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.     

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.     

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 in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.     

EphA2 bears plasticity to tumor invasion

The mammalian intestinal epithelium is one of the most actively self-renewing tissues, which is constantly replenished by pluripotent intestinal stem cells (ISCs). This remarkable characteristic seems to impact in its high propensity for malignant transformation. Indeed, many of the molecular pathways that regulate normal intestinal homeostasis appear involved in colorectal carcinogenesis. Inactivating mutations of the APC (Adenomatous Polyposis Coli) gene is a hallmark of colorectal cancer. The main tumor suppressive function of Apc is to negatively regulate Wnt signaling. Targeted deletion of Apc in the murine intestine, and more recently in the zebrafish gut, recapitulate many aspects of the human disease. Work in Drosophila now reveals that the role of APC in the intestine is ancient and highly conserved across species. In support of these findings, we present data which suggests that APC1 may be a marker for adult ISCs in Drosophila and is required specifically within the ISCs to regulate intestinal homeostasis. Here we discuss the similarities and differences between these model organisms in regards to the role of Wnt signaling and APC in intestinal homeostasis and transformation.     

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.     

Oncogenic Adenomatous Polyposis Coli Mutants Impair the Mitotic Checkpoint through Direct Interaction with Mad2

The majority of colorectal tumors are aneuploid because of the underlying chromosome instability (CIN) phenotype, in which a defective mitotic checkpoint is implicated. Adenomatous polyposis coli (APC), a tumor suppressor gene that is commonly mutated in colon cancers, has been suggested in causing CIN; however, the molecular mechanism remains unresolved. In this study, we report an interaction of tumor-associated N-terminal APC fragments (N-APC) with Mad2, an essential mitotic checkpoint protein, providing a direct molecular support for linking APC mutations to the generation of CIN. N-APC interacts with Mad2 in Xenopus egg extracts, colon cancer cells, and in vitro with purified components. The interaction between N-APC and Mad2 decreases the soluble pool of Mad2, which is essential for Mad2 cycling and releasing from unattached kinetochores to produce a diffusible |P`wait anaphase|P' signal. Addition of such an N-APC mutant of egg extracts inactivates the mitotic checkpoint. Expressing a tumor-associated N-APC mutant in mammalian cells with an intact mitotic checkpoint produces premature anaphase onset with missegregated chromosomes.     

Mutant KRAS in aberrant crypt foci (ACF): initiation of colorectal cancer?

Since aberrant crypt foci (ACF) were first described in 1987, they have been the subjects of hundreds of papers; however, the debate continues about their role in colorectal tumorigenesis. This review focuses on the many phenotypic, genetic and epigenetic alterations in ACF that support the hypothesis that ACF are putative precursors of colorectal cancer in both humans and experimental animals. Human ACF, both with and without dysplasia, are monoclonal and display evidence of chromosomal instability. Both of these characteristics are shared by colorectal cancers. While most ACF do not have APC mutations, a large proportion has KRAS mutations and methylated SFRP1 and SFRP2 genes. This epigenetic inactivation gives rise to constitutive Wnt signaling in these putative precursors of colorectal cancer.     

Characterisation of the human APC1, the largest subunit of the anaphase-promoting complex

Accurate segregation of sister chromatids during mitosis is necessary to avoid the aneuploidy found in many cancers. The spindle checkpoint, which monitors the metaphase to anaphase transition, has been shown to be defective in cancers with chromosomal instability. This checkpoint regulates the anaphase-promoting complex or cyclosome (APC/C), a cell cycle ubiquitin ligase regulating among other things sister chromatid separation. We have previously investigated the mouse Apc1 protein (previously also called Tsg24), the largest subunit of the APC/C. We have now sequenced a full-length human APC1 cDNA, mapped its chromosomal location, and analysed its intron-exon boundaries. We have also investigated the RNA and protein expression of the Apc1 and other APC/C components in normal and cancer cells and the relative occurrence of expressed sequence tags (ESTs) representing APC subunits from different tissues. The different APC/C subunits are expressed in most tissues and cell types at fairly constant levels relative to each other, suggesting that they perform their functions as part of a complex. A difference from this pattern is however seen for the APC6, which in some cases is more strongly expressed, suggesting a special function for this protein in certain tissues and cell types.     

Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene.

Ectopic expression of certain Wnt genes in mouse mammary tissue is tumorigenic, and mutations that stabilize beta-catenin are found in various human cancers including colorectal cancer. To determine the role of stabilized beta-catenin in intestinal tumorigenesis in mice, we constructed by embryonic stem (ES) cell-mediated homologous recombination, a mutant beta-catenin allele whose exon 3 was sandwiched by loxP sequences. When the germline heterozygotes were crossed with mice expressing Cre recombinase in the intestines, the serines and threonine encoded by exon 3 and to be phosphorylated by glycogen synthase kinase 3beta (GSK3beta) were deleted in the offspring intestines, which caused adenomatous intestinal polyps resembling those in Apc(Delta716) knockout mice. Some nascent microadenomas were also found in the colon. These results present experimental genetic evidence that activation of the Wnt signaling pathway can cause intestinal and colonic tumors.     

Retinoic acid inhibits beta-catenin through suppression of Cox-2: a role for truncated adenomatous polyposis coli

Mutations in adenomatous polyposis coli (APC) underlie the earliest stages of colorectal carcinogenesis. Consequences of APC mutation include stabilization of beta-catenin, dysregulation of cyclooxygenase-2 (COX-2) expression, and loss of retinoic acid production, events with poorly defined interactions. Here we showed that treatment of zebrafish expressing a truncated form of Apc with either retinoic acid or a selective COX-2 inhibitor decreased beta-catenin protein levels and downstream signaling events. Interestingly, the destruction of beta-catenin in apc mutant embryos following Cox-2 inhibition required the presence of truncated Apc. These findings support roles for retinoic acid and Cox-2 in regulating the stability of beta-catenin following Apc loss. Furthermore, truncated Apc appears to retain the ability to target beta-catenin for destruction, but only in the absence of Cox-2 activity. This novel function of truncated Apc may provide a molecular basis for the efficacy of COX-2 inhibitors in the treatment of colon cancer.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces genetic changes in murine intestinal tumours and cells with ApcMin mutation

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the mutagenic heterocyclic amines derived from cooked meat. In previous animal studies, spontaneous tumour formation in B6(Min/+) mice was associated with somatic loss of the wild-type Apc+ allele by loss of the entire chromosome 18 or by recombination. The objective of this study was to examine genetic changes caused by PhIP-exposure in a mouse intestinal cell line and in tumours from hybrid mice by keeping track of the chromosomes carrying the two Apc alleles. We transformed the SV40 T-immortalised intestinal epithelial cell line IMCE, derived from the B6(Min/+) mice by exposure to N-OH-PhIP, and studied the effect on Apc status and chromosome 18. Eighteen transformed cultures were obtained and all of them had retained the Apc+ allele. Five of seven transformed cultures were tumorigenic after implantation in nude mice. Chromosomal analysis of these five cultures and the parent IMCE cell line showed that the IMCE cells were near-tetraploid with an average of 77 chromosomes/cell, while the tumorigenic cell cultures were all triploid to hyper-triploid with a range of 61-69 chromosomes/cell. The number of copies of chromosome 18 was about four in the IMCE line and this copy number was retained in the transformed lines derived from IMCE. Changes in chromosome 18 and Apc during tumour development in vivo were examined in spontaneously formed and PhIP-induced intestinal tumours from two hybrid mice strains, i.e. B6(Min/+) - a murine FAP model - crossed with either AKR/J or A/J. We evaluated the allelic status of Apc, and the heterogenic microsatellite markers D18Mit19 and D18Mit4, located at the upper and lower ends of chromosome 18, respectively. In tumours from untreated animals, instability in the D18Mit19 and Apc was observed. Upon PhIP exposure, the B6(Min/A+) hybrid mouse tumours differed distinctly in genetic profile from those obtained from untreated animals and we detected three genetically different tumour groups, all of which had apparently retained Apc+. One group had allelic balance between the Apc(Min) and Apc+, the second had allelic imbalance between the Apc and D18Mit4 alleles, indicative of chromosomal stability in the first group and instability in the lower end of chromosome 18 in the second group, respectively. The third group showed variable allelic status of the three markers. A similar change in genetic profile was also seen in intestinal tumours of PhIP-exposed B6(Min/AKR+) hybrid mice, but it was less pronounced. Chromosomal breaks and/or recombinational events could be alternative explanations for the observed allelic imbalances in chromosome 18 markers in intestinal tumours from PhIP-exposed mice.     

Genomic instability and cancer

Tumorigenesis can be viewed as an imbalance between the mechanisms of cell-cycle control and mutation rates within the genes. Genomic instability is broadly classified into microsatellite instability (MIN) associated with mutator phenotype, and chromosome instability (CIN) recognized by gross chromosomal abnormalities. Three intracellular mechanisms are involved in DNA damage repair that leads to mutator phenotype. They include the nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). The CIN pathway is typically associated with the accumulation of mutations in tumor suppressor genes and oncogenes. Defects in DNA MMR and CIN pathways are responsible for a variety of hereditary cancer predisposition syndromes including hereditary non-polyposis colorectal carcinoma (HNPCC), Bloom syndrome, ataxia-telangiectasia, and Fanconi anaemia. While there are many genetic contributors to CIN and MIN, there are also epigenetic factors that have emerged to be equally damaging to cell-cycle control. Hypermethylation of tumor suppressor and DNA MMR gene promoter regions, is an epigenetic mechanism of gene silencing that contributes to tumorigenesis. Telomere shortening has been shown to increase genetic instability and tumor formation in mice, underscoring the importance of telomere length and telomerase activity in maintaining genomic integrity. Mouse models have provided important insights for discovering critical pathways in the progression to cancer, as well as to elucidate cross talk among different pathways. This review examines various molecular mechanisms of genomic instability and their relevance to cancer.     

Mechanisms of APC-driven tumorigenesis: lessons from mouse models.

Colorectal cancer still represents one of the most common causes of morbidity and mortality among Western populations. The adenomatous polyposis coli (APC) gene, originally identified as the gene responsible for familial adenomatous polyposis (FAP), an inherited predisposition to multiple colorectal tumors, is now considered as the true "gatekeeper" of colonic epithelial proliferation. It is mutated in the vast majority of sporadic colorectal tumors, and inactivation of both APC alleles occurs at early stages of tumor development in man and mouse. The study of FAP has also led to one of the most consistent genotype-phenotype correlations in hereditary cancer. However, great phenotypic variability is still observed not only among carriers of the identical APC mutation from unrelated families but also from within the same kindred. The generation of several mouse models carrying specific Apc mutations on the same inbred genetic background has confirmed the genotype-phenotype correlations initially established among FAP patients, as well as provided important insights into the mechanisms of colorectal tumor formation. Here we review the major features of the available animal models for FAP and attempt the formulation of a hypothetical model for APC-driven tumorigenesis based on the observed genetic and phenotypic variability in mouse and man.     

Possible causes of chromosome instability: comparison of chromosomal abnormalities in cancer cell lines with mutations in BRCA1, BRCA2, CHK2 and BUB1

A large proportion of epithelial cancers show the chromosome-instability phenotype, in which they have many chromosome abnormalities. This is thought to be the result of mutations that disrupt chromosome maintenance, but the causative mutations are not known. We identified cell lines known to have mutations that might cause chromosome instability, and examined their karyotypes. Two cell lines, the breast cancer line HCC1937 and the pancreatic cancer line CAPAN-1, that have mutations respectively in BRCA1 and BRCA2, had very abnormal karyotypes, with many structural and numerical chromosome changes and substantial variation between metaphases. However, two colorectal cancer lines with mutations in BUB1, a spindle checkpoint protein involved in chromosome segregation, had rather simple near-tetraploid karyotypes, with minimal loss or gain of chromosomes other than the endoreduplication event, and minimal structural change. Apart from tetraploidy, these karyotypes were typical of colorectal lines considered to be chromosomally stable. Two lines derived from the same tumour, DLD-1 and HCT-15, with bi-allelic mutation of CHK2, had karyotypes that were typical of near-diploid colorectal lines considered chromosomally stable. The karyotypes observed supported the proposed role for BRCA1 and BRCA2 mutations in chromosomal instability, but showed that the tested mutations in BUB1 and CHK2 did not result in karyotypes that would have been predicted if they were sufficient for chromosomal instability.     

APC mutations and other genetic and epigenetic changes in colon cancer

Relationships between adenomatous polyposis coli (APC) mutations, BRAF V600E mutations, and the CpG island methylator phenotype (CIMP) in colon cancer have not been explored. In addition, controversies exist about the proportion of tumors with APC mutations in the mutation cluster region (MCR); how commonly APC, Ki-ras, and p53 mutations occur in the same tumor; and whether APC mutations occur in sporadic microsatellite-unstable tumors. The APC gene was therefore sequenced in 90 colonic adenocarcinomas previously evaluated for CIMP, microsatellite instability, BRAF, Ki-ras, and p53. APC mutations were inversely related to BRAF mutations (P = 0.0003) and CIMP (P = 0.02) and directly related to p53 and Ki-ras mutations (P = 0.04). Slightly more than half of APC mutations occurred outside of the MCR, and frameshift mutations were more likely than nonsense mutations to occur in the MCR (21 of 28 versus 12 of 40, P = 0.0003). APC mutations were found in sporadic microsatellite-unstable tumors and were more likely to be frameshifts in short nucleotide repeats (P = 0.007). The occurrence of APC, Ki-ras, and p53 mutations together in the same tumor was uncommon (11.1%). In conclusion, an analysis restricted to the MCR will miss more than half of APC mutations as well as mischaracterize their mutational spectrum. The conventional wisdom that most colon cancers contain APC, Ki-ras, and p53 mutations is incorrect. Microsatellite instability may precede acquisition of APC mutations in sporadic microsatellite-unstable tumors. The relationships of APC mutations to other genetic and epigenetic alterations add to the already impressive genetic heterogeneity of colon cancer.     

Wnt pathway mutations selected by optimal beta-catenin signaling for tumorigenesis

Mutations in components of the Wnt/beta-catenin pathway are observed to be the earliest initiating event for most colorectal tumors. The majority of the mutations occur in the tumor suppressor adenomatous polyposis coli (APC), even though there are other genes that are capable of modulating the pathway activity. Moreover, the specific APC mutations associated in colon cancer indicate the possibility that the tumor selects for certain truncated forms of APC that partially retain its function, namely, inhibition of beta-catenin. We estimated the effects of various mutations in APC and other known mutations using a recent mathematical model of the Wnt pathway that was constructed to represent the conserved core molecular events. We provide evidence that APC mutations are selected not based on the maximal level of beta-catenin but rather based on distinct state of activity that appears to be optimal for the tissue-specific tumorigenesis. This optimal level is determined by balancing beta-catenin signaling and the induction of Axin2 that acts as a potent negative feedback. The predominant pattern of APC mutations may provide synergistic oncogenic effects that promote colorectal tumorigenesis: the optimal signaling for cell survival and renewal, disrupted cell adhesion, chromosomal instability, and altered asymmetric division of stem cells.     

Sixty years of follow-up of Hiroshima and Nagasaki survivors: current progress in molecular epidemiology studies

This article provides an overview of the on-going molecular epidemiology studies among atomic-bomb survivors conducted at the Radiation Effects Research Foundation in Japan. The focus is on: (a) inter-individual variations in sensitivity to radiation-induced somatic mutations (glycophorin A (GPA) mutations) and their potential relevance to differences in susceptibility to radiation-related cancers and (b) the role of specific mutations/rearrangements in radiation-induced thyroid and colorectal cancers. The glycophorin A mutant fractions showed large differences between the survivors at each of the estimated bone marrow doses. Of note is the finding at doses>or=1 Gy; that the slope of the mutant fraction was significantly higher in the 'cancer group' than in the 'non-cancer group'. This study provided the basis for validating the use of gammaH2AX and reticulocyte micronucleus assays for evaluating radiosensitivity differences and genetic instability, respectively, in our studies in the coming years. Preliminary results from our molecular oncology studies on adult-onset papillary thyroid cancer provide evidence for the induction of RET/PTC rearrangements and BRAF point mutation (both known to be early stage events in adult-onset papillary thyroid cancer) but with a difference: cases associated with the rearrangements were more frequent at high doses, and developed sooner than those with BRAF mutation. In the case of colorectal cancer, the results suggest that radiation exposure might influence microsatellite instability (MSI) status through MSI-related epigenetic and genetic alterations-processes that might occur in the early stage of colorectal carcinogenesis.