Loss of Uracil DNA Glycosylase Selectively Resensitizes P53-Mutant and -Deﬁcient Cells to 5-Fdu Yan Yan1, Yulan Qing2, John J

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. 2017 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 accumulation 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 2017 American Association for Cancer Research. cell death was dependent on UDG-mediated removal of uracil

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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- 80C. 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). Selection of FdU. In order to understand the underlying mechanism that positive UDG KD cells was assessed with puromycin (Sigma). contributes to the distinct response after UDG depletion, we utilized paired HCT116 cell lines with, or without, deletion of Clonogenic survival assay the TP53 gene and observed that loss of UDG selectively resensi- As described previously (24), cancer cells (200–300 cells/well) tized HCT116 cells with p53 deletion. This resensitization was were seeded in 6-well culture dishes and allowed to adhere also observed with pemetrexed, but to a lesser extent with 5-FU, overnight. For 5-FdU, cells were treated for 24 hours, then gently which mainly causes damage in RNA (21, 34–37). In the presence washed with PBS once, and incubated with fresh media for at least of WT p53, 5-FdU treatment induced activation of the apoptosis 10 days to allow individual colonies to form. For 5-FU or pathway in both UDG competent, or UDG-depleted cells at pemetrexed, cells were treated continuously for at least 10 days comparable levels. However, in the absence of WT p53, apoptosis to form colonies. After 10 to 18 days, the plates were stained with activation was compromised in UDG-expressing cells and dra- methylene blue. Colonies containing 50 cells were counted. The matically elevated in UDG-depleted cells. Collectively, these percentage of survival was determined relative to untreated con- findings suggest that loss, or mutation, of p53 is associated with trol averaged over three independent experiments. 5-FdU resistance, and UDG depletion can significantly restore sensitivity, indicating that UDG may serve as a therapeutic target Western blots and qPCR to improve the clinical effectiveness of 5-FdU. Western blots were performed as previously described (39). Twenty microgram of protein was loaded on SDS-polyacrylamide Materials and Methods gel. The following antibodies were used to detect proteins on the membrane: a-Tubulin (Calbiochem); GAPDH (Santa Cruz Cell lines and drugs Biotechnology); UDG (FL-313; Santa Cruz Biotechnology); HCT116 p53 knockout (KO) cells were a gift from Dr. Guang- cleaved PARP (Asp214)(19F4; Cell Signaling Technology); bin Luo (Department of Genetics, Case Western Reserve Univer- cleaved caspase 3 (Cell Signaling Technology); p53 (FL-393; Santa

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Cruz Biotechnology); and p21 (Santa Cruz Biotechnology). For Results qRT-PCR, total RNA from cells was extracted by using the RNeasy p53 mutation or deficiency affords 5-FdU resistance among Plus Mini Kit (Qiagen). cDNA synthesis was performed by using different types of cancer cells the SuperScript III First Strand Kit (Life Technologies). qPCR was Given that p53 mutations or deficiencies are frequently achieved with validated TaqMAN MGB FAMTM dye–labeled observed in cancers, studies have demonstrated that mutations probes (Applied Biosystems) for nuclear UDG on an ABI 7500 of p53 reduce 5-FU cytotoxicity (29–33). To understand Fast Real-time PCR System (Applied Biosystems). b-Actin was whether these mutations also altertheresponseto5-FdU,a used as an endogenous control, and relative gene expression was DD panel of human cancer cell lines from colon, lung, ovarian, calculated as 2 Ct. skin, and endometrium with intrinsically differing p53 status Flow cytometric assay of apoptosis was utilized in this study. The p53 status of each cell line is Cells were seeded in 6-well tissue culture plates (1.5 105 cells/ listed in Table 1. To determine p53 protein functionality in fi well) and allowed to attach overnight. Cells were then treated with p53 WT and p53-mutant (Mut) or -de cient cancer cell lines, 25 nmol/L 5-FdU for 24 hours, washed twice with PBS, and we assessed p53 levels and expression of p21, a widely accept- replenished with drug-free medium at 48, 72, and 96 hours. After ed initiator of p53-activated signaling (40), 24 hours after recovery, the cells floating in the medium were collected. The administration of 8 Gy gamma irradiation. All the p53 WT adherent cells were trypsinized, pelleted, washed in ice-cold PBS, cancer cell lines used in this work induced p21 expression after and resuspended in 1X binding buffer according to the manu- irradiation, indicating functional p53 in these cell lines (Sup- facturer's instructions (FITC Annexin V Apoptosis Detection Kit; plementary Fig. S1). In order to establish the relationship BD Pharmingen). Cells were then stained with FITC–Annexin V between p53 status and 5-FdU sensitivity, we evaluated the and propidium iodide (PI) for 15 minutes at room temperature in cytotoxicity of 5-FdU in these cell lines by clonogenic survival the dark. Annexin V–FITC detects translocation of phosphatidy- assay. As shown in Fig. 1A, the cell lines tested displayed a linositol from the inner to the out of cell membrane during early spectrum of 5-FdU sensitivities with IC50 values ranging from apoptosis, and PI can enter the cells in late apoptosis or necrosis. 1.32 0.33 to 269.55 0.73 nmol/L for A2780 and H1299 Untreated cells were used as control for the double staining. The lines, respectively. Importantly, we observed that, in general, fi cells were analyzed immediately after staining using an Attune cell lines with p53 mutation or de ciency (Fig. 1A, solid lines) fi NXT instrument and FlowJo software. For each measurement, at were signi cantly more resistant to 5-FdU than p53 WT least 20,000 cells were counted. cells (Fig. 1A, dashed lines), with the exception of A375 which has WT p53 but an IC50 of 110.81 1.80 nmol/L. In addition, Statistical analysis except for A375, the IC50 values for the p53 WT cancer lines Statistical significance between two treatment groups was ana- clustered together at a lower dose range (<10 nmol/L), whereas lyzed using unpaired two-tailed Student t test. Significance was p53-mutant or -deficient lines clustered at a higher range assigned for a P value < 0.05. Standard software GraphPad Prism (>100 nmol/L; Fig. 1B). These observations are consistent with and Excel 2013 (Microsoft Corp.) were used for all statistical the hypothesis that p53 mutation or deficiency is associated analysis. with resistance to 5-FdU.

Figure 1. 5-FdU resistance in different types of cancer cells with p53 mutation or deﬁciency. A, Clonogenic survival assay in cancer cell lines shown in Table 1 in response to increasing doses of 5-FdU. Cell lines with WT p53, dashed lines; cell lines with deﬁcient (or mutant) p53, solid lines. The results represent three independent experiments that were done in duplicate each time.

B, IC50 values of 5-FdU for cancer cell lines listed in Table 1 with WT p53 or deﬁcient (or mutant) p53 status, respectively.

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UDG Depletion Resensitizes p53-Mutant Cells to 5-FdU

UDG depletion sensitizes cancer cells with p53 mutation or loss of WT p53 protein expression can alter the response to 5-FdU. deficiency to 5-FdU exposure To address this, we utilized paired HCT116 colon cancer cell lines Previously, the discordant findings on sensitization to 5-FdU with or without genetic TP53 deletion and tested their sensitivity following UDG depletion were reported using cell models with to 5-FdU, and the loss of p53 expression was evaluated by Western differing p53 status (16, 18–25). To understand whether the blot (Fig. 3A). Using a clonogenic survival assay, we demonstrated divergent responses could be attributed to p53 status, we explored that p53 KO cells were more resistant to 5-FdU than p53 WT cells whether UDG depletion could sensitize p53-mutant or -deficient (Fig. 3B). KD of p53 by shRNA recapitulates the resistance cancer cells to 5-FdU differentially. For these experiments, we used observed in p53 KO cells (Fig. 3B), indicating that p53 status is shRNA to deplete UDG in various cancer cell lines with differing a key mediator of the response of HCT116 cells to 5-FdU. p53 status, as listed in Table 1. UDG stable KD was evaluated by To understand whether loss of p53 protein will affect the Western blot (Fig. 2A and B, insert). Based on a clonogenic response to 5-FdU after UDG depletion, we knocked down UDG survival assay, we observed that UDG depletion selectively sen- by shRNA in both HCT116 p53 WT and p53 KO cells. UDG KD sitized cells with p53 mutation or deficiency to 5-FdU exposure levels were shown to be greater than 90% as evaluated by Western (Fig. 2A). However, in p53 WT cell lines, UDG depletion did not blot and qPCR (Fig. 3C and D). In agreement with our data using alter the cytotoxicity of 5-FdU (Fig. 2B). Collectively, these results p53-mutant cells, UDG depletion greatly enhanced cytotoxicity of demonstrate that UDG depletion resensitizes p53-mutant or 5-FdU in p53 KO cells but did not significantly affect p53 WT cells -deficient cancer cells, providing a novel therapeutic target for (Fig. 3E and F), indicating that p53 is involved in regulating the patients with p53-mutant tumors. response to 5-FdU following UDG depletion. To exclude the off- target effect of a single shRNA, we also utilized two other shRNAs 5-FdU resistance in p53 KO or KD cells is reversed by UDG that target UDG in HCT116 p53 WT and p53 KO cells and depletion observed similar effect (Supplementary Fig. S2). In addition, Because many studies have identified gain of various functions depletion of UDG also potentiated 5-FdU cytotoxicity in two for specific p53-mutated proteins (41, 42), we next asked whether HCT116 cancer cells with different shRNAs targeted to p53

Figure 2. UDG depletion selectively sensitizes cells with p53 mutation or deﬁciency to 5-FdU. Stable cancer cell lines infected with nontargeted scramble control shRNA (shSCR) or UDG-directed shRNA (shUDG) were analyzed by Western blot to examine UDG levels (insert). Clonogenic survival assays of UDG-expressing (shSCR) and UDG-depleted (shUDG) cancer cells with (A) mutant or deﬁcient p53, or (B) WT p53 that are treated with increasing doses of 5-FdU. Values indicate mean values S.E.M. The results represent three independent experiments that were done in duplicate each time (, P < 0.01).

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Figure 3. 5-FdU resistance due to loss of p53 is reversed by UDG depletion. A, p53 expression levels were analyzed by Western blot in HCT116 cells with wild- type p53 (p53WT), knockout of p53 (p53KO), shGFP-expressing vector (shGFP), and shp53-expressing vector (shp53); , nonspeciﬁc bands. B, Clonogenic survival assay for increasing doses of 5-FdU in HCT116 p53WT, p53KO, shGFP-infected, and shp53-infected cells. HCT116 p53WT and p53KO cells stably infected with nontargeted scramble control shRNA (shSCR) or UDG-directed shRNA (shUDG) were analyzed by Western blot (C) and qPCR (D) to examine UDG levels. Clonogenic survival assay for increasing doses of 5-FdU in HCT116 (E) p53WT cells, with shSCR, or with shUDG; and (F) p53KO cells, with shSCR, or with shUDG. Viable colonies (>50 cells) stained with methylene blue after 10 days of culture were counted. The results represent three independent experiments that were done in duplicate each time (, P < 0.01).

(Fig. 4A–E). Collectively, these results confirm that loss of p53 UDG-expressing and UDG-depleted cells in the presence of p53 protein renders cells resistant to 5-FdU, and UDG depletion after pemetrexed or 5-FU treatment (Fig. 5A and B). However, in selectively resensitizes p53 KO and KD cells to 5-FdU. the absence of p53, UDG depletion sensitized cells to pemetrexed (Fig. 5C), whereas loss of UDG only moderately sensitized cells to UDG depletion selectively sensitizes p53 KO cancer cells to 5-FU at high concentrations (Fig. 5D), reaffirming that the pri- pemetrexed and 5-FU mary cytotoxic effect of 5-FU depends on RNA incoporation. Although all TS inhibitors have the ability to block TS, disrupt- Together, these results indicate that UDG depletion also sensitizes ing DNA replication and leading to uracil incorporation into cells without p53 to other TS inhibitors, mainly through gener- DNA, differences among distinct TS inhibitors have been reported ation of DNA damage. in terms of their other metabolism-mediated cytotoxic pathways (2). For example, pemetrexed polyglutamate derivatives also 5-FdU activates cell death in p53 KO cancer cells with depleted demonstrate inhibitory activity for other folate-dependent UDG enzymes such as glycinamide ribonucleotide, but to a lesser extent To understand whether 5-FdU resistance observed in p53 KO (10–13). Moreover, unlike 5-FdU, which mainly exerts its cyto- cells is due to a failure to activate cell death pathways, we toxicity due to effects at the DNA level (24), studies have revealed monitored cell death progression by Annexin V and PI staining. that the cytotoxicity of 5-FU is primarily RNA-mediated, as 5-FU is Cells were exposed to 5-FdU for 24 hours, washed with PBS, metabolized to fluorouridine triphosphate which affects multiple and then allowed to recover in drug-free medium for a total of RNA processes following its incorporation into RNAs (21, 34–37). 48, 72, and 96 hours (Fig. 6A). In cells with WT p53, 5-FdU In order to address the question of whether p53 status is respon- caused significant cell death (Annexin V and PI positive) at sible for the differences in sensitivity to other TS inhibitors, 48 hours which was retained at 72 and 96 hours in both including pemetrexed and 5-FU, in UDG-depleted cells, we eval- UDG-expressing and UDG-depleted cells (Fig. 6B and C). uated cell viability following drug exposure in UDG-depleted p53 However, in the absence of p53, cell death caused by 5-FdU WT and p53 KO cancer cells. Similar to our observations with was significantly lower in UDG-expressing cells, whereas in 5-FdU, no significant survival differences were found between UDG-depleted cells, cell death was detected at 24 hours and

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Figure 4. p53 KD resensitizes cancer cells with UDG depletion to 5-FdU. A, HCT116 cells stably infected with shGFP or shp53 (shp53-THM or shp53-Bleo) shRNAs were analyzed by the Western blot to examine p53 KD levels (, nonspecific bands). B, HCT116 cells expressing shGFP or shp53 (shp53-THM or shp53-Bleo) were further infected with nontargeted scramble control shRNA (shSCR) or UDG-directed shRNA (shUDG). UDG mRNA levels were determined by qPCR. Clonogenic survival assay for increasing doses of 5-FdU in (C) shGFP, (D) shp53-THM, and (E) shp53-Bleo–infected HCT116 cells alone, with shSCR, or with shUDG. Viable colonies (>50 cells) stained with methylene blue after 10 days of culture were counted. The results represent three independent experiments that were done in duplicate each time (, P < 0.01). significantly elevated at 48 to 96 hours (Fig. 6B and C). These to loss of p53 is responsible for the enhanced cell survival datasuggestthat5-FdU–induced cell death is dependent upon observed in p53 KO cells. However, in p53 KO cell with coinci- p53, supporting the observation that drug resistance can be dent UDG depletion, 5-FdU selectively activates a p53-indepen- observed as a result of abrogation of the p53-mediated cell dent apoptotic pathway through a mechanism which needs death pathway. Importantly, UDG depletion significantly further investigation. potentiates death of cells lacking WT p53 activity through a p53-independent pathway. Discussion To further elucidate whether the cell death caused by 5-FdU is due to apoptosis, we examined expression of proteins involved in In this study, we utilized multiple cancer cells bearing differing the activation of the apoptotic pathway. In WT p53 cells, we p53 status with or without UDG expression. We observed that loss observed that p53 expression was induced at 24 hours, and the of UDG selectively resensitized cancer cells with p53 mutation or induction remained for 96 hours in both shSCR and shUDG cells deficiency to 5-FdU, but did not alter the response of p53 WT cells. following 5-FdU exposure (Fig. 6D). The expression of cleaved These results demonstrate that UDG, through its function of PARP, a hallmark of apoptotic cell death, was induced at 48 hours removing uracil or 5-FU, plays a major role in the effect of 5-FdU and persisted through 72 and 96 hours in p53 WT cells regardless on the response of cells lacking WT p53 activity. Our findings of whether UDG was present or not (Fig. 6D). In addition, cleaved resolve the unexplained discrepancy observed in a number of caspase 3 was also detected in both UDG-expressing or -depleted prior studies regarding the role of UDG in sensitivity to TS p53 WT cells (Fig. 6D). In the absence of p53, induction of cleaved inhibitors. Prior studies revealed that either loss of UDG PARP or caspase 3 was not readily detected in cells expressing enhanced the cytotoxicity of 5-FdU or pemetrexed in cancer cells UDG after 5-FdU exposure (Fig. 6E), whereas both were robustly (24, 25), or overexpression or inhibition of UDG had no effect induced from 48 to 96 hours in cells depleted of UDG (Fig. 6E). on the sensitivity of human or mouse cells to TS inhibition Taken together, our results suggest that 5-FdU–induced apoptosis (16, 18–20, 22). The difference, we propose, is dependent on is mediated through p53, and the lack of apoptosis activation due p53 status.

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Figure 5. UDG depletion selectively sensitizes p53 KO cells to pemetrexed and 5-FU. Clonogenic survival assay in HCT116 p53 WT cells (shSCR and shUDG) treated with increasing doses of (A) pemetrexed and (B) 5-FU. Clonogenic survival assay in HCT116 p53 KO cells (shSCR and shUDG) treated with increasing doses of (C) pemetrexed and (D) 5-FU. Viable colonies (>50 cells) stained with methylene blue after 10 days of culture were counted. The results represent three independent experiments that were done in duplicate (, P < 0.01).

p53 plays a key role in determining the sensitivity of cells to Our results demonstrated that inhibition of UDG selectively 5-FU. A number of studies reported that enhanced 5-FU resistance sensitized p53-mutant and -deficient cancer cells to 5-FdU, but has been observed in cells bearing TP53 deletion or mutations did not alter the response in p53 WT cells. Importantly, we have (29–33). However, unlike other TS inhibitors, 5-FU exposure observed that apoptosis following 5-FdU is efficiently induced in caused only slightly potentiated cytotoxicity at higher doses in the presence of p53 but highly compromised in cells lacking p53, UDG-depleted, p53 KO cell lines. Recently, several studies have indicating that the activation of the 5-FdU–induced cell death observed that the cytotoxicity of 5-FU is more dependent on its pathway is dependent on p53. Further studies with different p53 incorporation into RNA than its inhibition of TS, diminishing its WT cell lines also revealed cells highly sensitive to 5-FdU with IC50 effect on DNA (21, 34–37). In addition, activation of p53 fol- values lower than 10 nmol/L. One exception we observed was in lowing 5-FU exposure has been identified as working through the A375 melanoma cells line, which has a WT TP53 gene. A375 RNA mechanisms (43, 44). Because UDG recognizes only DNA was relatively insensitive to 5-FdU and had an IC50 of 110.81 lesions, it is not surprising that depletion of UDG does not 1.80 nmol/L. Clearly, more knowledge is needed regarding the significantly alter cellular responses to agents that primarily affect p53-mediated cell death pathway and how 5-FdU, with or with- RNA function. Together, this suggests that the increased cytotox- out UDG, causes damage and triggers cell death. In response to icity of 5-FdU and pemetrexed observed in UDG-depleted cells is 5-FdU, cells lacking WT p53, combined with UDG depletion, primarily due to uracil and 5-FU incorporation into DNA. activate cell death in a p53-independent manner, which reverses The present results illustrate that cells with p53 mutation or chemoresistance and selectively resensitizes these cancer cells to deficiency are significantly resistant to 5-FdU in comparison with 5-FdU. p53 WT cells. It is clear that many different mutant p53s also The current findings of this article focus on the role of p53 in acquire oncogenic functions that are distinct from the activities of apoptosis and show that in the presence of p53, both UDG WT– WT p53 (41, 42). Some p53 mutants provide enhanced resistance and UDG-depleted cells activate apoptosis at similar levels; how- to apoptosis induced by a variety of treatments, including certain ever, in the absence of p53, apoptosis induction is compromised chemotherapeutic drugs (27, 45). In particular, one study iden- in the UDG WT cells but significantly increased in UDG-depleted tified that p53 mutants activate expression of dUTPase (46), which cells. These data explain that p53 WT cells have 5-FdU IC50 values has been related to the resistance to TS inhibitors (47–49). Our less than 10 nmol/L, whereas p53-mutant or -deficient cells have results on a select group of p53 mutants as well as p53 KO cell lines IC50 values higher than 100 nmol/L. The result is consistent with a revealed resistance to 5-FdU treatment. However, the p53 KO cell previous publication from Janet Houghton's group (30) that cells line is much less resistant to 5-FdU than other p53-mutant cell with WT p53 displayed acute apoptosis, whereas cells with mutant lines, suggesting the potential for enhanced resistance due to p53 showed delayed or compromised apoptosis following fluor- gained functions for certain p53 mutants. opyrimidine treatment. Based on these results, we propose the

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UDG Depletion Resensitizes p53-Mutant Cells to 5-FdU

Figure 6. UDG depletion induces cell death caused by 5-FdU in p53 KO cancer cells. A, Schematic diagram of the treatment for HCT116 p53WT (shSCR and shUDG) and p53KO (shSCR and shUDG) cells with 25 nmol/L 5-FdU for 24 hours, washed, and replenished with drug-free medium at indicated time points. B, Untreated (Unt) or treated cells were subjected to FITC– Annexin V and PI staining and analyzed by flow cytometry. Representative flow plots of three independent experiments are shown. C, Cell death is expressed as the percentage of Annexin V-positive cells. Values indicate mean values SD. All experiments were performed independently for 3 times (, P < 0.01). Protein expression involved in regulation of apoptotic cell death in response to 5-FdU was detected in HCT116 (D) p53 WT (shSCR and shUDG) and (E) p53 KO (shSCR and shUDG) cells (, nonspecific bands).

mechanism of cell death that: ﬁrst, DNA damage due to loss of already been activated in the presence of WT p53 following UDG enhanced the apoptosis in p53-mutant or -deﬁcient cancer 5-FdU–induced stress. Second, loss of UDG increases levels of cells, but did not change the apoptosis in p53 WT cells which has persistent uracil and 5-FU incorporation at the replication forks

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Yan et al.

that without p53 results in early S phase arrest and disintegration Authors' Contributions of the replication fork progression, as we have shown previously Conception and design: Y. Yan, Y. Qing, S.L. Gerson (24). Further support for this mechanism comes from an article Development of methodology: S.L. Gerson published recently by Swati Palit Deb's lab (50), which has shown Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Yan that lack of WT p53 increases DNA origin firing compared with fi Analysis and interpretation of data (e.g., statistical analysis, biostatistics, p53 WT cells. In our system, more DNA origin rings would result computational analysis): Y. Yan, Y. Qing, J.J. Pink, S.L. Gerson in both more 5-FU and uracil incorporation and further disrup- Writing, review, and/or revision of the manuscript: Y. Yan, Y. Qing, J.J. Pink, tion of these replication forks that improve the killing effect in S.L. Gerson p53-mutant or -deficient cancer cells. Taken together, these results Study supervision: S.L. Gerson provide an explanation for the discordant findings in previous published data regarding the role of UDG in mediating the The costs of publication of this article were defrayed in part by the cytotoxicity of TS inhibitors and suggest that UDG is an attractive payment of page charges. This article must therefore be hereby marked therapeutic target in cancer cells with p53 mutation or deficiency, advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate to enhance their response to TS inhibitors. this fact.

Disclosure of Potential Conﬂicts of Interest Received April 21, 2017; revised August 2, 2017; accepted October 26, 2017; No potential conﬂicts of interest were disclosed. published OnlineFirst November 8, 2017.

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Loss of Uracil DNA Glycosylase Selectively Resensitizes p53-Mutant and -Deficient Cells to 5-FdU

Yan Yan, Yulan Qing, John J. Pink, et al.

Mol Cancer Res 2018;16:212-221. Published OnlineFirst November 8, 2017.

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