Initiation of Genome Instability and Preneoplastic Processes through Loss of Fhit Expression

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.

WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response

“…Similar results highlight the contribution of the FHIT gene, which encompasses FRA3B and encodes a 14-kDa protein with tumor suppressor functions (55). Deficiency of the FHIT gene as early as in preneoplastic lesions induced global genome instability and clonal expansion (56,57). Likewise, the product of FRA15A (RORA) and FRA8I (SPIDR), which is inactivated in multiple tumors, has been shown to be involved in cellular stress response, DNA damage repair, and maintenance of chromosomal integrity (58,59), suggesting that their impaired activity may contribute to genomic instability in cancer cells.…”

Reduction in squamous cell carcinomas in mouse skin by dietary zinc supplementation

“…We employed Fhit − / − mice because of their sensitivity to carcinogen induction of various tumor types, and B6 mice as wt control cohort. We have previously reported that Fhit deficiency causes genome instability that is undetected by cellular checkpoints 24, providing an optimal environment for selection and clonal expansion to survive various stressors 25. For instance, we have shown that Fhit − / − mouse kidney cells survive acute DMBA treatment and chronic severe nutritional stress whereas wt cells do not 25.…”

“…Also, loss of expression of Fhit tumor suppressor protein occurs in >50% of human cancers 23. Fhit also serves as a genome caretaker preventing the onset of global genome instability 24, 25. Fhit‐deficient and haploinsufficient mice spontaneously develop lung tumors, lymphomas and liver hemangiomas at slightly higher incidences than wt mice 21 and 100% of Fhit knockout mice developed NMBA‐induced forestomach tumors, significantly more than wt mice 20, 21.…”

APOBEC3 genes: retroviral restriction factors to cancer drivers

“…Replication stress and A3-induced mutagenesis have recently been linked to loss of a chromosomal fragile site gene, FHIT [63]. FHIT is frequently lost very early in tumor development, causing replication stress due to deoxythymidine triphoshate (dTTP) depletion [64]. When genomic sequences from lung adenocarcinomas were stratified by A3B and FHIT expression, those with high A3B and FHIT loss showed significantly higher A3 signature mutation loads than high A3B expressers with normal FHIT levels, thus the mutagenic potential of A3B may be unleashed in the absence of FHIT [63].…”