfunction causes CIN and might lead to the development of cancer.     

Preventing aneuploidy: the contribution of mitotic checkpoint proteins

Aneuploidy, an abnormal number of chromosomes, is a trait shared by most solid tumors. Chromosomal instability (CIN) manifested as aneuploidy might promote tumorigenesis and cause increased resistance to anti-cancer therapies. The mitotic checkpoint or spindle assembly checkpoint is a major signaling pathway involved in the prevention of CIN. We review current knowledge on the contribution of misregulation of mitotic checkpoint proteins to tumor formation and will address to what extent this contribution is due to chromosome segregation errors directly. We propose that both checkpoint and non-checkpoint functions of these proteins contribute to the wide array of oncogenic phenotypes seen upon their misregulation.     

The wages of CIN

Aneuploidy and chromosome instability (CIN) are hallmarks of the majority of solid tumors, but the relationship between them is not well understood. In this issue, Thompson and Compton (Thompson, S.L., and D.A. Compton. 2008. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell. Biol. 180:665-672) investigate the mechanism of CIN in cancer cells and find that CIN arises primarily from defective kinetochore-spindle attachments that evade detection by the spindle checkpoint and persist into anaphase. They also explore the consequences of artificially elevating chromosome missegregation in otherwise karyotypically normal cells. Their finding that induced aneuploidy is rapidly selected against suggests that the persistence of aneuploid cells in tumors requires not only chromosome missegregation but also additional, as yet poorly defined events.     

Aneuploidy: cells losing their balance

A change in chromosome number that is not the exact multiple of the haploid karyotype is known as aneuploidy. This condition interferes with growth and development of an organism and is a common characteristic of solid tumors. Here, we review the history of studies on aneuploidy and summarize some of its major characteristics. We will then discuss the molecular basis for the defects caused by aneuploidy and end with speculations as to whether and how aneuploidy, despite its deleterious effects on organismal and cellular fitness, contributes to tumorigenesis.     

Causes and consequences of centrosome abnormalities in cancer

Centrosome amplification is a hallmark of cancer. However, despite significant progress in recent years, we are still far from understanding how centrosome amplification affects tumorigenesis. Boveri''s hypothesis formulated more than 100 years ago was that aneuploidy induced by centrosome amplification promoted tumorigenesis. Although the hypothesis remains appealing 100 years later, it is also clear that the role of centrosome amplification in cancer is more complex than initially thought. Here, we review how centrosome abnormalities are generated in cancer and the mechanisms cells employ to adapt to centrosome amplification, in particular centrosome clustering. We discuss the different mechanisms by which centrosome amplification could contribute to tumour progression and the new advances in the development of therapies that target cells with extra centrosomes.     

2n or not 2n: Aneuploidy,polyploidy and chromosomal instability in primary and tumor cells

Mitotic defects leading to aneuploidy have been recognized as a hallmark of tumor cells for over 100 years. Current data indicate that ∼85% of human cancers have missegregated chromosomes to become aneuploid. Some maintain a stable aneuploid karyotype, while others consistently missegregate chromosomes over multiple divisions due to chromosomal instability (CIN). Both aneuploidy and CIN serve as markers of poor prognosis in diverse human cancers. Despite this, aneuploidy is generally incompatible with viability during development, and some aneuploid karyotypes cause a proliferative disadvantage in somatic cells. In vivo, the intentional introduction of aneuploidy can promote tumors, suppress them, or do neither. Here, we summarize current knowledge of the effects of aneuploidy and CIN on proliferation and cell death in nontransformed cells, as well as on tumor promotion, suppression, and prognosis.     

Examining the link between chromosomal instability and aneuploidy in human cells

Solid tumors can be highly aneuploid and many display high rates of chromosome missegregation in a phenomenon called chromosomal instability (CIN). In principle, aneuploidy is the consequence of CIN, but the relationship between CIN and aneuploidy has not been clearly defined. In this study, we use live cell imaging and clonal cell analyses to evaluate the fidelity of chromosome segregation in chromosomally stable and unstable human cells. We show that improper microtubule-chromosome attachment (merotely) is a cause of chromosome missegregation in unstable cells and that increasing chromosome missegregation rates by elevating merotely during consecutive mitoses generates CIN in otherwise stable, near-diploid cells. However, chromosome missegregation compromises the proliferation of diploid cells, indicating that phenotypic changes that permit the propagation of nondiploid cells must combine with elevated chromosome missegregation rates to generate aneuploid cells with CIN.     

Defining 'chromosomal instability'

Most scientists agree that the majority of human solid malignant tumors are characterized by chromosomal instability (CIN) involving gain or loss of whole chromosomes or fractions of chromosomes. CIN is thought to be an early event during tumorigenesis and might therefore be involved in tumor initiation. Despite its frequent occurrence in tumors and its potential importance in tumor evolution, CIN is poorly defined and is used inconsistently and imprecisely. Here, we provide criteria to define CIN and argue that few experimental approaches are capable of assessing the presence of CIN. Accurate assessment of CIN is crucial to elucidate whether CIN is a driving force for tumorigenesis and whether a chromosomally unstable genome is necessary for tumor progression.     

Rapid proliferation of daughter cells lacking particular chromosomes due to multipolar mitosis promotes clonal evolution in colorectal cancer cells

Aneuploidy and chromosome instability (CIN) are hallmarks of the vast majority of solid tumors. However, the origins of aneuploid cells are unknown. The aim of this study is to improve our understanding of how aneuploidy and/or CIN arise and of karyotype evolution in cancer cells. By using fluorescence in situ hybridization (FISH) on cells after long-term live cell imaging, we demonstrated that most (> 90%) of the newly generated aneuploid cells resulted from multipolar divisions. Multipolar division occurred in mononucleated and binucleated parental cells, resulting in variation of chromosome compositions in daughter cells. These karyotypes can have the same chromosome number as their mother clone or lack a copy of certain chromosomes. Interestingly, daughter cells that lost a chromosome were observed to survive and form clones with shorter cell cycle duration. In our model of cancer cell evolution, the rapid proliferation of daughter cells from multipolar mitosis promotes colonal evolution in colorectal cancer cells.