Review
Large common fragile site genes and cancer

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Abstract

The common fragile sites are large regions of genomic instability that are found in all individuals and are hot spots for chromosomal rearrangements and deletions. A number of the common fragile sites have been found to span genes that are encoded by very large genomic regions. Two of these genes, FHIT and WWOX, have already been demonstrated to function as tumor suppressors. In this review we will discuss the large common fragile site genes that have been identified to date, and the role that these genes appear to play both in cellular responses to stress and in the development of cancer.

Introduction

The common fragile sites (CFSs) are regions of profound genomic instability that are distributed throughout the genome [1], [2]. In contrast to the rare fragile sites (RFSs), which are seen in less than 5% of the population [3], CFSs are found in all individuals [4]. In addition, unlike the RFSs which by and large are caused by expansion of unstable repeat sequences [5], [6], there is no evidence that the CFSs are caused by expanded repeat sequences [2]. The CFSs are also found to be large regions of genomic instability sometimes extending for over 10 Mbs [7], [8], [9].

There are 90 described CFSs throughout the human genome, and these sites vary in their frequency of expression [4]. Fragile site expression is generally observed when cells are cultured in the presence of inhibitors of replication such as aphidicolin (which induces expression of 83 of the 90 described CFSs) [4], 5-azacytidine, and bromo-deoxyuridine [10]. The four most frequently expressed CFSs are the aphidicolin-sensitive sites FRA3B (3p14.2), FRAXB (Xp21.1), FRA16D (16q23.2), and FRA6E (6q26) [4]. Other fragile sites are expressed at a lower frequency. CFSs have also been observed in other species, including the great apes (including chimpanzees) [11], [12], bovine [13], and mice [14].

The scientific interest in the CFSs stems primarily from the fact that these chromosomal regions are hot-spots for deletions and other alterations in cancer cells [2]. This has prompted researchers to focus on these regions of instability searching for candidate tumor suppressor genes that might be contained within these regions. The first CFS to be analyzed in detail was FRA3B, as it's the most frequently expressed CFS in the human genome [15]. The FRA3B region was first cloned by Boldog et al., utilizing the large insert YAC clones constructed for the characterization of the human genome [16]. FRA3B was found to be a region of instability, and several years later the very large FHIT gene was described within this unstable region [17]. FHIT was found to be a target of alterations in multiple cancers [18], [19]. Interestingly, FHIT had a very unusual structure. In spite of the fact that this gene spanned 1.5 Mbs within 3p14.2, the final processed transcript of this gene was only 1.1 kb [20], therefore 99.99% of this gene corresponded to intronic sequences.

The second CFS region to be characterized was FRA16D; this was also found to be a large region of instability, spanning 2.0 Mbs within chromosomal band 16q23.2 [21]. Spanning the center of this unstable region was another large gene, WWOX [22], [23]. WWOX spanned 1.0 Mbs and similar to FHIT, it also has a relatively small final processed transcript (2.1 kbs). This gene was also found to be a frequent target of alterations in multiple cancers [24], [25], [26]. Other CFS regions were identified that did not span large genes, including FRA7G (7q31.2) [27], FRA7H [28] and the frequently expressed FRAXB region [29]; thus, not all the CFS regions contain such large genes. However, the FRA6E CFS was cloned and characterized, and this region also spans a very large gene, the 1.36 Mbs Parkin gene [30]. This was the first large CFS gene that had a definite association with neurological development, as this gene is mutated in some patients with autosomal recessive juvenile Parkinsonism [31].

The finding that three of the four most frequently expressed CFS regions contained very large genes prompted us, as well as others, to begin to examine whether there were other CFSs associated with very large genes. In this review we will summarize the CFS regions that have already been described to span very large genes with a similar structure to FHIT, WWOX and Parkin. A number of these genes have already been demonstrated to function as tumor suppressors. We will also discuss the known roles that several of these play in the cellular response to stress, suggesting that the CFS regions and the large genes contained within them might be part of a stress response system within cells. Finally, we will examine the potential role that many of these large CFS genes play in normal neurological development, suggesting a very interesting relationship between the processes involved in neurological development and the development of cancer.

Section snippets

FRA3B and FHIT

FRA3B (3p14.2) is the most frequently expressed CFS region in the human genome [15]. This region is also frequently deleted in multiple cancers including those of the lungs [32], [33], kidney [34], [35], and cervix [36], [37]. In addition, a family was described by Cohen et al. [38] that had a balanced reciprocal translocation (t(3;8)(3p14.2;8q23.2). Individuals who inherited this balanced reciprocal translocation had an 80% probability of developing renal cell carcinoma. For this reason, this

FRA16D and WWOX

FRA16D (16q23.2) is the second most frequently expressed CFS region in the human genome [4]. This chromosomal region is also frequently deleted in multiple cancers [70], [71], [72]. In addition, in about 25% of multiple myelomas there is a balanced reciprocal translocation between this region and the chromosomal band that contains the immunoglobulin genes (14q23.2) [73]. This suggests that this region might also contain an important tumor suppressor gene similar to FHIT.

The FRA16D region was

FRA6E and Parkin

The fourth most frequently expressed CFS is FRA6E (6q26) [4]. This is derived from a chromosomal region frequently deleted in multiple cancers also including cancers of the ovary, breast, kidney, and lung [91], [92], [93], [94] Cesari et al. described another large gene from this chromosomal region, the 1.36 Parkin gene [95]. This gene also has a similar genomic organization to FHIT and WWOX because the final processed transcript of this gene is only 2 kb. While mutation analysis revealed no

FRA4G and GRID2

The next large CFS gene to be identified was GRID2. This gene encodes the ionotropic glutamate receptor delta 2 gene which was shown to be allelic to the mouse mutant hotfoot [104]. The first hotfoot allele was described in 1960 [105], and since then at least 16 new alleles have been reported in the mouse genome database. Hotfoot is a spontaneous deletion of chromosomal sequences in mouse chromosomal band 6C1. This is a region that has frequent chromosomal rearrangements and the target of these

FRA15A and RORA

We then began a systematic examination of large genes and their potential association with CFS regions of instability. We obtained from our colleague, Dr. Robert Kuhn (who works in the UCSC Genome Database), a list of the largest human genes. When this list was carefully annotated, we found that there were 40 human genes that spanned greater than 1.0 Mb of genome sequence and a total of 240 genes that spanned over 500 kb. A number of these genes were derived from chromosomal regions known to

FRA13A and neurobeachin (NBEA)

The FRA13A CFS is derived from within chromosomal band 13q13.2. Dr. Manfred Schwab's group was working to characterize this CFS, and the strategy that they employed was based upon previous findings that demonstrated that the CFSs were hot-spots for the integration of linear fragments of DNA when cells are cultured in the presence of aphidicolin [116]. Utilizing the selectable marker pSV2neo, they found integration of this marker into 13q12–14. They then employed six-colour chromosomal

FRA2F and LRP1B

We have used a number of different strategies to precisely localize many of the 90 known CFS regions. This includes using large insert clones and a FISH-based assay to triangulate to CFS regions of instability. We have also found that genes which were consistently down-regulated as observed through transcriptional profiling of ovarian carcinomas were frequently located within CFS regions [119]. However, the strategy that resulted in the localization of over 25 CFS regions was based upon the

Common fragile sites that don’t span large genes

We have, therefore, summarized what is currently published about the relationship between CFSs and large genes that may play an important role in cancer development. However, not all the CFSs are associated with very large genes. This was first seen when we cloned and characterized the FRA7G (7q31.2) CFS [27]. This CFS is also derived from a chromosomal region frequently deleted in multiple cancers, but when we localized and characterized this region of instability, we only found several small

Large CFS genes as a stress response system?

What is the function of the CFSs and the large genes that are contained within many of them? The fact that there appears to be co-conservation of these large genes and the CFS regions spanning them suggests that together they might serve some function within cells. The observation that there is conservation of genomic organization and considerable conservation of even intronic sequences within these large genes also suggests that there is some function to even the introns of these large genes.

Large common fragile site genes and neurological development

Many of the large CFS genes appear to be involved in normal neurological development. At present there is no evidence that FHIT is involved in this process, but this is certainly the case with RORA, GRID2, NBEA, Parkin, and also potentially with WWOX. The fact that these genes also appear to be strong tumor suppressor candidates then provides additional support for connections between the processes of development and carcinogenesis.

There are also a number of other potential large CFS genes

Summary

At least half of the CFS regions appear to be associated with very large genes with a structure similar to FHIT and WWOX. Both FHIT and WWOX have been demonstrated to function as tumor suppressors and the inactivation of expression of these genes is associated with a worse clinical prognosis. These two genes also appear to be involved in the cellular response to stress. To date there have been a total of 9 published large CFS genes; FHIT and the adjacent PTPRG gene, WWOX, Parkin, GRID2 and its

Acknowledgements

The work that we have done on the characterization of large CFS genes was supported by Department of Defense grant no. DAMD 17-00-1-0296, a grant from the Minnesota Ovarian Cancer Alliance, and a grant from the Eagle's Foundation.

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