Chromosomal Instability In Colorectal Cancer Research Articles

Deep learning-based histological predictions of chromosomal instability in colorectal cancer.

Colorectal cancer (CRC) is a lethal malignancy and a leading cause of cancer-related mortality worldwide. Chromosomal instability (CIN) is a key driver of genomic instability in CRC and is characterized by aneuploidy and somatic copy-number alterations. This study aimed to predict CIN in CRC using histological data from whole slide images (WSIs). CRC samples from TCGA were analyzed, with tumor regions segmented into tiles and nuclei for feature extraction using convolutional neural network (CNN) and morphologic analysis. Binary classification models were developed to distinguish high and low aneuploidy scores (AS) based on slide-level features. The analysis included 313 patients with 315 WSIs, resulting in over 350,000 tumor tiles and nearly 2.7 million tumor cell nuclei. The ResNet18-SSL model, pre-trained on histopathological images, demonstrated superior accuracy in tile-based AS prediction, while DenseNet121 excelled in nucleus-based prediction. Combining CNN-based and morphological features enhanced the classification accuracy of nucleus-based predictions. Additionally, significant correlations were observed between morphological features and copy-number signatures. Unsupervised clustering of nuclear features revealed that distinct groups are significantly correlated with CIN and TP53 mutations. This study underscores the potential of histological features from WSIs to predict CIN in CRC samples. Nuclear feature analysis, combined with deep-learning techniques, offers a robust method for CIN prediction, highlighting the importance of further research into the relationships between histological and molecular phenotypes.

Genomic aberrations after short-term exposure to colibactin-producing E. coli transform primary colon epithelial cells

Genotoxic colibactin-producing pks+ Escherichia coli induce DNA double-strand breaks, mutations, and promote tumor development in mouse models of colorectal cancer (CRC). Colibactin’s distinct mutational signature is reflected in human CRC, suggesting a causal link. Here, we investigate its transformation potential using organoids from primary murine colon epithelial cells. Organoids recovered from short-term infection with pks+ E. coli show characteristics of CRC cells, e.g., enhanced proliferation, Wnt-independence, and impaired differentiation. Sequence analysis of Wnt-independent organoids reveals an enhanced mutational burden, including chromosomal aberrations typical of genomic instability. Although we do not find classic Wnt-signaling mutations, we identify several mutations in genes related to p53-signaling, including miR-34a. Knockout of Trp53 or miR-34 in organoids results in Wnt-independence, corroborating a functional interplay between the p53 and Wnt pathways. We propose larger chromosomal alterations and aneuploidy as the basis of transformation in these organoids, consistent with the early appearance of chromosomal instability in CRC.

MAP9 Loss Triggers Chromosomal Instability, Initiates Colorectal Tumorigenesis, and Is Associated with Poor Survival of Patients with Colorectal Cancer.

Chromosomal instability (CIN) is a common phenomenon in colorectal cancer, but its role and underlying cause remain unknown. We have identified that mitotic regulator microtubule-associated protein 9 (MAP9) is a critical regulator of CIN in colorectal cancer. We thus studied the effect of MAP9 loss on colorectal cancer in Map9-knockout mice and in cell lines. We generated colon epithelial-specific Map9-knockout mice and evaluated colorectal cancer development. Effect of Map9 knockout on colorectal cancer progression was determined in chemical or ApcMin /+ -induced colorectal cancer. Molecular mechanism of MAP9 was determined using spectral karyotyping, microtubule assays, and whole-genome sequencing (WGS). Clinical significance of MAP9 was examined in 141 patients with CRC. Spontaneous colonic tumors (9.1%) were developed in colon epithelium-specific Map9-knockout mice at 17 months, but none was observed in wild-type littermates. Map9 deletion accelerated colorectal cancer formation both in ApcMin /+ mice and azoxymethane-treated mice, and reduced survival in ApcMin /+ mice. Mechanistically, MAP9 stabilized microtubules and mediated mitotic spindle assembly. MAP9 also maintained the spindle pole integrity and protected K-fiber from depolymerization at spindle poles. MAP9 loss induced severe mitosis failure, chromosome segregation errors, and aneuploidy, leading to transformation of normal colon epithelial cells. WGS confirmed enhanced CIN in intestinal tumors from Map9 knockout ApcMin /+ mice. In patients with colorectal cancer, MAP9 was frequently silenced and its downregulation was associated with poor survival. MAP9 is a microtubule stabilizer that contributes to spindle stability and inhibits colorectal tumorigenesis, supporting the role of MAP9 as a tumor suppressor for preventing CIN in colorectal cancer.

Abstract 1743: Evaluating the effect of USP22 depletion on chromosome instability in colorectal cancer

Abstract Chromosome instability (CIN) is an aberrant phenotype defined as an increase in the rate at which whole chromosomes or large chromosomal fragments are gained or lost, and is observed in virtually all cancer types. Although CIN is associated with aggressive tumors, multi-drug resistance and poor patient prognosis, the genetic defects underlying CIN remain poorly understood. Preliminary data from our laboratory suggest that diminished USP22 (Ubiquitin-Specific Peptidase 22) expression induces CIN. Importantly, USP22 deletions and reduced USP22 expression are frequently observed in multiple cancer types, including colorectal cancer, suggesting diminished USP22 expression/function may be a pathogenic event underlying oncogenesis. Therefore, the present study seeks to evaluate the impact diminished USP22 expression has on CIN by determining whether karyotypically stable colorectal cancer (HCT116) and immortalized fibroblast (hTERT) cell lines can be converted into karyotypically unstable lines following the reduction of USP22 expression. Diminished USP22 expression was achieved by siRNA-based silencing or CRISPR-Cas9-mediated gene knockout (KO) and quantitative image-based single-cell analyses were employed to monitor changes in surrogate markers for CIN, including nuclear areas and chromosome numbers. USP22 silencing induced significant changes in nuclear area as well as increased frequencies of chromosomes gains and losses, which suggest USP22 is a CIN gene. Next, to assess the long-term impact diminished USP22 expression has on CIN, two HCT116 USP22-KO clones were monitored for 10 weeks and revealed dynamic changes in nuclear areas and chromosome numbers over time relative to controls. Collectively, these data identify USP22 as a novel CIN gene and indicate that USP22 deletions in cancer may have long-term impact on CIN and may thereby promote oncogenesis. Further analysis is ongoing to investigate the mechanism through which USP22 depletion induces CIN, and in particular its effect on H2B monoubiquitination. Characterizing the effect diminished USP22 expression has on CIN is the first step towards the development of therapeutic strategies targeting USP22-deficient cancers. Citation Format: Lucile M. Jeusset, Kirk J. McManus. Evaluating the effect of USP22 depletion on chromosome instability in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1743.

MACROD2, an Original Cause of CIN?

The fundamental cause of chromosomal instability in colorectal cancer remains elusive. Genetic deletions of the MACROD2 tumor suppressor could play a major role. Cancer Discov; 8(8); 921-3. ©2018 AACRSee related article by Sakthianandeswaren et al., p. 988.

2030 Chromosomal instability in colorectal cancer according to the age of onset

2030 Chromosomal instability in colorectal cancer according to the age of onset

Deregulation of the Replisome Factor MCMBP Prompts Oncogenesis in Colorectal Carcinomas through Chromosomal Instability

Genetic instability has emerged as an important hallmark of human neoplasia. Although most types of cancers exhibit genetic instability to some extent, in colorectal cancers genetic instability is a distinctive characteristic. Recent studies have shown that deregulation of genes involved in sister chromatid cohesion can result in chromosomal instability in colorectal cancers. Here, we show that the replisome factor minichromosome maintenance complex–binding protein (MCMBP), which is directly involved in the dynamics of the minichromosome maintenance complex and contributes to maintaining sister chromatid cohesion, is transcriptionally misregulated in different types of carcinomas. Cellular studies revealed that both MCMBP knockdown and overexpression in different breast and colorectal cell lines is associated with the emergence of a subpopulation of cells with abnormal nuclear morphology that likely arise as a consequence of aberrant cohesion events. Association analysis integrating gene expression data with clinical information revealed that enhanced MCMBP transcript levels correlate with an increased probability of relapse risk in colorectal cancers and different types of carcinomas. Moreover, a detailed study of a cohort of colorectal tumors showed that the MCMBP protein accumulates to high levels in cancer cells, whereas in normal proliferating tissue its abundance is low, indicating that MCMBP could be exploited as a novel diagnostic marker for this type of carcinoma.

Lamin B2 prevents chromosome instability by ensuring proper mitotic chromosome segregation

The majority of human cancer shows chromosomal instability (CIN). Although the precise mechanism remains largely uncertain, proper progression of mitosis is crucial. B-type lamins were suggested to be components of the spindle matrix of mitotic cells and to be involved in mitotic spindle assembly; thus, B-type lamins may contribute to the maintenance of chromosome integrity. Here, using a proteomic approach, we identified lamin B2 as a novel protein involved in CIN. Lamin B2 expression decreased in colorectal cancer cell lines exhibiting CIN, as compared with colorectal cancer cell lines exhibiting microsatellite instability (MIN), which is mutually exclusive to CIN. Importantly, lamin B2 knockdown in MIN-type colorectal cancer cells induced CIN phenotypes such as aneuploidy, chromosome mis-segregation and aberrant spindle assembly, whereas ectopic expression of lamin B2 in CIN-type colorectal cancer cells prevented their CIN phenotypes. Additionally, immunohistochemical analysis showed a lower expression of lamin B2 in cancer tissues extracted from patients with sporadic colorectal cancer (CIN-type) than that from patients with hereditary non-polyposis colorectal cancer (HNPCC; MIN type). Intriguingly, mitotic lamin B2 in MIN cancer cells was localized outside the spindle poles and mitotic lamin B2 localization was diminished in CIN cancer cells, suggesting an important role of lamin B2 in proper mitotic spindle formation. The obtained results suggest that lamin B2 maintains chromosome integrity by ensuring proper spindle assembly and that its downregulation causes CIN in colorectal cancer.

NIPBL, a Cohesion Loading Factor, Is Somatically Mutated in Gastric and Colorectal Cancers with High Microsatellite Instability

Timely segregation of sister chromatids during the cell cycle is essential for maintaining euploidy [1]. Aberrant segregation into daughter cells promotes aneuploidy and chromosome instability, which are features of many tumors [1]. Chromatid cohesion, a physical linkage of replicated sister chromatids, is mediated by a multiprotein complex called cohesin [1]. NIPBL is a cohesion loading factor that is required for cohesin to interact with chromosomes [2, 3]. NIPBL mutations (missense, nonsense, frameshift and deletion mutations) account for approximately 60 % of Cornelia de Lange syndrome, a congenital disorder with multisystem developmental abnormalities [3]. In Cornelia de Lange syndrome, mutated NIPBL interferes with its interaction with its partners in the cohesion complex [3]. Components in the cohesin complex and cohesin regulatory proteins are frequently altered at expression and mutation levels in human cancers [1]. For example, cohesin protector SGOL1 down-regulation leads to chromosomal instability in colorectal cancers (CRC) [4]. Somatic mutations of cohesion regulatory factor PDS5B have been identified in prostate cancers [5]. These data suggest that cohesin-related genes could be considered potential tumor suppressor genes (TSG) [1]. Somatic mutations of the NIPBL gene have been found in CRC with stable miscrosatellite instability (MSI) that may underlie chromosomal instability in CRC [6]. Together, these data suggest a possibility that NIPBL is a candidate TSG. In a public genome database (http://genome.cse.ucsc. edu/), we found that the human NIPBL gene had mononucleotide repeats in its coding sequences that could be targets for frameshift mutation in cancers with high MSI (MSI-H) [7]. To data, however, the data on NIPBL somatic mutation in gastric (GC) and CRC with MSI-H are lacking. To see whether the mononucleotide repeats in the NIPBL gene are mutated in GC and CRC, we analyzed two A7 and one A8 repeats in NIPBL exon 10 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay with three primer pairs. For this, we used 32 GC with MSI-H, 59 GC with MSS, 47 CRC with MSI-H and 53 CRC with MSS in methacarnfixed tissues. All of the patients of the cancers were Koreans. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosinstained slides by microdissection [8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP. On the SSCP, we observed aberrant bands of the NIPBL gene in 11 cancers (Fig. 1). DNA from normal tissue Min Sung Kim and Chang Hyeok An contributed equally to this study.

A Genome-Wide Study of Cytogenetic Changes in Colorectal Cancer Using SNP Microarrays: Opportunities for Future Personalized Treatment

In colorectal cancer (CRC), chromosomal instability (CIN) is typically studied using comparative-genomic hybridization (CGH) arrays. We studied paired (tumor and surrounding healthy) fresh frozen tissue from 86 CRC patients using Illumina's Infinium-based SNP array. This method allowed us to study CIN in CRC, with simultaneous analysis of copy number (CN) and B-allele frequency (BAF) - a representation of allelic composition. These data helped us to detect mono-allelic and bi-allelic amplifications/deletion, copy neutral loss of heterozygosity, and levels of mosaicism for mixed cell populations, some of which can not be assessed with other methods that do not measure BAF. We identified associations between CN abnormalities and different CRC phenotypes (histological diagnosis, location, tumor grade, stage, MSI and presence of lymph node metastasis). We showed commonalities between regions of CN change observed in CRC and the regions reported in previous studies of other solid cancers (e.g. amplifications of 20q, 13q, 8q, 5p and deletions of 18q, 17p and 8p). From Therapeutic Target Database, we identified relevant drugs, targeted to the genes located in these regions with CN changes, approved or in trials for other cancers and common diseases. These drugs may be considered for future therapeutic trials in CRC, based on personalized cytogenetic diagnosis. We also found many regions, harboring genes, which are not currently targeted by any relevant drugs that may be considered for future drug discovery studies. Our study shows the application of high density SNP arrays for cytogenetic study in CRC and its potential utility for personalized treatment.

Abstract 95: Increased hypomethylation of LINE-1 and Alu in human colorectal cancer metastasis

Abstract Background: Global hypomethylation of cytosines within CpG dinucleotides is one of the distinguishing features of the neoplastic cells in human cancers. More specifically, hypomethylation of evolutionarily conserved repetitive elements (Alu and LINE-1) is associated with increased chromosomal instability in colorectal cancer (CRC). Recent data indicates that transcription start sites of certain proto-oncogenes are located within these repeat elements, and increased hypomethylation of these regions may induce the expression of illegitimate oncogenic transcripts. Primary CRCs demonstrate frequent global Alu and LINE-1 hypomethylation; however, it is unclear whether hypomethylation of these repeat sequences may be associated with a metastatic phenotype Aim: This study was aimed to determine the role of increased hypomethylation of Alu and LINE-1 sequences in CRC metastasis development. Materials and Methods: We analyzed a panel of CRC cells with different metastatic potential, as well as tissues from 50 CRC patients with matched primary colon cancer and corresponding liver metastasis tissues. Alu and LINE-1 methylation levels were determined by quantitative bisulfite pyrosequencing. Results: Lower levels of Alu and LINE-1 methylation were observed in CRC cell lines that came from metastatic foci. When we analyzed Alu and LINE-1 methylation levels in clinical specimens, the levels of Alu methylation in liver metastatis were significantly lower compared to the matched primary CRC tissues (77.2%±8.3 and 80.9%±10.7, respectively; P<0.01). Similarly, the levels of LINE-1 methylation in metastasized liver foci was significantly lower compared to the corresponding matched primary CRC (61.2%±9.7 vs 65.8%±7.0 (P<0.01). The relative demethylation level was higher in LINE-1 (4.6%) than Alu (3.7%) sequences. In further support of our results, methylation analysis of surrounding normal liver tissue showed higher methylation levels for Alu (82.2%±4.7) and LINE1 (71.3%±8.3) compared with both primary CRC and liver metastasis. Conclusion: Our results provide evidence that increased Alu and LINE-1 hypomethylation is a novel feature of liver metastasis from CRC. We speculate that hypomethylation of these repeat elements may inadvertently permit activation of previously silenced proto-oncogenes, which may facilitate a more aggressive malignant phenotype in these advanced stage cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 95. doi:10.1158/1538-7445.AM2011-95

Aurora-A Expression Is Independently Associated with Chromosomal Instability in Colorectal Cancer

AURKA (the official symbol for Aurora-A, STK15, or BTAK) regulates the function of centrosomes, spindles, and kinetochores for proper mitotic progression. AURKA overexpression is observed in various cancers including colon cancer, and a link between AURKA and chromosomal instability (CIN) has been proposed. However, no study has comprehensively examined AURKA expression in relation to CIN or prognosis using a large number of tumors. Using 517 colorectal cancers in two prospective cohort studies, we detected AURKA overexpression (by immunohistochemistry) in 98 tumors (19%). We assessed other molecular events including loss of heterozygosity (LOH) in 2p, 5q, 17q, and 18q, the CpG island methylation phenotype (CIMP), and microsatellite instability (MSI). Prognostic significance of AURKA was evaluated by Cox regression and Kaplan-Meier method. In both univariate and multivariate logistic regressions, AURKA overexpression was significantly associated with CIN (defined as the presence of LOH in any of the chromosomal segments; multivariate odds ratio, 2.97; 95% confidence interval, 1.40–6.29; P = .0045). In multivariate analysis, AURKA was associated with cyclin D1 expression (P = .010) and inversely with PIK3CA mutation (P=.014), fatty acid synthase expression (P=.028), and family history of colorectal cancer (P = .050), but not with sex, age, body mass index, tumor location, stage, CIMP, MSI, KRAS, BRAF, BMI, LINE-1 hypomethylation, p53, p21, β-catenin, or cyclooxygenase 2. AURKA was not significantly associated with clinical outcome or survival. In conclusion, AURKA overexpression is independently associated with CIN in colorectal cancer, supporting a potential role of Aurora kinase-A in colorectal carcinogenesis through genomic instability (rather than epigenomic instability).

Human Sgo1 downregulation leads to chromosomal instability in colorectal cancer

Background and aims:Chromosomal instability (CIN) is recognised as a hallmark of cancer and is caused by a spindle assembly checkpoint disorder or chromosome mis-segregation during mitosis. Although the recent identification...

Mitotic origins of chromosomal instability in colorectal cancer

Mitosis is a crucial part of the cell cycle. A successful mitosis requires the proper execution of many complex cellular behaviors. Thus, there are many points at which mitosis may be disrupted. In cancer cells, chronic disruption of mitosis can lead to unequal segregation of chromosomes, a phenomenon known as chromosomal instability. A majority of colorectal tumors suffer from this instability, and recent studies have begun to reveal the specific ways in which mitotic defects promote chromosomal instability in colorectal cancer.

Association of JC Virus T-Antigen Expression With the Methylator Phenotype in Sporadic Colorectal Cancers

JC virus (JCV) is a polyomavirus that ubiquitously infects humans and has been implicated in various human cancers. JCV encodes a "transforming" gene, T-antigen (T-Ag), which is believed to mediate the oncogenic potential of the virus. We have previously shown that JCV DNA sequences are usually present in human colorectal cancers (CRCs), and we have provided in vitro evidence that JCV can induce chromosomal instability (CIN) in CRC cells. This study tests the hypothesis that JCV T-Ag expression correlates with one or more forms of genomic or epigenetic instability in sporadic CRCs. We characterized 100 sporadic CRCs for microsatellite instability (MSI) and CIN. PCR amplifications were performed for T-Ag sequences, and immunohistochemical (IHC) staining was performed to detect T-Ag expression. De novo methylation of the promoter regions of nine putative tumor suppressor genes thought to play a role in colorectal carcinogenesis was studied by methylation-specific PCR. JCV T-Ag DNA sequences were found in 77% of the CRCs and 56% of these cancers (or 43% of the total) expressed T-Ag by IHC. Significant associations were observed between T-Ag expression and CIN in CRCs (P = .017) and between T-Ag expression and promoter methylation of multiple genes (P = .01). The association between T-Ag expression and promoter methylation in CRC suggests that this viral oncogene may induce methylator phenotype and that JCV may be involved in CRC through multiple mechanisms of genetic and epigenetic instability.

Combined Array-Comparative Genomic Hybridization and Single-Nucleotide Polymorphism-Loss of Heterozygosity Analysis Reveals Complex Changes and Multiple Forms of Chromosomal Instability in Colorectal Cancers

Cancers with chromosomal instability (CIN) are held to be aneuploid/polyploid with multiple large-scale gains/deletions, but the processes underlying CIN are unclear and different types of CIN might exist. We investigated colorectal cancer cell lines using array-comparative genomic hybridization (CGH) for copy number changes and single-copy number polymorphism (SNP) microarrays for allelic loss (LOH). Many array-based CGH changes were not found by LOH because they did not cause true reduction-to-homozygosity. Conversely, many regions of SNP-LOH occurred in the absence of copy number change, comprising an average per cell line of 2 chromosomes with complete LOH; 1-2 terminal regions of LOH (mitotic recombination); and 1 interstitial region of LOH. SNP-LOH detected many novel changes, representing possible locations of uncharacterized tumor suppressor loci. Microsatellite unstable (MSI+) lines infrequently showed gains/deletions or whole-chromosome LOH, but their near-diploid karyotypes concealed mitotic recombination frequencies similar to those of MSI- lines. We analyzed p53 and chromosome 18q (SMAD4) in detail, including mutation screening. Almost all MSI- lines showed LOH and/or deletion of p53 and 18q; some near-triploid lines had acquired three independent changes at these loci. We found consistent results in primary colorectal cancers. Overall, the distributions of mitotic recombination and whole-chromosome LOH in the MSI- cell lines differed significantly from random, with some lines having much higher than expected levels of these changes. Moreover, lines with more LOH changes had significantly fewer copy number changes. These data suggest that CIN is not synonymous with copy number change and some cancers have a specific tendency to whole-chromosome deletion and regain or to mitotic recombination.

Genomic instability and colorectal cancer

Colorectal cancer results from the accumulation of genetic and epigenetic alterations that transform colonic epithelial cells into colon adenocarcinoma cells. The loss of genomic stability appears to be a key molecular and pathogenetic step that occurs early in the tumorigenesis process and permits the acquisition of sufficient alterations in tumor suppressor genes and oncogenes in a clone of cells to result in cancer. Three predominant forms of genomic instability have been identified in colon cancer: 1) microsatellite instability (MSI), 2) chromosome instability (CIN), and 3) chromosomal translocations. Substantial progress has been made recently regarding potential causes of MSI and CIN in colorectal cancer. Furthermore, in addition to genomic instability, a form of epigenomic instability has been recently identified that results in the aberrant methylation of tumor suppressor genes. The discovery of the cause and role of genomic and epigenomic instability in colon cancer formation has the potential to yield more effective chemotherapy strategies for colon cancer.

APC and chromosome instability in colorectal cancer

Colon cancer is a common disease that can be sporadic or familial. An inactivated adenomatous polyposis coli (APC) suppressor gene is found in over 80% of colorectal tumors, this being an early alteration in the development of adenomatous polyps. APC function is not only critical for tumor initiation and progression, and chromosome instability (CIN) is another characteristic dependent at least partly on APC mutations.

No mutations of the Bub1 gene in human gastric carcinomas.

Chromosomal instability in colorectal cancers is associated with functional loss of a mitotic check point partly due to mutations of the Bub1, one of the mitotic check point genes. However, mutation of coding sequences of human Bub1 gene has not been fully elucidated in gastric carcinomas. We performed sequencing analysis on reverse transcriptase-polymerase chain reaction (RT-PCR) product of the Bub1 cDNA (entire coding sequence) from 5 human gastric carcinomas as well as on genomic PCR products of Bub1 kinase domain from 7 gastric carcinoma tissues. Although sequencing analysis of the Bub1 cDNA revealed several point mutations in 2 gastric carcinoma cases, we could not confirm the mutations by analyzing genomic DNA. Furthermore, genomic DNA sequencing revealed no mutations in the kinase domain of the Bub1 gene in any gastric carcinoma examined. These results suggest that mutational inactivation of the Bub1 gene might not play a key role in human stomach carcinogenesis.

Mutations in the APC tumour suppressor gene cause chromosomal instability.

Two forms of genetic instability have been described in colorectal cancer: microsatellite instability and chromosomal instability. Microsatellite instability results from mutations in mismatch repair genes; chromosomal instability is the hallmark of many colorectal cancers, although it is not completely understood at the molecular level. As truncations of the Adenomatous Polyposis Coli (APC) gene are found in most colorectal tumours, we thought that mutations in APC might be responsible for chromosomal instability. To test this hypothesis, we examined mouse embryonic stem (ES) cells homozygous for Min (multiple intestinal neoplasia) or Apc1638T alleles. Here we show that Apc mutant ES cells display extensive chromosome and spindle aberrations, providing genetic evidence for a role of APC in chromosome segregation. Consistent with this, APC accumulates at the kinetochore during mitosis. Apc mutant cells form mitotic spindles with an abundance of microtubules that inefficiently connect with kinetochores. This phenotype is recapitulated by the induced expression of a 253-amino-acid carboxy-terminal fragment of APC in microsatellite unstable colorectal cancer cells. We conclude that loss of APC sequences that lie C-terminal to the beta-catenin regulatory domain contributes to chromosomal instability in colorectal cancer.