Exome‐wide single‐base substitutions in tissues and derived cell lines of the constitutive Fhit knockout mouse

Abstract

Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit-deficient mouse models are exquisitely susceptible to carcinogen induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit-deficient tissues and cultured cells, we find 300 to >1000 single-base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the ‘age at diagnosis’ signature identified in human cancers. The Fhit-deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in −/− exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of Thymidine Kinase 1 in Fhit-deficient cells. Fhit-deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally exhibited increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen-induced preneoplastic changes.

Fragile Genes That Are Frequently Altered in Cancer: Players Not Passengers

“…T>C mutations may be due to the misincorporation of dUTP in place of dTTP, a consequence of TK1 downregulation in FHIT-deficient cells. This FHIT loss mutation profile is comparable to previously published mutation signatures reported for bladder cancer, human papillary kidney cancer, and an "age at diagnosis" signature, or aging signature, that represents accumulation of mutations in normal tissues over time [Alexandrov et al, 2013;Paisie et al, 2016]. As FHITdeficient genomes accumulate mutations, it is not surprising then that many of these mutations are found within important genes which, when altered, contribute to cellular transformation and preneoplastic changes.…”

“…FHIT-deficient liver tissues exhibited almost a 4-fold increase in total SBSs compared to wild-type liver tissue. Further examination of the types of transition and transversion mutations observed among these SBSs demonstrated that FHIT-deficient cells and tissues displayed prominent increases in C>T and T>C mutations, as well as slightly elevated levels of C>A, C>G, and T>A mutations [Paisie et al, 2016]. C>T mutations can be generated by spontaneous deamination or DNA replication errors.…”

“…This criterion is not met by FHIT loss-induced replication stress. Therefore, FHIT loss causes mild replication stress that persists through each cell division and contributes to accumulation of genome-wide instability Karras et al, 2014;Paisie et al, 2016]. Coupled with the fact that loss of FHIT expression is one of the earliest changes in the preneoplasia process, FHIT deletion is a strong candidate for being an initiator of genome instability that fuels the development of many cancers.…”

“…As FHITdeficient genomes accumulate mutations, it is not surprising then that many of these mutations are found within important genes which, when altered, contribute to cellular transformation and preneoplastic changes. Such genes include Mutyh , Tpx2 , Nek9 , Zfat , and Dlc1 , which are essential for DNA repair, chromosomal stability, replication stress response, cell survival, and regulation of the Wnt/β-catenin signaling pathway [Paisie et al, 2016].…”

Tumor Suppressor Genes within Common Fragile Sites Are Active Players in the DNA Damage Response

“…In a recent report, the Huebner group analyzed whole exome sequences of Fhit -deficient tissues and cells and compared them to a control C57Bl6 genome, identifying hundreds of single-base substitutions associated with FHIT deficiency. The mutation signature is characterized by increased C>T and T>C mutations, in accordance with the signature identified in human malignancies [66]. …”