Fhit and Wwox loss-associated genome instability: A genome caretaker one-two punch

Abstract

Expression of Fhit and Wwox protein is frequently lost or reduced in many human cancers. In this report, we provide data that further characterizes the molecular consequences of Fhit loss in the initiation of DNA double-strand breaks (DSBs), and of Wwox loss in altered repair of DSBs. We show that loss of Fhit initiates mild genome instability in early passage mouse kidney cells, confirming that DNA damage associated with Fhit-deficiency is not limited to cancer cells. We also demonstrate that the cause of Fhit-deficient DSBs: thymidine deficiency-induced replication stress, can be resolved with thymidine supplementation in early passage mouse kidney cells before extensive genome instability occurs. As for consequences of Wwox loss in cancer, we show in a small panel of breast cancer cells and mouse embryonic fibroblasts that Wwox expression predicts response to radiation and mitomycin C, all agents that cause DSBs. In addition, loss of Wwox significantly reduced progression free survival in a cohort of ovarian cancer patients treated with platin-based chemotherapies. Finally, stratification of a cohort of squamous lung cancers by Fhit expression reveals that Wwox expression is significantly reduced in the low Fhit-expressing group, suggesting that loss of Fhit is quickly succeeded by loss of Wwox. We propose that Fhit and Wwox loss work synergistically in cancer progression and that DNA damage caused by Fhit could be targeted early in cancer initiation for prevention, while DNA damage caused by Wwox loss could be targeted later in cancer progression, particularly in cancers that develop resistance to genotoxic therapies.

Keywords: Chromosome fragile site; DNA double-strand breaks; Fhit; Genome caretaker; Wwox.

Fig. 1.. Thymidine supplementation prevents ongoing DNA damage.

A) Immunoblot of Vinculin and TK1 in −/− and +/+ mouse kidney cells. B) Indirect immunofluorescence of γH2AX, before thymidine supplementation. C) Quantification of γH2AX-positive cells before thymidine supplementation. Bar graph indicates the means, and error bars represent the standard error. D) Neutral comet assay of mouse kidney cells before thymidine supplementation. Box plots of tail moments include data (WT, n = 285; Fhit −/−, n = 435) from 3 separate experiments. E) Representative photos of Fhit−/− and Fhit +/+ comet tails F) Neutral comet assay of mouse kidney cells 40 days post 10 μM thymidine supplementation. Box plots of tail moments include data (WT untreated, n = 344; WT with thymidine, n = 228; Fhit −/− untreated, n = 341; Fhit −/− with thymidine, n = 286) from 3 separate experiments. dT, thymidine.

Fig. 2.. TK1 reactivation in Fhit-negative cancer cells.

A) Western blot analysis of TK1 expression in mouse kidney cells. In +/+ cells, TK1 expression decreases in parallel to loss of Fhit expression. In −/− cells, loss of Fhit results in transient TK1 downregulation, as TK1 expression increases at late passage. B) Assessment of DNA damage via neutral comet assay in mouse kidney cells. Fhit−/− NS3 is a nutritionally stressed cell line that has undergone cellular transformation in vitro and over-expresses TK1. Box plots of tail moments include: Fhit+/+ without thymidine, n=72; Fhit+/+ with thymidine, n=103; Fhit−/− without thymidine, n=282; Fhit−/− with thymidine, n=331; NS3 without thymidine, n=144; NS3 with thymidine, n=161. Thymidine supplementation (10μM) for 16 days and includes data from two separate experiments. No significant difference in levels of damage between mock and dT of Fhit+/+ (p=0.4095) and Fhit−/− NS3 (p=0.5729). dT, thymidine. C) RNA-seq data obtained from The Cancer Genome Atlas shows a negative correlation between Fhit expression and expression of enzymes involved in dTTP synthesis (TK1, RRM2, and TYMS). Red, up-regulated. Green, down-regulated. We conclude that during cancer progression, there is selective pressure to increase expression of these proteins needed for balanced dNTP pools and for optimal DNA replication.

Fig. 3.. Model for Fhit as a scavenger decapping enzyme regulating translation of TK1 mRNA.

A) Structure of diadenosine triphosphate (Ap3A), the first recognized in vitro substrate for Fhit, and the 5’ 7-methyl-guanosine cap. B) In the 3‟ to 5‟ mRNA decay pathway, the exosome generates free m7GpppN dinucleotides that can be hydrolyzed by a scavenger decapping enzyme. The model hypothesizes that In the presence of Fhit, Fhit binds and hydrolyzes m7GpppN into m7GDP and m7GMP, which are cleared from the cell. In the absence of Fhit, free m7GpppN caps accumulate. Preferential binding of translation initiation factors to these free caps instead of capped TK1 mRNAs leads to deregulated translation of TK1 mRNA. As the model proposes, Fhit is thus a scavenger-decapping enzyme that eliminates residual cap structures to promote ribosomal binding and translation of cap-bearing mRNAs.

Fig. 4.. Wwox-deficient cells are significantly more resistant to various genotoxic agents.

A) Graph of % survival of a small panel of five breast cancer cell lines following exposure to various doses of gamma IR. B) Graph of % survival of mouse epithelial cell line, kd2, at early and late passage following various doses of gamma IR. C) Line graph depicting % survival of Wwox MEFs following exposure to MMC for 4 hrs. D) Line graph depicting % survival of Wwox MEFs following exposure to MMC for 4 hours + ABT-888 for 24 hours. Embedded graph depicts cell survival of MEFs with ABT-888 treatment alone. (A-B) Embedded western blots depict Wwox expression in the cell lines. (A-D) Error bars depict standard error and are the results of three independent experiments.

Fig. 5.. Fhit and Wwox expression affect cancer patient survival.

A) Kaplan-Meier plot of ovarian cancers treated with Platinum-based agents and separated into Wwox-reduced or Wwox normal/high groups. Patients with Wwox-deficient cancers have significantly shorter progression-free survivals (P< 0.001, n=1185). B) Box plot depicting relative Wwox expression in lung squamous cell carcinomas categorized into two groups: Fhit normal and Fhit low.