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Published OnlineFirst November 8, 2017; DOI: 10.1158/1541-7786.MCR-17-0215 Cell Death and Survival Molecular Cancer Research Loss of Uracil DNA Glycosylase Selectively Resensitizes p53-Mutant and -Deficient Cells to 5-FdU Yan Yan1, Yulan Qing2, John J. Pink2, and Stanton L. Gerson2 Abstract Thymidylate synthase (TS) inhibitors including fluoropyri- significantly sensitizes p53 KO cells.Thissensitizationcanalso midines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine be recapitulated by UDG depletion in cells with p53 KD by (5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) are shRNAs. In addition, sensitization is also observed with peme- widely used against solid tumors. Previously, we reported that trexed in p53 KO cells, but not with 5-FU, most likely due to shRNA-mediated knockdown (KD) of uracil DNA glycosylase RNA incorporation. Importantly, in p53 WT cells, the apoptosis (UDG) sensitized cancer cells to 5-FdU. Because p53 has also pathway induced by 5-FdU is activated independent of UDG been shown as a critical determinant of the sensitivity to TS status. However, in p53 KO cells, apoptosis is compromised in inhibitors, we further interrogated 5-FdU cytotoxicity after UDG-expressing cells, but dramatically elevated in UDG- UDG depletion with regard to p53 status. By analyzing a panel depleted cells. Collectively, these results provide evidence that of human cancer cells with known p53 status, it was deter- loss of UDG catalyzes significant cell death signals only in mined that p53-mutated or -deficient cells are highly resistant cancer cells mutant or deficient in p53. to 5-FdU. UDG depletion resensitizes 5-FdU in p53-mutant and -deficient cells, whereas p53 wild-type (WT) cells are not Implications: This study reveals that UDG depletion restores affected under similar conditions. Utilizing paired HCT116 p53 sensitivity to TS inhibitors and has chemotherapeutic potential WT and p53 knockout (KO) cells, it was shown that loss of p53 in the context of mutant or deficient p53. Mol Cancer Res; 16(2); improves cell survival after 5-FdU, and UDG depletion only 212–21. Ó2017 AACR. Introduction Fluoropyrimidines are widely used in the treatment of various types of malignancies for their broad antitumor activity. Once Thymidylate synthase (TS) is a key enzyme that catalyzes the taken into cells, fluoropyrimidines can be metabolized into fluor- only means for de novo synthesis of deoxythymidine monopho- odeoxyuridine monophosphate (FdUMP) and fluorodeoxyuri- sphate (dTMP; ref. 1). TS utilizes 5,10-methylenetetrahydrofolate dine triphosphate (FdUTP; refs. 2–5). The metabolite FdUMP (5,10-CH THF) as the methyl-group donor and catalyzes the 2 inhibits TS by forming a stable ternary complex with TS and reductive methylation of deoxyuridine monophosphate (dUMP) CH THF (6–8), which ultimately leads to the depletion of dTTP to dTMP (1). dTMP is subsequently phosphorylated to deoxythy- 2 and accumulation of deoxyuridine triphosphate (dUTP). The midine triphosphate (dTTP), a critical precursor for DNA repli- resulting imbalance of deoxynucleotide pools favors the utiliza- cation and repair. As TS contains binding sites for the substrate tion of dUTP and FdUTP during DNA replication and leads to the nucleotide (dUMP) and the cofactor folate (5,10-CH THF), two 2 accumulationofbothuraciland5-FUinDNA (2–5).Multitargeted structurally different classes of inhibitors, nucleotide, or folate antifolates such as pemetrexed have been approved as compo- analogs block the activity of TS (2). The class of fluoropyrimidines nents of first-line therapy in combination with cisplatin for the including 5-fluorouracil (5-FU) and floxuridine (5-FdU) target treatment of advanced non–small cell lung cancer (9). Pemetrexed the nucleotide-binding site, whereas the antifolates such as peme- inhibits several folate-dependent enzymes; however, TS is its trexed target the folate-binding site of TS. predominant target (10–13). Administration of pemetrexed leads to a global reduction in nucleotide synthesis as well as accumu- lation of dUTP (14). As a result, dUTP is used in DNA synthesis in place of dTTP, generating uracil misincorporation into DNA (15). 1Department of Pharmacology, Case Western Reserve University, Cleveland, 2 Misincorporated uracil and 5-FU are both primarily recognized Ohio. Case Comprehensive Cancer Center, Division of General Medical and repaired by the uracil DNA glycosylase (UDG)–initiated base Sciences-Oncology, Case Western Reserve University, Cleveland, Ohio. excision repair pathway (16). Although incorporation of uracil Note: Supplementary data for this article are available at Molecular Cancer and 5-FU into DNA is well documented as a consequence of Research Online (http://mcr.aacrjournals.org/). exposure to TS inhibitors (15), the impact of the downstream Corresponding Author: Stanton L. Gerson, Case Comprehensive Cancer Center, repair pathway directed by UDG on cell survival is not consistent. Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106. It has been hypothesized that thymine-less futile cycles of uracil Phone: 216-844-8562; Fax: 216-844-4975; E-mail: [email protected] misincorporation, excision by UDG, and further dUTP reinsertion doi: 10.1158/1541-7786.MCR-17-0215 result in DNA strand breaks and cell death (17). If thymine-less Ó2017 American Association for Cancer Research. cell death was dependent on UDG-mediated removal of uracil 212 Mol Cancer Res; 16(2) February 2018 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Published OnlineFirst November 8, 2017; DOI: 10.1158/1541-7786.MCR-17-0215 UDG Depletion Resensitizes p53-Mutant Cells to 5-FdU and 5-FU, one would expect a correlation between the cytotoxicity Table 1. Cell lines and strains used in this work of TS inhibitors and UDG expression. However, the majority of Cell line Origin p53 status studies reported that neither overexpression, nor inhibition, of A375 Melanoma wt UDG affected the sensitivity to TS inhibitors in human, mouse, or LoVo Colon cancer wt – RKO Colon cancer wt chicken DT40 cells (16, 18 23). In contrast, recently both our and A2780 Ovarian cancer wt the Karnitz group observed that loss of UDG highly potentiated H460 Large cell lung cancer wt the cytotoxicity of 5-FdU in several cancer cell lines, indicating H1299 Non–small cell lung cancer null that uracil and 5-FU incorporation played a key role in cell killing OVCAR8 Ovarian cancer del 126-132 (24, 25). DLD1 Colon cancer S241F As the mediators of cell killing due to persistent uracil and 5-FU HEC1A Endometrial cancer R248Q lesions in DNA are not clear, we assessed the likely pathways and noted that one of the major differences in these disparate findings is that cancer cells bearing p53 mutations were used in our and sity, Cleveland, OH). Other cancer cell lines were purchased from Karnitz's experimental system, whereas nontransformed or p53 the American Type Culture Collection. Details of the cell lines (wild-type) WT cancer cells were used in the majorities of others used in this study are listed in Table 1. All cells were maintained in (16, 18–20, 22). Mutation of TP53 is the most frequently observed DMEM (Corning 15-017-CV) supplemented with 10% dialyzed gene alteration in cancers (26). Mutations in p53 have been FBS, 2 mmol/L L-glutamine, 1% MEM NEAA, 100 U/mL penicil- shown to influence cellular response to chemotherapeutic agents lin, and 100 mg/mL streptomycin. Cells were incubated at 37Cin such as cisplatin, etoposide, and 5-FU (27, 28). Notably, sub- a humidified atmosphere of 95% air and 5% CO2. 5-FdU and 5- stantial evidence reveals that loss of p53, or p53 mutations, is FU were purchased from Sigma-Aldrich, dissolved respectively in linked to resistance to 5-FU due to inability to activate apoptosis Milli-Q water and DMSO, and stored as a 10 mmol/L stock at pathway. For example, a study using isogenic cell systems dem- À80C. Pemetrexed was purchased from LC laboratories and onstrated that deletion of p53 from a p53 WT colon cancer cell prepared fresh for each experiment by dissolving in Milli-Q water. line (HCT116) rendered cells remarkably resistant to apoptosis induced by 5-FU (29). In addition, 5-FU resistance was also Lentiviral shRNA knockdown described in a variety of p53-mutated cancer cells, including p53 or UDG knockdown (KD) was achieved via shRNA trans- colon, bladder, pancreatic, and gastric cancer (30–33). However, duction. Lentiviral vectors LV-THM-shp53 (which also expresses a few studies have reported on the link of p53 status with the GFP reporter) or LV-Bleo-shp53 to perform p53 KD in WT response to other TS inhibitors such as 5-FdU. HCT116 cells were obtained from Dr. Mark Jackson's laboratory Given the divergent cell models with different p53 status used at Case Western Reserve University, Cleveland, OH (38). Lenti- in our and other studies, the following questions remain unan- viral vector targeting GFP (sh-GFP) was used as control. UDG swered: (1) does loss or mutation of p53 render cells resistant to 5- shRNA vectors (shUDG: NM_003362.2-656s21c1, shUDG-2: FdU, and (2) is the potentiated cytotoxicity of 5-FdU after UDG NM_003362.2-758s21c1, and shUDG-3: NM_003362.2- depletion reliant upon p53 status? To gain insight into these 893s21c1) were purchased from Sigma, and a scramble targeting questions, we tested the impact of UDG depletion on 5-FdU shRNA vector (Sigma) was used as paired control. The lentiviral cytotoxicity in a number of cancer cell lines with differing p53 production and infection were performed as previously described status. We found that, in general, loss or mutation of p53 remark- (24). Cells stably infected with LV-THM-p53 were isolated by cell ably reduced the sensitivity to 5-FdU, and depletion of UDG sorting on the basis of their GFP expression. Cells stably infected selectively resensitized p53-deficient or -mutated cancer cells to 5- with LV-Bleo-p53 were selected with zeocin (Sigma).

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