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Initiation of genome instability and preneoplastic processes through loss of Fhit expression
Joshua C Saldivar1, Satoshi Miuma, Jessica Bene
1Biomedical Sciences Graduate Program, Ohio State University, Columbus, Ohio, United States of America.
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.
Insights
Loss of Fhit protein expression causes DNA replication stress and double-strand breaks, initiating genomic instability in early preneoplasias. This instability, driven by replication fork defects, facilitates cancer transformation.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Genomic instability is a hallmark of cancer, but its initiation in sporadic tumors remains unclear.
- Alterations at chromosome fragile sites, like FRA3B/FHIT, are early genetic events in preneoplasias, often involving Fhit protein loss.
- Replication stress is implicated in fragile site instability, but the role of specific gene products is debated.
Purpose of the Study:
- To investigate the role of Fhit protein in maintaining genomic stability during DNA replication.
- To elucidate the mechanism by which Fhit loss contributes to replication stress and DNA damage.
- To determine if Fhit depletion initiates genomic instability and facilitates cancer transformation.
Main Methods:
- Utilized normal, transformed, and cancer-derived cell lines with Fhit depletion.
- Employed DNA combing to analyze replication fork progression in Fhit-deficient cells.
- Assessed DNA damage response, cell cycle progression, and nucleotide pool balance.
- Examined Fhit knockout mouse tissues and derived cell lines for genomic aberrations and transformation potential.
Main Results:
- Fhit depletion induced replication stress and DNA double-strand breaks, primarily due to replication fork stalling and collapse.
- Fhit deficiency impaired replication fork progression, linked to altered Thymidine kinase 1 expression and nucleotide pools.
- Restoring nucleotide balance rescued replication defects and suppressed DNA breakage in Fhit-deficient cells.
- Fhit depletion did not trigger DNA damage response or cell cycle arrest, promoting chromosomal instability.
- Fhit knockout tissues and cells exhibited significant DNA copy number aberrations, rapid immortalization, and gene amplifications (e.g., Mdm2).
Conclusions:
- Loss of Fhit expression is a critical early event that initiates genomic instability by causing replication stress and DNA breakage.
- Fhit's role in regulating nucleotide metabolism is crucial for maintaining replication fork integrity.
- Fhit loss-induced genomic instability provides a foundation for cellular transformation and tumorigenesis.
- This study links alterations at common fragile sites to the origin of genome instability in cancer.
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