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

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

Fhit Deficiency-Induced Global Genome Instability Promotes Mutation and Clonal Expansion

Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome \u27caretaker\u27 whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit-/- MEFs escape senescence to become immortal more rapidly than Fhit+/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a \u27mutator\u27 phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive

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

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

Fhit activates TK1 expression.

<p>(A) Western blot analysis of TK1 and TYMS expression in siRNA transfected HEK293 cells. Western blots were performed on 5 independent experiments, and a representative blot is shown. (B) Western blot analysis of TK1 expression in siRNA transfected HEK293 cells with or with exogenous Fhit overexpression. pRcCMV expression plasmid carrying <i>FHIT</i> cDNA was co-transfected with <i>FHIT</i> siRNAs to achieve exogenous Fhit overexpression. A representative blot is shown. (C) Western blot analysis of TK1 expression in <i>Fhit^+/+</i> and <i>Fhit^−/−</i> mouse kidney epithelial cells. A representative blot is shown. (D) Western blot analysis of TK1 expression in A549 cells with Fhit stably knocked down for 7–9 weeks. A representative blot is shown.</p

Genomic instability in Fhit-deficient cells correlates with onset of rapid proliferation and immortalization.

<p>(A) Analysis of <i>Fhit</i>^+/+ and <i>Fhit</i>^−/− 3T3 MEF cell lines (n = 3, cell lines established from 3 embryos for each mouse strain). Arrows mark the passage numbers when MEFs became immortalized. (B) Western blot of <i>Fhit</i>^+/+ MEFs for Fhit and GAPDH expression. Immunoblots were performed on lysates obtained at the indicated passage number. (C) Summary of copy number aberrations (CNAs) in pre- and post-senescence MEFs from <i>Fhit</i>^+/+ and <i>Fhit</i>^−/− mice. (D) The Fhit loss–induced genome instability model. Deletions in <i>FHIT</i> alleles occur due to FRA3B fragility causing loss of Fhit protein expression. Fhit loss causes dTTP pool insufficiency triggering replication stress, followed by stress-induced chromosomal instability. Chromosomal instability increases the likelihood of activating mutations in oncogenes and/or inactivating mutations in tumor suppressors, which are then selected for, facilitating cell transformation.</p

Loss of Fhit causes replication stress.

<p>(A) Cyclin A and γH2AX indirect immunofluorescence after Fhit knockdown. Representative images are shown; bars, 10 µm. (B) Data obtained in (A) were quantified from 3 independent experiments, and statistical significance was determined using a 2-sided T-test. Bar graphs represent the means, and error bars mark the standard deviations. (C) pATR immunofluorescence 2 days after Fhit knockdown in HEK293 cells. Representative images are shown; bars, 5 µm. (D) Quantification of cells positive for more than 5 pATR foci/cell from 3 independent experiments; statistical significance was determined using a 2-sided T-test. (E) Neutral comet assays in siRNA transfected HEK293 cells treated with 2 mM hydroxyurea for 4 h. Box plots show quantification of Tail moments. P-values were determined using the Mann-Whitney rank sum test. (F) Neutral comet assays in H1299 E1 and D1 cells with ponasterone A-induction treated with 2 mM hydroxyurea for 4 h. Box plots show quantification of Tail moments. P-values were determined using the Mann-Whitney rank sum test.</p

Fhit-deficient cells exhibit spontaneous DNA breaks.

<p>(A) Neutral comet assays of HEK293 cells 2 days after transfections with siRNAs and pRcCMV-<i>FHIT</i>-flag or pRcCMV-empty-flag plasmids. Representative nuclei are shown; bars, 20 µm. (B) Box plots of Tail moments include data (siCtrl, n = 183; si<i>FHIT</i>, n = 142; si<i>FHIT</i>+CMV-ev, n = 135; si<i>FHIT</i>+CMV-Fhit, n = 132) from 3 separate experiments. Statistical significance was determined using the Kruskal-Wallis rank sum test. (C) Indirect immunofluorescence of γH2AX and 53BP1, 2 days after Fhit knockdown. Representative nuclei are shown; bars, 10 µm. (D and E) Quantification of γH2AX-positive cells (D) and 53BP1-positive cells (E). Bar graphs indicate the means, and error bars represent the standard deviations. Data were collected from 3 independent experiments. Statistical significance was assessed using a 2-sided Student's T-test. (F) Neutral comet assays of <i>Fhit</i>^+/+ or <i>Fhit</i>^−/− mouse kidney cells 48 h after transfection with pRcCMV-<i>FHIT</i>-flag or pRcCMV-empty-flag plasmids. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test. (G) Neutral comet assays of Fhit-deficient H1299 lung carcinomas cells, with or without induction of Fhit expression. Comet assays were performed 48 h after ponasterone A-induction of Fhit expression. Box plots of Tail moments are shown. Statistical significance was determined using the Mann-Whitney rank sum test.</p

Loss of Fhit expression causes fork stalling.

<p>(A) siRNA transfected HEK293 cells were pulse-labeled with CldU for 30 min, washed and pulse-labeled with IdU for 30 min. Representative indirect immunofluorescence images of labeled fibers are shown; bars, 10 µm. (B) Quantification of fork velocity in HEK293 cells. Fork velocity was determined by measuring lengths (µm) of IdU-labeled fibers and converting to kbp using a conversion factor of 1 µm = 2.59 kbp. Bars extending through boxplots indicate mean velocity, and bars contained within boxplots indicate median velocity. Statistical significance was determined using a 2-sided Student's T-test (n = 238 for siCtrl; n = 320 for siFHIT). (C) Representative images of sister forks proceeding outward from a common origin in siRNA transfected HEK293 cells; bars, 10 µm. DNA fibers from siRNA transfected HEK293 cells were prepared as in (A). (D) Scatter plots of distances traveled by left and right sister forks during pulse-labeling with IdU. The central area marked by red lines represents sister forks with less than 25% length difference. The percentages of asymmetric sister forks are indicated at the upper left region of plots. (E) Fork asymmetry is calculated as the ratio of the longest IdU tract to the shortest for each pair of sister forks. P-value was determined using the Mann-Whitney rank sum test. (F) DNA fiber analysis of fork velocity in <i>Fhit</i>^+/+, <i>Fhit</i>^−/− and <i>Fhit</i>^−/− pRcCMV-<i>FHIT</i>-flag plasmid transfected mouse kidney cells. Quantification and statistical analysis was performed as described in (B). (G) DNA fiber analysis of fork velocity in H1299 E1 and D1 cells 48 h after ponasterone A treatment. Quantification and statistical analysis was performed as described in (B).</p