Abstract
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.
Highlights
Genomic instability drives tumorigenesis by expediting the acquisition of mutations that provide for selective clonal expansion and escape of normal cellular restraints [1]
A fragile region, FRA3B, lies within the FHIT gene, and deletions within FRA3B are common in precancer cells, causing loss of Fhit protein expression
We find that loss of Fhit protein causes defective DNA replication, leading to further DNA breaks
Summary
Genomic instability drives tumorigenesis by expediting the acquisition of mutations that provide for selective clonal expansion and escape of normal cellular restraints [1]. Chromosomal instability is the most commonly observed form of genome instability, occurs in the majority of sporadic cancers and includes structural chromosome aberration (translocations, inversions, deletions and duplications) or numerical abnormality (aneuploidy, triploidy, tetraploidy.) [2]. Because of its occurrence in most cancers, the molecular events causing chromosome and genome instability have been the subject of intense investigation. Chromosome and genome instability terminology is used interchangeably in this study to refer to chromosome structural and numerical abnormalities. In sporadic cancers the known DNA caretaker genes are rarely mutated before the rise of genome instability [2]. It has been proposed that in early stages of sporadic tumorigenesis, activated oncogenes induce replication stress through deregulation of cell cycle progression, causing chromosomal instability, first at common fragile sites, and later throughout the genome [4,5]. Exogenously supplied nucleosides suppressed oncogenesis in the model systems studied
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