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Common fragile sites: genomic hotspots of DNA damage and carcinogenesis.

Ma K, Qiu L, Mrasek K, Zhang J, Liehr T, Quintana LG, Li Z - Int J Mol Sci (2012)

Bottom Line: However, despite tremendous efforts, identifying a cancer-associated CFS gene (CACG) remains a challenge and little is known about the function of CACGs at most CFS loci.Recent studies of FATS (for Fragile-site Associated Tumor Suppressor), a new CACG at FRA10F, reveal an active role of this CACG in regulating DNA damage checkpoints and suppressing tumorigenesis.The identification of FATS may inspire more discoveries of other uncharacterized CACGs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China; E-Mails: nmgdxmark@126.com (K.M.); nangongningchen@163.com (L.Q.); doctorjunzhang@163.com (J.Z.) ; Key Laboratory of Ministry of Education for Breast Cancer Prevention and Treatment, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.

ABSTRACT
Genomic instability, a hallmark of cancer, occurs preferentially at specific genomic regions known as common fragile sites (CFSs). CFSs are evolutionarily conserved and late replicating regions with AT-rich sequences, and CFS instability is correlated with cancer. In the last decade, much progress has been made toward understanding the mechanisms of chromosomal instability at CFSs. However, despite tremendous efforts, identifying a cancer-associated CFS gene (CACG) remains a challenge and little is known about the function of CACGs at most CFS loci. Recent studies of FATS (for Fragile-site Associated Tumor Suppressor), a new CACG at FRA10F, reveal an active role of this CACG in regulating DNA damage checkpoints and suppressing tumorigenesis. The identification of FATS may inspire more discoveries of other uncharacterized CACGs. Further elucidation of the biological functions and clinical significance of CACGs may be exploited for cancer biomarkers and therapeutic benefits.

Show MeSH

Related in: MedlinePlus

Experimental design for identification of genomic regions susceptible to DNA damage. This approach may be useful to discover evolutionarily conserved CFS genes. IR, ionizing radiation; CGH, comparative genomic hybridization; BAC, bacterial artificial chromosome; SNP, single nucleotide polymorphism.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC3472787&req=5

f1-ijms-13-11974: Experimental design for identification of genomic regions susceptible to DNA damage. This approach may be useful to discover evolutionarily conserved CFS genes. IR, ionizing radiation; CGH, comparative genomic hybridization; BAC, bacterial artificial chromosome; SNP, single nucleotide polymorphism.

Mentions: CFSs are specific genomic loci susceptible to DNA damage induced by replication stress and genotoxic agents. Consistently, CFSs are preferentially involved in sister chromatid exchange [9], an indication that some type of repair has occurred at the site. Ionizing radiation (IR) is a well-known carcinogen that is able to induce DNA damage and initiate neoplastic progression. In addition, IR-induced tumors exhibit a high frequency of localized deletion/amplification events [102]. It is therefore practical to identify evolutionarily conserved CFS genes through genome-wide dissecting allelic imbalances in IR-induced tumors. To this end, Li et al. [82] analyzed CGH profiling of IR-induced mouse lymphomas in comparison with those of spontaneous tumors (Figure 1), using the same approach as described before [102]. All the tumors had the same genetic background (p53+/−), which facilitates time-consuming collection of tumor samples [103] and high quality of CGH analysis with less interference of repetitive sequences in mouse than that in human. Using microarrays containing over 19,200 bacterial artificial chromosome (BAC) clones with insert size in the range of 170–210 kb and maximized coverage of mouse genome [104], the results of CGH analysis reveal a deletion site close to the end of mouse chromosome 7 in IR-induced tumors. This DNA region, corresponding to the contig of RP23-35I7, was deleted in 15% of spontaneous tumors (n = 20). Remarkably, the same region was deleted in 21 out of 29 (72.4%, p < 0.001) IR-induced tumor samples, indicating that this genomic region, which likely harbors an uncharacterized candidate tumor suppressor gene D7Ertd443e (Genbank accession number: GQ499374, NM_001199941), is highly susceptible to DNA damage [82]. Its human counterpart, C10orf90, a functionally uncharacterized gene, is mapped to a CFS FRA10F at 10q26, spanning a LOH region associated with cancers [105,106]. The co-localization of this new gene with FRA10F was confirmed by fluorescence in situ hybridization (FISH), using a BAC probe RP11-179O22 that spans 170 kb and contains all exons of C10orf90 (Table 2). Therefore, this gene, encoding an uncharacterized and evolutionarily conserved protein, is named FATS (for Fragile-site Associated Tumor Suppressor) [82]. Non-coding sequences of FATS gene are AT-rich. Interestingly, the largest exon of mouse FATS, encoding the NH2-terminal domain of FATS that is evolutionarily conserved, is surrounded by AT-rich sequences inserted with AT-dinucleotide repeats, which is a feature of CFSs instability. Unusually, additional dinucleotide repeats such as (CA)n and (TG)n, are distributed in AT-rich sequences both upstream and downstream of this coding exon, which may form a succession of stem-loop structures with a tendency to induce replication pausing and cause DNA fragility [82]. These dinucleotide repeats inserted in AT-rich sequences thus confer genetic instability to the FATS locus. Moreover, FATS expression is deficient or silent not only in mouse lymphomas and multiple human cancer cell lines [82], but also in clinical tumor samples from patients with ovarian, breast and lung cancer [107–109], demonstrating that FATS is a CFS gene at FRA10F. Fine-mapping of FRA10F by FISH using five BAC probes defines the boundary of FRA10F (Table 2), which spans 2.8 Mb at the junction of 10q26.13–26.2 (Figure 2 and 3). Although 30 genes are associated with FRA10F, only FATS is functionally validated as a CACG. The approach to FATS identification is practical for discovering some, if not all, other uncharacterized CACGs, especially when a genome-wide high-density SNP-oligonucleotide array is commercially available. Although IR does not cause gaps and breaks at CFSs in culture cells, IR-induced DNA damage, which is associated with deletion or amplification, occurs at relatively specific genomic loci in the mouse tumor genome. The FATS locus is just one of the most repetitively detected loci susceptible to DNA damage [82,102]. Although there are 30 genes associated with FRA10F spanning 2.8 Mb (Figure 2), only the FATS gene locus is highly susceptible to DNA damage-induced deletion in p53+/− tumors [82,102]. Similarly, aphidicolin-induced replication stress leads to DNA damage, and the number of CFS induced by aphidicolin can be as many as 230, dependent on the dose of aphidicolin treatment [110]. Notably, chromosomal lesions induced by chemical mutagens involve large regions of chromosomes, and changes in spontaneous tumors often involve whole chromosomes [102]. In contrast, IR-induced tumors exhibit a high frequency of localized deletion/amplification events [102], facilitating the identification of cancer-related genes using the approach illustrated in Figure 1. Because of very high numbers of recurrent gains and losses induced by IR, the cancer-related genes identified by this approach may consist of CACGs and other oncogenes or tumor suppressor genes. In the post-genomics era, identifying CFS-associated genes and cloning CFSs is less important than identifying new CACGs with important physiological functions and a causal role in tumorigenesis. Given that CFS instability, at least FRA3B instability, exhibits tissue specificity [28], cancer-related CFSs and CACGs need to be reassessed in the cell type from which each tumor originates. The frequent deletion of T-cell-receptor-α (TCR-α) gene locus is both detected in both spontaneous and IR-induced tumors [102], using the approach we propose in Figure 1. In addition, microarray-based studies of mouse tumor models (Figure 1) also reveals the tumor suppressor gene CDKN2A (p16) at a frequently deleted region in tumors induced by IR (data not shown). These results demonstrated the reliability of the microarray-based approach in mouse tumor models, which led to the discovery of the FATS tumor suppressor gene. Recently, Bignell et al. [111] identified 2428 somatic homozygous deletions (HDs) in 746 cancer cell lines. Interestingly, CDKN2A (p16) exhibits more HDs than other recessive cancer genes, and TCR-α genes are located at the deepest HD in lymphoid cells in their dataset [111]. The microarray-based screening of murine tumor models (Figure 1) therefore may be helpful to discover new cancer-related genes, including some evolutionarily conserved CACGs whose gene loci are susceptible to DNA damage. After functional validation, the localization of a CACG at a CFS should be verified by FISH analysis using BAC clones spanning the whole gene locus. Similarly, Tsantoulis et al. [112] performed a genome-wide array CGH study on preneoplastic mouse models, in addition to analyzing 56 aphidicolin-type CFSs in growth-factor-induced human skin hyperplasia, showing that genomic alterations are more common within CFSs in epidermal and urothelial preneoplastic lesions as well as in cancer. Further functional study based on these microarray data may provide new insights into the molecular nature of uncharacterized CFSs, leading to identification of new CACGs.


Common fragile sites: genomic hotspots of DNA damage and carcinogenesis.

Ma K, Qiu L, Mrasek K, Zhang J, Liehr T, Quintana LG, Li Z - Int J Mol Sci (2012)

Experimental design for identification of genomic regions susceptible to DNA damage. This approach may be useful to discover evolutionarily conserved CFS genes. IR, ionizing radiation; CGH, comparative genomic hybridization; BAC, bacterial artificial chromosome; SNP, single nucleotide polymorphism.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3472787&req=5

f1-ijms-13-11974: Experimental design for identification of genomic regions susceptible to DNA damage. This approach may be useful to discover evolutionarily conserved CFS genes. IR, ionizing radiation; CGH, comparative genomic hybridization; BAC, bacterial artificial chromosome; SNP, single nucleotide polymorphism.
Mentions: CFSs are specific genomic loci susceptible to DNA damage induced by replication stress and genotoxic agents. Consistently, CFSs are preferentially involved in sister chromatid exchange [9], an indication that some type of repair has occurred at the site. Ionizing radiation (IR) is a well-known carcinogen that is able to induce DNA damage and initiate neoplastic progression. In addition, IR-induced tumors exhibit a high frequency of localized deletion/amplification events [102]. It is therefore practical to identify evolutionarily conserved CFS genes through genome-wide dissecting allelic imbalances in IR-induced tumors. To this end, Li et al. [82] analyzed CGH profiling of IR-induced mouse lymphomas in comparison with those of spontaneous tumors (Figure 1), using the same approach as described before [102]. All the tumors had the same genetic background (p53+/−), which facilitates time-consuming collection of tumor samples [103] and high quality of CGH analysis with less interference of repetitive sequences in mouse than that in human. Using microarrays containing over 19,200 bacterial artificial chromosome (BAC) clones with insert size in the range of 170–210 kb and maximized coverage of mouse genome [104], the results of CGH analysis reveal a deletion site close to the end of mouse chromosome 7 in IR-induced tumors. This DNA region, corresponding to the contig of RP23-35I7, was deleted in 15% of spontaneous tumors (n = 20). Remarkably, the same region was deleted in 21 out of 29 (72.4%, p < 0.001) IR-induced tumor samples, indicating that this genomic region, which likely harbors an uncharacterized candidate tumor suppressor gene D7Ertd443e (Genbank accession number: GQ499374, NM_001199941), is highly susceptible to DNA damage [82]. Its human counterpart, C10orf90, a functionally uncharacterized gene, is mapped to a CFS FRA10F at 10q26, spanning a LOH region associated with cancers [105,106]. The co-localization of this new gene with FRA10F was confirmed by fluorescence in situ hybridization (FISH), using a BAC probe RP11-179O22 that spans 170 kb and contains all exons of C10orf90 (Table 2). Therefore, this gene, encoding an uncharacterized and evolutionarily conserved protein, is named FATS (for Fragile-site Associated Tumor Suppressor) [82]. Non-coding sequences of FATS gene are AT-rich. Interestingly, the largest exon of mouse FATS, encoding the NH2-terminal domain of FATS that is evolutionarily conserved, is surrounded by AT-rich sequences inserted with AT-dinucleotide repeats, which is a feature of CFSs instability. Unusually, additional dinucleotide repeats such as (CA)n and (TG)n, are distributed in AT-rich sequences both upstream and downstream of this coding exon, which may form a succession of stem-loop structures with a tendency to induce replication pausing and cause DNA fragility [82]. These dinucleotide repeats inserted in AT-rich sequences thus confer genetic instability to the FATS locus. Moreover, FATS expression is deficient or silent not only in mouse lymphomas and multiple human cancer cell lines [82], but also in clinical tumor samples from patients with ovarian, breast and lung cancer [107–109], demonstrating that FATS is a CFS gene at FRA10F. Fine-mapping of FRA10F by FISH using five BAC probes defines the boundary of FRA10F (Table 2), which spans 2.8 Mb at the junction of 10q26.13–26.2 (Figure 2 and 3). Although 30 genes are associated with FRA10F, only FATS is functionally validated as a CACG. The approach to FATS identification is practical for discovering some, if not all, other uncharacterized CACGs, especially when a genome-wide high-density SNP-oligonucleotide array is commercially available. Although IR does not cause gaps and breaks at CFSs in culture cells, IR-induced DNA damage, which is associated with deletion or amplification, occurs at relatively specific genomic loci in the mouse tumor genome. The FATS locus is just one of the most repetitively detected loci susceptible to DNA damage [82,102]. Although there are 30 genes associated with FRA10F spanning 2.8 Mb (Figure 2), only the FATS gene locus is highly susceptible to DNA damage-induced deletion in p53+/− tumors [82,102]. Similarly, aphidicolin-induced replication stress leads to DNA damage, and the number of CFS induced by aphidicolin can be as many as 230, dependent on the dose of aphidicolin treatment [110]. Notably, chromosomal lesions induced by chemical mutagens involve large regions of chromosomes, and changes in spontaneous tumors often involve whole chromosomes [102]. In contrast, IR-induced tumors exhibit a high frequency of localized deletion/amplification events [102], facilitating the identification of cancer-related genes using the approach illustrated in Figure 1. Because of very high numbers of recurrent gains and losses induced by IR, the cancer-related genes identified by this approach may consist of CACGs and other oncogenes or tumor suppressor genes. In the post-genomics era, identifying CFS-associated genes and cloning CFSs is less important than identifying new CACGs with important physiological functions and a causal role in tumorigenesis. Given that CFS instability, at least FRA3B instability, exhibits tissue specificity [28], cancer-related CFSs and CACGs need to be reassessed in the cell type from which each tumor originates. The frequent deletion of T-cell-receptor-α (TCR-α) gene locus is both detected in both spontaneous and IR-induced tumors [102], using the approach we propose in Figure 1. In addition, microarray-based studies of mouse tumor models (Figure 1) also reveals the tumor suppressor gene CDKN2A (p16) at a frequently deleted region in tumors induced by IR (data not shown). These results demonstrated the reliability of the microarray-based approach in mouse tumor models, which led to the discovery of the FATS tumor suppressor gene. Recently, Bignell et al. [111] identified 2428 somatic homozygous deletions (HDs) in 746 cancer cell lines. Interestingly, CDKN2A (p16) exhibits more HDs than other recessive cancer genes, and TCR-α genes are located at the deepest HD in lymphoid cells in their dataset [111]. The microarray-based screening of murine tumor models (Figure 1) therefore may be helpful to discover new cancer-related genes, including some evolutionarily conserved CACGs whose gene loci are susceptible to DNA damage. After functional validation, the localization of a CACG at a CFS should be verified by FISH analysis using BAC clones spanning the whole gene locus. Similarly, Tsantoulis et al. [112] performed a genome-wide array CGH study on preneoplastic mouse models, in addition to analyzing 56 aphidicolin-type CFSs in growth-factor-induced human skin hyperplasia, showing that genomic alterations are more common within CFSs in epidermal and urothelial preneoplastic lesions as well as in cancer. Further functional study based on these microarray data may provide new insights into the molecular nature of uncharacterized CFSs, leading to identification of new CACGs.

Bottom Line: However, despite tremendous efforts, identifying a cancer-associated CFS gene (CACG) remains a challenge and little is known about the function of CACGs at most CFS loci.Recent studies of FATS (for Fragile-site Associated Tumor Suppressor), a new CACG at FRA10F, reveal an active role of this CACG in regulating DNA damage checkpoints and suppressing tumorigenesis.The identification of FATS may inspire more discoveries of other uncharacterized CACGs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China; E-Mails: nmgdxmark@126.com (K.M.); nangongningchen@163.com (L.Q.); doctorjunzhang@163.com (J.Z.) ; Key Laboratory of Ministry of Education for Breast Cancer Prevention and Treatment, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China.

ABSTRACT
Genomic instability, a hallmark of cancer, occurs preferentially at specific genomic regions known as common fragile sites (CFSs). CFSs are evolutionarily conserved and late replicating regions with AT-rich sequences, and CFS instability is correlated with cancer. In the last decade, much progress has been made toward understanding the mechanisms of chromosomal instability at CFSs. However, despite tremendous efforts, identifying a cancer-associated CFS gene (CACG) remains a challenge and little is known about the function of CACGs at most CFS loci. Recent studies of FATS (for Fragile-site Associated Tumor Suppressor), a new CACG at FRA10F, reveal an active role of this CACG in regulating DNA damage checkpoints and suppressing tumorigenesis. The identification of FATS may inspire more discoveries of other uncharacterized CACGs. Further elucidation of the biological functions and clinical significance of CACGs may be exploited for cancer biomarkers and therapeutic benefits.

Show MeSH
Related in: MedlinePlus