Inhibition of uracil DNA glycosylase sensitizes cancer cells to 5-fluorodeoxyuridine through replication fork collapse-induced DNA damage

Abstract

5-fluorodeoxyuridine (5-FdU, floxuridine) is active against multiple cancers through the inhibition of thymidylate synthase, which consequently introduces uracil and 5-FU incorporation into the genome. Uracil DNA glycosylase (UDG) is one of the main enzymes responsible for the removal of uracil and 5-FU. However, how exactly UDG mediates cellular sensitivity to 5-FdU, and if so whether it is through its ability to remove uracil and 5-FU have not been well characterized. In this study, we report that UDG depletion led to incorporation of uracil and 5-FU in DNA following 5-FdU treatment and significantly enhanced 5-FdU's cytotoxicity in cancer cell lines. Co-treatment, but not post-treatment with thymidine prevented cell death of UDG depleted cells by 5-FdU, indicating that the enhanced cytotoxicity is due to the retention of uracil and 5-FU in genomic DNA in the absence of UDG. Furthermore, UDG depleted cells were arrested at late G1 and early S phase by 5-FdU, followed by accumulation of sub-G1 population indicating cell death. Mechanistically, 5-FdU dramatically reduced DNA replication speed in UDG depleted cells. UDG depletion also greatly enhanced DNA damage as shown by γH2AX foci formation. Notably, the increased γH2AX foci formation was not suppressed by caspase inhibitor treatment, suggesting that DNA damage precedes cell death induced by 5-FdU. Together, these data provide novel mechanistic insights into the roles of UDG in DNA replication, damage repair, and cell death in response to 5-FdU and suggest that UDG is a target for improving the anticancer effect of this agent.

Administration of a Nucleoside Analog Promotes Cancer Cell Death in a Telomerase-Dependent Manner

“…However, previous studies have recently expanded the mechanism of action and cytotoxicity of 5-FU to include its incorporation into RNA products (Silverstein et al, 2011). Subsequent studies have similarly shown that 5-FdU is phosphorylated to serve as a direct substrate for DNA misincorporation, thereby exacerbating its cytotoxic effects (Yan et al, 2016). Our data support this latter role of 5-FdU in mediating carcinoma cytotoxicity, as we demonstrate that the low-fidelity telomerase reverse-transcriptase readily misincorporates non-native nucleotide analogs, including 5-FdU, into its telomere DNA products.…”

“…In these cases, the remaining cytotoxic effects are likely due to inhibition of thymidylate synthase and/or misincorporation of 5-FdU or deoxyuridine (a by-product of thymidylate synthase inhibition) into genomic DNA. Indeed, both uracil and 5-FU moieties are detected in genomic DNA after treating cells with 5-FdU (Yan et al, 2016). These dual effects are likely recapitulated at the telomere upon treatment of 5-FdU.…”

Genome-wide alterations of uracil distribution patterns in human DNA upon chemotherapeutic treatments

“…As shown in Supplementary Figure S1B-C, UGI expression and drug (5FdUR or RTX) treatment led to significantly increased uracil content in genomic DNA. It is important to note that either UGI expression or treatments with drugs targeting de novo thymidylate biosynthesis pathways on their own do not lead to elevated U-DNA level (Luo, Walla, & Wyatt, 2008; Róna et al, 2016; Yan et al, 2016). Following U-DNA immunoprecipitation, successful enrichment of U-DNA could be confirmed by dot blot assay in the case of drug-treated cells (5FdUR_UGI or RTX_UGI, Figure 1B).…”

“…Scale bar represents 40 μm. Note that the nuclei of the treated cells (5FdUR_UGI) are enlarged as compared to the non-treated ones (NT_UGI) presumably due to cell cycle arrest (Huehls et al, 2016; Yan et al, 2016).…”

Advances in therapeutic targeting of the DNA damage response in cancer

“…Inhibitors of Pol β have also been developed but demonstrated only mild preclinical efficacy, and new compounds suffer from the fear of toxicity due to overlapping specificity with additional DNA polymerases [29]. Finally, recently our lab and others demonstrated that inhibition of uracil DNA glycosylase (UDG) sensitized cancer cells to thymidylate synthase (TS) inhibitors including floxuridine and pemetrexed in the preclinical setting, providing rationale for further development of UDG inhibitors [30,31]. …”