Candidate tumor suppressor genes at FRA7G are coamplified with MET and do not suppress malignancy in a gastric cancer

Common fragile sites predispose to specific chromosomal breakage associated with deletion, amplification, and/or translocation in certain forms of cancer. Chromosomal fragile sites not only are susceptible to DNA instability in cancer cells, but may also be associated with genes that contribute to the neoplastic process. FRA7G is a common fragile site containing the candidate tumor suppressor genes CAV1, CAV2, and TESTIN (TES). The human gastric cancer cell line GTL-16 has an amplification of this genomic region and was used to seek evidence for the suppressor candidacy of one of these genes. Our results demonstrate that CAV1, CAV2, and TESTIN are coamplified with the MET oncogene and overexpressed in GTL-16. Somatic mutation was not detected in the coding regions of these genes, although they were each overexpressed. The results show that CAV1, CAV2, and TESTIN are not tumor suppressor genes in this gastric cancer.     

Characterization of the human common fragile site FRA2G

Common fragile sites are nonrandom loci that show gaps and breaks when cells are exposed to specific compounds. They are preferentially involved in recombination, chromosomal rearrangements, and foreign DNA integration. These sites have been suggested to play a role in chromosome instability observed in cancer. In this work we used a FISH-based approach to identify a BAC contig that spans the FRA2G fragile site located at the 2q31 region. Our observations indicate that a very fragile region spanning at least 450 kb is present within a large fragile region that extends over 1 Mb. At least seven genes are mapped in the fragile region. One of these seems to be a good candidate as a potential tumor suppressor gene impaired by the recurrent deletions observed at the 2q31 region in some neoplasms. In the fragile region, a considerable number of regions of high flexibility that may be related to the fragility are present.     

Common fragile genes

Common chromosome fragile sites show susceptibility to DNA damage, leading to alterations that contribute to cancer development. The cloning and characterization of fragile sites have demonstrated that fragile sites are associated with genes that relate to tumorigenesis. Identification of the basis of instability at fragile sites and the related genes provides an entree to understanding of important aspects of chromosomal instability, a prominent feature of neoplastic genomes. FHIT/FRA3B and WWOX/FRA16D, the most sensitive common fragile genes in the human genome, function as tumor suppressor genes. The common features of these two common fragile genes are summarized, and suggest clues to understanding the relation between genomic instability and tumor biology.     

Sites of genetic instability in mitosis and cancer

Certain chromosomal regions called common fragile sites are prone to difficulty during replication. Many tumors have been shown to contain alterations at fragile sites. Several models have been proposed to explain why these sites are unstable. Here we describe work to investigate models of fragile site instability using a yeast artificial chromosome carrying human DNA from a common fragile site region. In addition, we describe a yeast system to investigate whether repair of breaks at a naturally occurring fragile site in yeast, FS2, involves mitotic recombination between homologous chromosomes, leading to loss of heterozygosity (LOH). Our initial evidence is that repair of yeast fragile site breaks does lead to LOH, suggesting that human fragile site breaks may similarly contribute to LOH in cancer. This work is focused on gaining understanding that may enable us to predict and prevent the situations and environments that promote genetic changes that contribute to tumor progression.     

DNA instability at chromosomal fragile sites in cancer

Human chromosomal fragile sites are specific genomic regions which exhibit gaps or breaks on metaphase chromosomes following conditions of partial replication stress. Fragile sites often coincide with genes that are frequently rearranged or deleted in human cancers, with over half of cancer-specific translocations containing breakpoints within fragile sites. But until recently, little direct evidence existed linking fragile site breakage to the formation of cancer-causing chromosomal aberrations. Studies have revealed that DNA breakage at fragile sites can induce formation of RET/PTC rearrangements, and deletions within the FHIT gene, resembling those observed in human tumors. These findings demonstrate the important role of fragile sites in cancer development, suggesting that a better understanding of the molecular basis of fragile site instability is crucial to insights in carcinogenesis. It is hypothesized that under conditions of replication stress, stable secondary structures form at fragile sites and stall replication fork progress, ultimately resulting in DNA breaks. A recent study examining an FRA16B fragment confirmed the formation of secondary structure and DNA polymerase stalling within this sequence in vitro, as well as reduced replication efficiency and increased instability in human cells. Polymerase stalling during synthesis of FRA16D has also been demonstrated. The ATR DNA damage checkpoint pathway plays a critical role in maintaining stability at fragile sites. Recent findings have confirmed binding of the ATR protein to three regions of FRA3B under conditions of mild replication stress. This review will discuss recent advances made in understanding the role and mechanism of fragile sites in cancer development.     

Common fragile sites

Aphidicolin-induced common fragile sites are site-specific gaps or breaks seen on metaphase chromosomes after partial inhibition of DNA synthesis. These fragile sites were first recognized during the early studies of the fragile X syndrome and are induced by the same conditions of folate or thymidylate stress used to induce the fragile X site. Common fragile sites are normally stable in cultured human cells. However, following induction with replication inhibitors, they display a number of characteristics of unstable and highly recombinogenic DNA. From the many studies that have cloned and characterized fragile sites, it is now known that these sites extend over large regions, are associated with genes, exhibit late or delayed replication, and contain regions of high flexibility but are otherwise unremarkable in sequence. Studies showing that fragile sites and their associated genes are frequently deleted or rearranged in cancer cells have clearly demonstrated their importance in genome instability in tumorigenesis. Yet until recently, very little was known about the molecular mechanisms involved in their stability. Recent findings showing that the key checkpoint genes ATR and BRCA1 are critical for genome stability at fragile sites have shed new light on these mechanisms and on the biological significance of common fragile sites.     

Fragile Site Instability in Saccharomyces cerevisiae Causes Loss of Heterozygosity by Mitotic Crossovers and Break-Induced Replication

Loss of heterozygosity (LOH) at tumor suppressor loci is a major contributor to cancer initiation and progression. Both deletions and mitotic recombination can lead to LOH. Certain chromosomal loci known as common fragile sites are susceptible to DNA lesions under replication stress, and replication stress is prevalent in early stage tumor cells. There is extensive evidence for deletions stimulated by common fragile sites in tumors, but the role of fragile sites in stimulating mitotic recombination that causes LOH is unknown. Here, we have used the yeast model system to study the relationship between fragile site instability and mitotic recombination that results in LOH. A naturally occurring fragile site, FS2, exists on the right arm of yeast chromosome III, and we have analyzed LOH on this chromosome. We report that the frequency of spontaneous mitotic BIR events resulting in LOH on the right arm of yeast chromosome III is higher than expected, and that replication stress by low levels of polymerase alpha increases mitotic recombination 12-fold. Using single-nucleotide polymorphisms between the two chromosome III homologs, we mapped the locations of recombination events and determined that FS2 is a strong hotspot for both mitotic reciprocal crossovers and break-induced replication events under conditions of replication stress.     

Two Distinct Categories of Focal Deletions in Cancer Genomes

One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we’ve addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.     

Common fragile sites as targets for chromosome rearrangements

Common fragile sites are large chromosomal regions that preferentially exhibit gaps or breaks after DNA synthesis is partially perturbed. Fragile site instability in cultured cells is well documented and includes gaps and breaks on metaphase chromosomes, translocation and deletions breakpoints, and sister chromosome exchanges. In recent years, much has been learned about the genomic structure at fragile sites and the cellular mechanisms that monitor their stability. The study of fragile sites has merged with that of cell cycle checkpoints and DNA repair, with multiple proteins from these pathways implicated in fragile site stability, including ATR, BRCA1, CHK1, and RAD51. Since their discovery, fragile sites have been implicated in constitutional and cancer chromosome rearrangements in vivo and recent studies suggest that common fragile sites may serve as markers of chromosome damage caused by replication stress during early tumorigenesis. Here we review the relationship of fragile sites to chromosome rearrangements, particularly in tumor cells, and discuss the mechanisms that may be involved.     

Inhibition of topoisomerase I prevents chromosome breakage at common fragile sites

Common fragile sites are loci that preferentially form gaps and breaks on metaphase chromosomes when DNA synthesis is perturbed, particularly after treatment with the DNA polymerase inhibitor, aphidicolin. We and others have identified several cell cycle checkpoint and DNA repair proteins that influence common fragile site stability. However, the initial events underlying fragile site breakage remain poorly understood. We demonstrate here that aphidicolin-induced gaps and breaks at fragile sites are prevented when cells are co-treated with low concentrations of the topoisomerase I inhibitor, camptothecin. This reduction in breakage is accompanied by a reduction in aphidicolin-induced RPA foci, CHK1 and RPA2 phosphorylation, and PCNA monoubiquitination, indicative of reduced levels of single stranded DNA. Furthermore, camptothecin reduces spontaneous fragile site breakage seen in cells lacking ATR, even in the absence of aphidicolin. These data from cultured human cells demonstrate that topoisomerase I activity is required for DNA common fragile site breaks and suggest that polymerase–helicase uncoupling is a key initial event in this process.     

Molecular parameters of genome instability: roles of fragile genes at common fragile sites

Common chromosome fragile sites occur at specific sequences within mammalian genomes that exhibit apparent single-stranded regions in mitotic chromosomes on exposure of cells to replication stress. Recent progress in the characterization of sequences, and more precise mapping of common fragile sites in mammalian and yeast genomes, has led to the exact placement of large common fragile regions straddling the borders of chromosomal G and R bands, with early and late replicating genomic regions, respectively, and could lead to breakthroughs in understanding the function of these evolutionarily conserved but highly recombinogenic chromosome elements. Deficiency of genes involved in DNA damage checkpoint responses, such as ATR, CHK1, HUS1 leads to increased frequency of fragile site instability. Some of these fragile sites, particularly FRA3B, encode genes that are themselves involved in the protection of cells from DNA damage through various mechanisms. Protection of mammalian genomes from accumulation of DNA damage in somatic cells is critical during development, puberty and during the reproductive lifespan, and occurs through mechanisms involving surveillance of the genome for damage, signals to the cell cycle machinery to stop cell cycle progression, signals to repair machinery to repair damage, signals to resume cycling or initiate apoptotic programs, depending on the extent of damage and repair. When genes involved in these processes are altered or deleted, cancer can occur. The tumor suppressor gene, FHIT at the FRA3B locus, and possibly other fragile genes, is a common target of damage and paradoxically encodes a protein with roles in protection from DNA damage.     

Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

Genomic instability drives tumorigenesis, but how it is initiated in sporadic neoplasias is unknown. In early preneoplasias, alterations at chromosome fragile sites arise due to DNA replication stress. A frequent, perhaps earliest, genetic alteration in preneoplasias is deletion within the fragile FRA3B/FHIT locus, leading to loss of Fhit protein expression. Because common chromosome fragile sites are exquisitely sensitive to replication stress, it has been proposed that their clonal alterations in cancer cells are due to stress sensitivity rather than to a selective advantage imparted by loss of expression of fragile gene products. Here, we show in normal, transformed, and cancer-derived cell lines that Fhit-depletion causes replication stress-induced DNA double-strand breaks. Using DNA combing, we observed a defect in replication fork progression in Fhit-deficient cells that stemmed primarily from fork stalling and collapse. The likely mechanism for the role of Fhit in replication fork progression is through regulation of Thymidine kinase 1 expression and thymidine triphosphate pool levels; notably, restoration of nucleotide balance rescued DNA replication defects and suppressed DNA breakage in Fhit-deficient cells. Depletion of Fhit did not activate the DNA damage response nor cause cell cycle arrest, allowing continued cell proliferation and ongoing chromosomal instability. This finding was in accord with in vivo studies, as Fhit knockout mouse tissue showed no evidence of cell cycle arrest or senescence yet exhibited numerous somatic DNA copy number aberrations at replication stress-sensitive loci. Furthermore, cells established from Fhit knockout tissue showed rapid immortalization and selection of DNA deletions and amplifications, including amplification of the Mdm2 gene, suggesting that Fhit loss-induced genome instability facilitates transformation. We propose that loss of Fhit expression in precancerous lesions is the first step in the initiation of genomic instability, linking alterations at common fragile sites to the origin of genome instability.     

Secondary structure formation and DNA instability at fragile site FRA16B

Human chromosomal fragile sites are specific loci that are especially susceptible to DNA breakage following conditions of partial replication stress. They often are found in genes involved in tumorigenesis and map to over half of all known cancer-specific recurrent translocation breakpoints. While their molecular basis remains elusive, most fragile DNAs contain AT-rich flexibility islands predicted to form stable secondary structures. To understand the mechanism of fragile site instability, we examined the contribution of secondary structure formation to breakage at FRA16B. Here, we show that FRA16B forms an alternative DNA structure in vitro. During replication in human cells, FRA16B exhibited reduced replication efficiency and expansions and deletions, depending on replication orientation and distance from the origin. Furthermore, the examination of a FRA16B replication fork template demonstrated that the majority of the constructs contained DNA polymerase paused within the FRA16B sequence, and among the molecules, which completed DNA synthesis, 81% of them underwent fork reversal. These results strongly suggest that the secondary-structure-forming ability of FRA16B contributes to its fragility by stalling DNA replication, and this mechanism may be shared among other fragile DNAs.     

普通型脆性位点的研究进展

脆性位点是DNA合成被部分抑制之后,在中期染色体上发生的位点特异的缺口或断裂区域,它也是基因组不稳定的区域。通常脆性位点在体细胞中是稳定的,但它们在许多癌细胞中经常发生缺失或重排。在已发现的80多种脆性位点基因中,研究最多的是FHIT和WWOX,它们与肿瘤的发生发展密切相关。简要综述了普通型脆性位点的研究进展及其与肿瘤的关系。     

Myc oncogene-induced genomic instability: DNA palindromes in bursal lymphomagenesis

Genetic instability plays a key role in the formation of naturally occurring cancer. The formation of long DNA palindromes is a rate-limiting step in gene amplification, a common form of tumor-associated genetic instability. Genome-wide analysis of palindrome formation (GAPF) has detected both extensive palindrome formation and gene amplification, beginning early in tumorigenesis, in an experimental Myc-induced model tumor system in the chicken bursa of Fabricius. We determined that GAPF-detected palindromes are abundant and distributed nonrandomly throughout the genome of bursal lymphoma cells, frequently at preexisting short inverted repeats. By combining GAPF with chromatin immunoprecipitation (ChIP), we found a significant association between occupancy of gene-proximal Myc binding sites and the formation of palindromes. Numbers of palindromic loci correlate with increases in both levels of Myc over-expression and ChIP-detected occupancy of Myc binding sites in bursal cells. However, clonal analysis of chick DF-1 fibroblasts suggests that palindrome formation is a stochastic process occurring in individual cells at a small number of loci relative to much larger numbers of susceptible loci in the cell population and that the induction of palindromes is not involved in Myc-induced acute fibroblast transformation. GAPF-detected palindromes at the highly oncogenic bic/miR-155 locus in all of our preneoplastic and neoplastic bursal samples, but not in DNA from normal and other transformed cell types. This finding indicates very strong selection during bursal lymphomagenesis. Therefore, in addition to providing a platform for gene copy number change, palindromes may alter microRNA genes in a fashion that can contribute to cancer development.     

Chromosomal instability at common fragile sites in Seckel syndrome

Seckel syndrome (SCKL) is a rare, genetically heterogeneous disorder, with dysmorphic facial appearance, growth retardation, microcephaly, mental retardation, variable chromosomal instability, and hematological disorders. To date, three loci have been linked to this syndrome, and recently, the gene encoding ataxia-telangiectasia and Rad3-related protein (ATR) was identified as the gene mutated at the SCKL1 locus. The ATR mutation affects splicing efficiency, resulting in low levels of ATR in affected individuals. Elsewhere, we reported increased instability at common chromosomal fragile sites in cells lacking the replication checkpoint gene ATR. Here, we tested whether cells from patients carrying the SCKL1 mutation would show increased chromosome breakage following replication stress. We found that, compared with controls, there is greater chromosomal instability, particularly at fragile sites, in SCKL1-affected patient cells after treatment with aphidicolin, an inhibitor of DNA polymerase alpha and other polymerases. The difference in chromosomal instability between control and patient cells increases at higher levels of aphidicolin treatment, suggesting that the low level of ATR present in these patients is not sufficient to respond appropriately to replication stress. This is the first human genetic syndrome associated with increased chromosome instability at fragile sites following replication stress, and these findings may be related to the phenotypic findings in patients with SCKL1.     

BRCA1 is required for common-fragile-site stability via its G2/M checkpoint function

Common fragile sites are loci that form chromosome gaps or breaks when DNA synthesis is partially inhibited. Fragile sites are prone to deletions, translocations, and other rearrangements that can cause the inactivation of associated tumor suppressor genes in cancer cells. It was previously shown that ATR is critical to fragile-site stability and that ATR-deficient cells have greatly elevated fragile-site expression (A. M. Casper, P. Nghiem, M. F. Arlt, and T. W. Glover, Cell 111:779-789, 2002). Here we demonstrate that mouse and human cells deficient for BRCA1, due to mutation or knockdown by RNA interference, also have elevated fragile-site expression. We further show that BRCA1 functions in the induction of the G(2)/M checkpoint after aphidicolin-induced replication stalling and that this checkpoint function is involved in fragile-site stability. These data indicate that BRCA1 is important in fragile-site stability and that fragile sites are recognized by the G(2)/M checkpoint pathway, in which BRCA1 plays a key role. Furthermore, they suggest that mutations in BRCA1 or interacting proteins could lead to rearrangements at fragile sites in cancer cells.     

The role of recombinational hotspots in genome instability in mammalian cells

Genome instability has been associated with progression of transformed cells to high tumorigenicity. Although genome instability may result from a variety of factors, some studies suggest that DNA in the region of a chromosome rearrangement can subsequently have much higher rates of DNA deletions or gene amplification. One approach to studying the factors that produce these high rates of DNA rearrangement is by analysis of unstable integration sites for DNA transfected into mammalian cells. Integrated sequences commonly show a temporary instability, and at rare locations this instability is continuous and can be observed even after multiple subclonings. These continuously unstable locations undergo DNA amplification of both the integrated sequences and the surrounding cell DNA, and it can occur either at the original site or on episomes after looping out from the chromosome. Because the adjacent cell DNA plays a role in this instability, and the region can be shown to be stable before integration, the results indicate that these recombinational hotspots can be formed de novo by the process of integration. Current studies are attempting to determine which sequences are responsible for the high rates of recombination and whether similar types of event are involved in the instability associated with endogenous cellular genes in cancer cells.     

ATR regulates fragile site stability

Conditions that partially inhibit DNA replication induce expression of common fragile sites. These sites form gaps and breaks on metaphase chromosomes and are deleted and rearranged in many tumors. Yet, the mechanism of fragile site expression has been elusive. We demonstrate that the replication checkpoint kinase ATR, but not ATM, is critical for maintenance of fragile site stability. ATR deficiency results in fragile site expression with and without addition of replication inhibitors. Thus, we propose that fragile sites are unreplicated chromosomal regions resulting from stalled forks that escape the ATR replication checkpoint. These findings have important implications for understanding both the mechanism of fragile site instability and the consequences of stalled replication in mammalian cells.