Research on understanding the significance of human chromosomal fragile sites in cancer has been very active. Fragile sites are most commonly associated with deletion breakpoints in tumor cells, while few translocations involving these sites have been reported. A limited number of translocation breakpoints have been reported near fragile sites, suggesting that chromosome fragility at these sites may contribute to these rearrangements [29
]. Our study herein provides a comprehensive survey of all cancer-specific translocations to date. Therefore, by demonstrating that breakpoints in over half (52%) of the gene pairs co-map to fragile sites, these results provide strong evidence to support a role for fragile sites in the generation of cancer-specific translocations. It is important to note that we have chosen to focus on translocations and deletions leading to fusion transcripts, and have not included other types of rearrangements such as single gene deletions, insertions, or complex translocations. We have found that many of the genes examined in this study are commonly involved in these other types of rearrangements, like deletion of the FHIT gene located within fragile site FRA3B, and in some cases, the same set of genes is involved in multiple translocations observed in a variety of cancers. An interesting observation was that some genes located near fragile sites, such as NUP98
(11p15.4) which participates in twenty-two different translocations examined, could not be included in Additional file 1
, because the gene was not located directly at a fragile site. Recently described fragile sites, like FRA6H at 6p21 [30
], have been identified after discovering an association between the chromosome location and sites of recurrent aberrations in disorders. This suggests that 11p15.4 could be a fragile site that has not yet been identified, or that the proximal fragile sites FRA11C and FRA11I at 11p15.1 are larger than previously determined. Therefore, it is appropriate to assume that the total number of chromosomal aberrations in cancer associated with fragile sites could be even greater than presented in this study, arguing for the significance of the involvement of fragile sites in tumorigenesis.
In addition to solidifying the role of fragile sites participating in cancer development, this study also supports the common hypothesis for the molecular basis of fragility at these sites. We have shown that the DNA sequences within and surrounding three pairs of translocation-prone genes exhibit features of fragility. On average, peaks of significantly high flexibility occur more often than in random DNA, which is consistent with previous results [17
]. We also found these peaks to have a high A/T content and to be rich in AT-dinucleotides to the same extent as established in fragile sites [18
]. Furthermore, our data from the MFOLD program indicate that the sequences have the potential to form highly stable secondary structures, another distinct characteristic of fragile sites [18
], which could disturb progression of the replication fork. Based on our results, and the proposed mechanism of fragile site expression, it is likely that the AT-rich flexibility islands within or flanking translocation-prone genes are able to stall replication by the formation of secondary structures, which may then lead to DNA strand breakage, and ultimately to chromosome rearrangements.
Several proteins involved in the replication checkpoint pathway are essential for maintaining stability at fragile sites [14
]. These include the S phase and G2
/M checkpoint kinase ATR [25
], and its downstream targets BRCA1 [31
], FACD2 [32
], and CHK1 [33
]. ATR is a major component of the checkpoint pathway, where it functions by sensing and responding to DNA damage, including stalled and collapsed replication forks [34
]. It is hypothesized that ATR maintains fragile site stability by sensing and binding to single-stranded DNA resulting from stalled replication forks [25
]. However, in the absence of ATR, the main transducer of the DNA double-strand break (DSB) signal, which is ATM, has been shown to regulate fragile site stability [36
], indicating that DSBs also occur at fragile sites. Following breakage, chromosome rearrangements may take place via the homologous recombination (HR), nonhomologous end-joining (NHEJ) DSB repair pathways [37
], or microhomology-mediated single-strand annealing (SSA) [39
]. The repair of lesions at fragile sites is still not clear, but evidence suggests that all three pathways may be involved. Based on recent observations made by Lieber et al
., it is hypothesized that the sequence-specific RAG complex involved in V(D)J recombination, which is an important process of the NHEJ type, may also recognize and cleave at non-B DNA structures [40
], a feature shared by all fragile sites examined to date. Schwartz et al
] have shown that induction of fragile sites leads to RAD51 focus formation, and phosphorylation of DNA-PKcs, key components of the HR and NHEJ pathways, respectively. Furthermore, they found that down-regulation of RAD51 and DNA-PKcs increases fragile site instability, suggesting that both HR and NHEJ DSB repair pathways mediate break repair at fragile sites. In addition, the majority of breakpoints in a papillary thyroid carcinoma rearrangement mapped to fragile sites occur at microhomology patches, indicating that fragile site-associated rearrangements can also arise by microhomology-mediated SSA [42
]. Although the underlying basis of chromosome rearrangements is still unclear, our results show that 39% of the translocations examined have only one breakpoint mapped to a fragile site, suggesting that gene rearrangements could be achieved with strand breakage at only one gene, as well as at both participating genes. To support this hypothesis, additional studies are needed to obtain a greater understanding of the mechanisms of chromosome rearrangements.
The most intriguing finding from this study is that the majority (65%) of fragile sites mapped to translocation breakpoints are common fragile sites, which are present in all individuals, and can be induced by a variety of environmental factors and chemical agents. Interestingly, most cancers associated with the translocations examined in this study have little or no genetic component. These observations suggest that exposure to fragile site-inducing chemicals and/or reduced levels of proteins critical for the maintenance of fragile sites may confer a risk for cancer-specific rearrangements. It will be important to identify factors that contribute to chromosome fragility, such as DNA sequences, proteins and environmental/dietary agents, since fragile sites are sensitive to a range of chemicals. Understanding the molecular basis of fragile sites could therefore allow development of a prognostic assay for cancer risk.