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Characterization of Uracil DNA Glycosylase as a Therapeutic Target for Sensitization of Floxuridine in Cancer with P53 Mutation or Deficiency

Yan, Yan
http://rave.ohiolink.edu/etdc/view?acc_num=case1499644338014377

Abstract Details

, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
Cancer is a collection of genetic diseases which occur via a step-by-step mutagenic process whereby cancer cells gain a selective growth advantage over normal epithelial cells. Mutation in the TP53 tumor suppressor gene is one of the most frequent events in tumorigenesis, as p53 acts as a master regulator of numerous signaling pathways that drive tumor suppressive activities. In particular, mutant p53 proteins not only lose their tumor suppressor function but often endow cells with novel abilities that promote tumor progression as well as chemoresistance. Chemotherapeutic agents such as thymidylate synthase (TS) inhibitors stand out as some of the most successful drugs in the management of cancer. Floxuridine (5-fluorodeoxyuridine, 5-FdU) is a classic TS inhibitor that can impede DNA metabolism and ultimately introduce uracil and 5-FU incorporation into the genome. According to in vitro kinetic studies, uracil DNA glycosylase (UDG) is the predominant enzyme that removes genomic uracil and 5-FU lesions. However, little is known about how uracil and 5-FU are processed in cancer cells, and whether UDG is a determinant of floxuridine sensitivity that can be exploited as a therapeutic target for potentiation of its cytotoxicity. In the present study, we first demonstrate that UDG plays a key role in limiting uracil and 5-FU incorporation into genomic DNA following 5-FdU treatment. The persistent uracil and 5-FU incorporation due to loss of UDG highly enhance the sensitization of cancer cells to 5-FdU. UDG is required for recovery from cell cycle arrest after 5-FdU, while UDG depleted cells arrest at late G1 and early S phase. Mechanistically, 5-FdU significantly delays replication fork progression in UDG depleted cells. Importantly, the sustained DNA damage, likely due to replication fork collapse, contributes to the genotoxicity of 5-FdU in the absence of UDG, which cannot be rescued by a caspase inhibitor. Together, these results indicate a critical role for UDG in regulating the cell killing effect of 5-FdU. Since p53 has been shown previously to be a critical determinant of sensitivity to TS inhibitors, we further interrogate the cytotoxicity of 5-FdU after UDG depletion with regard to p53 status. By analyzing a panel of cancer cells with differing p53 status, we find that cells with p53 mutations or deficiencies are highly resistant to 5-FdU. UDG depletion re-sensitizes p53 mutant or deficient cancer cells to 5-FdU, whereas p53 wild-type cells are not affected by loss of UDG. Utilizing paired HCT116 p53 wild-type (WT) and p53 knockout (KO) cell lines, we report that loss of p53 improves cell survival after 5-FdU, and UDG depletion only sensitizes p53 KO cells. Additionally, re-sensitization is also detected after treatment with the TS inhibitors pemetrexed and 5-FU in p53 KO cells. In p53 WT cells, the apoptosis pathway induced by 5-FdU is activated similarly regardless of whether UDG is present or not. However, in p53 KO cells, apoptosis is compromised in UDG expressing cells, but dramatically elevated in UDG depleted cells. Collectively, these findings provide evidence that UDG greatly impacts the cytotoxicity of TS inhibitors in p53 mutant and deficient cancer cells, which are commonly refractory to cell death in response to TS-targeted chemotherapy. Therefore, targeting UDG can be manipulated for the therapeutic purposes to augment the cell killing effect of TS inhibitors selectively in p53 mutant and deficient cancers.
Stanton Gerson (Advisor)
Pharmacology

Recommended Citations

Citations

  • Yan, Y. (n.d.). Characterization of Uracil DNA Glycosylase as a Therapeutic Target for Sensitization of Floxuridine in Cancer with P53 Mutation or Deficiency [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1499644338014377

    APA Style (7th edition)

  • Yan, Yan. Characterization of Uracil DNA Glycosylase as a Therapeutic Target for Sensitization of Floxuridine in Cancer with P53 Mutation or Deficiency. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1499644338014377.

    MLA Style (8th edition)

  • Yan, Yan. "Characterization of Uracil DNA Glycosylase as a Therapeutic Target for Sensitization of Floxuridine in Cancer with P53 Mutation or Deficiency." Doctoral dissertation, Case Western Reserve University. Accessed APRIL 25, 2024. http://rave.ohiolink.edu/etdc/view?acc_num=case1499644338014377

    Chicago Manual of Style (17th edition)

case1499644338014377
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.