Aneuploidy,cell delamination and tumorigenesis in Drosophila epithelia

Chromosomal instability (CIN) is a common feature in human cancer, and highly aneuploid tumors are frequently associated with poor prognosis; however, the molecular and cellular mechanisms underlying CIN-induced tumorigenesis are poorly understood. Here we review recent findings about the role of CIN in driving tumor-like growth and host invasiveness in Drosophila epithelia and discuss the commonalities of CIN-induced tumors with other Drosophila-based cancer models. We also discuss possible scenarios that can account for the participation of CIN in tumorigenesis and propose that, alternatively to the classical role of aneuploidy in promoting the accumulation of mutations in cancer cells, aneuploidy can be a source of stress that may contribute to cancer initiation and/or progression.     

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.     

Chromosomal instability and human cancer

Genetic instability is a defining feature of human cancer. The main type of genetic instability, chromosomal instability (CIN), enhances the rate of gross chromosomal changes during cell division. CIN is brought about by mutations of CIN genes, i.e. genes that are involved in maintaining the genomic integrity of the cell. A major question in cancer genetics is whether genetic instability is a cause and hence a driving force of tumorigenesis. A mathematical framework for studying the somatic evolution of cancer sheds light onto the causal relations between CIN and human cancer.     

Cancer chromosomal instability: therapeutic and diagnostic challenges

Chromosomal instability (CIN)-which is a high rate of loss or gain of whole or parts of chromosomes-is a characteristic of most human cancers and a cause of tumour aneuploidy and intra-tumour heterogeneity. CIN is associated with poor patient outcome and drug resistance, which could be mediated by evolutionary adaptation fostered by intra-tumour heterogeneity. In this review, we discuss the clinical consequences of CIN and the challenges inherent to its measurement in tumour specimens. The relationship between CIN and prognosis supports assessment of CIN status in the clinical setting and suggests that stratifying tumours according to levels of CIN could facilitate clinical risk assessment.     

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.     

Aneuploid colon cancer cells have a robust spindle checkpoint

Colon cancer cells frequently display minisatellite instability (MIN) or chromosome instability (CIN). While MIN is caused by mismatch repair defects, the lesions responsible for CIN are unknown. The observation that CIN cells fail to undergo mitotic arrest following spindle damage suggested that mutations in spindle checkpoint genes may account for CIN. However, here we show that CIN cells do undergo mitotic arrest in response to spindle damage. Although the maximum mitotic index achieved by CIN lines is diminished relative to MIN lines, CIN cells clearly have a robust spindle checkpoint. Consistently, mutations in spindle checkpoint genes are rare in human tumours. In contrast, the adenomatous polyposis coli (APC) gene is frequently mutated in CIN cells. Significantly, we show here that expression of an APC mutant in MIN cells reduces the mitotic index following spindle damage to a level observed in CIN cells, suggesting that APC dysfunction may contribute to CIN.     

Reduced ATR or Chk1 Expression Leads to Chromosome Instability and Chemosensitization of Mismatch Repair–deficient Colorectal Cancer Cells

Genomic instability in colorectal cancer is categorized into two distinct classes: chromosome instability (CIN) and microsatellite instability (MSI). MSI is the result of mutations in the mismatch repair (MMR) machinery, whereas CIN is often thought to be associated with a disruption in the APC gene. Clinical data has recently shown the presence of heterozygous mutations in ATR and Chk1 in human cancers that exhibit MSI, suggesting that those mutations may contribute to tumorigenesis. To determine whether reduced activity in the DNA damage checkpoint pathway would cooperate with MMR deficiency to induce CIN, we used siRNA strategies to partially decrease the expression of ATR or Chk1 in MMR-deficient colorectal cancer cells. The resultant cancer cells display a typical CIN phenotype, as characterized by an increase in the number of chromosomal abnormalities. Importantly, restoration of MMR proficiency completely inhibited induction of the CIN phenotype, indicating that the combination of partial checkpoint blockage and MMR deficiency is necessary to trigger CIN. Moreover, disruption of ATR and Chk1 in MMR-deficient cells enhanced the sensitivity to treatment with the commonly used colorectal chemotherapeutic compound, 5-fluorouracil. These results provide a basis for the development of a combination therapy for those cancer patients.     

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.     

Linear Model of Colon Cancer Initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

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.     

Linear model of colon cancer initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

Chronic oxidative DNA damage due to DNA repair defects causes chromosomal instability in Saccharomyces cerevisiae

Oxidative DNA damage is likely to be involved in the etiology of cancer and is thought to accelerate tumorigenesis via increased mutation rates. However, the majority of malignant cells acquire a specific type of genomic instability characterized by large-scale genomic rearrangements, referred to as chromosomal instability (CIN). The molecular mechanisms underlying CIN are not entirely understood. We utilized Saccharomyces cerevisiae as a model system to delineate the relationship between genotoxic stress and CIN. It was found that elevated levels of chronic, unrepaired oxidative DNA damage caused chromosomal aberrations at remarkably high frequencies under both selective and nonselective growth conditions. In this system, exceeding the cellular capacity to appropriately manage oxidative DNA damage resulted in a “gain-of-CIN” phenotype and led to profound karyotypic instability. These results illustrate a novel mechanism for genome destabilization that is likely to be relevant to human carcinogenesis.     

Haploinsufficiency of SGO1 results in deregulated centrosome dynamics,enhanced chromosomal instability and colon tumorigenesis

Chromosome instability (CIN) is found in 85% of colorectal cancers. Defects in mitotic processes are implicated in high CIN and may be critical events in colorectal tumorigenesis. Shugoshin-1 (SGO1) aids in the maintenance of chromosome cohesion and prevents premature chromosome separation and CIN. In addition, integrity of the centrosome may be compromised due to the deficiency of Cohesin and Sgo1 through the disengagement of centrioles. We report here the generation and characterization of SGO1-mutant mice and show that haploinsufficiency of SGO1 leads to enhanced colonic tumorigenesis. Complete disruption of SGO1 results in embryonic lethality, whereas SGO1^+/- mice are viable and fertile. Haploinsufficiency of SGO1 results in genomic instability manifested as missegregation of chromosomes and formation of extra centrosomal foci in both murine embryonic fibroblasts and adult bone marrow cells. Enhanced CIN observed in SGO1-deficient mice resulted in an increase in formation of aberrant crypt foci (ACF) and accelerated development of tumors after exposure to azoxymethane (AOM), a colon carcinogen. Together, these results suggest that haploinsufficiency of SGO1 causes enhanced CIN, colonic preneoplastic lesions and tumorigenesis in mice. SGO1 is essential for the suppression of CIN and tumor formation.     

Tumorigenicity analysis of heterogeneous dental stem cells and its self-modification for chromosome instability

Heterogeneity demonstrates that stem cells are constituted by several sub-clones in various differentiation states. The heterogeneous state is maintained by cross-talk among sub-clones, thereby ensuring stem cell adaption. In this study, we investigated the roles of heterogeneity on genetic stability. Three sub-clones (DF2, DF8 and DF18) were isolated from heterogeneous dental stem cells (DSCs), and were proved to be chromosome instability (CIN) after long term expansion. Cell apoptosis were not detected in sub-clones, which exhibited strong tumorigenesis tendency, coupled with weak expression of p53 and aberrant ultra-structure. However, 3 sub-clones did not overexpress tumor related markers or induce tumorigenesis in vivo. The mixed-culture study suggested that 3-clone-mixed culturing cells (DF1) presented apparent decrease in the ratio of aneuploidy. The screening experiment further proved that 3 sub-clones functioned separately in this modification procedure but only mixed culturing all 3 sub-clones, simulated heterogeneous microenvironment, could achieve complete modification. Additionally, osteogenesis capability of 3 sub-clones was partially influenced by CIN while DSCs still kept stronger osteogenesis than sub-clones. These results suggested aberrant sub-clones isolated from heterogeneous DSCs were not tumorigenesis and could modify CIN by cross-talk among themselves, indicating that the heterogeneity played a key role in maintaining genetic stability and differentiation capability in dental stem cells.     

Exploring the role of CENP-A Ser18 phosphorylation in CIN and Tumorigenesis

Chromosome instability (CIN) contributes to the development of many cancer. In this paper, we summarize our recent finding that a novel pathway by which FBW7 loss promotes Centromere Protein A (CENP-A) phosphorylation on Serine 18 through Cyclin E1/CDK2, therefore promoting CIN and tumorigenesis. Our finding demonstrates the importance of CENP-A post-translational modification on modulating centromere and mitotic functions in cancer.