Loss of Uracil DNA Glycosylase Selectively Resensitizes p53-Mutant … · 2017. 11. 28. · DNA...

DNA Damage and Repair

Loss of Uracil DNA Glycosylase SelectivelyResensitizes p53-Mutant and -Deficient Cellsto 5-FdUYan Yan1, Yulan Qing2, John J. Pink2, and Stanton L. Gerson2

Abstract

Thymidylate synthase (TS) inhibitors including fluoropyri-midines [e.g., 5-Fluorouracil (5-FU) and 5-Fluorodeoxyuridine(5-FdU, floxuridine)] and antifolates (e.g., pemetrexed) arewidely used against solid tumors. Previously, we reported thatshRNA-mediated knockdown (KD) of uracil DNA glycosylase(UDG) sensitized cancer cells to 5-FdU. Because p53 has alsobeen shown as a critical determinant of the sensitivity to TSinhibitors, we further interrogated 5-FdU cytotoxicity afterUDG depletion with regard to p53 status. By analyzing a panelof human cancer cells with known p53 status, it was deter-mined that p53-mutated or -deficient cells are highly resistantto 5-FdU. UDG depletion resensitizes 5-FdU in p53-mutantand -deficient cells, whereas p53 wild-type (WT) cells are notaffected under similar conditions. Utilizing paired HCT116 p53WT and p53 knockout (KO) cells, it was shown that loss of p53improves cell survival after 5-FdU, and UDG depletion only

significantly sensitizes p53 KO cells. This sensitization can alsobe recapitulated by UDG depletion in cells with p53 KD byshRNAs. In addition, sensitization is also observed with peme-trexed in p53 KO cells, but not with 5-FU, most likely due toRNA incorporation. Importantly, in p53 WT cells, the apoptosispathway induced by 5-FdU is activated independent of UDGstatus. However, in p53 KO cells, apoptosis is compromised inUDG-expressing cells, but dramatically elevated in UDG-depleted cells. Collectively, these results provide evidence thatloss of UDG catalyzes significant cell death signals only incancer cells mutant or deficient in p53.

Implications: This study reveals that UDG depletion restoressensitivity to TS inhibitors and has chemotherapeutic potentialin the context of mutant or deficient p53. Mol Cancer Res; 1–10.�2017 AACR.

IntroductionThymidylate synthase (TS) is a key enzyme that catalyzes the

only means for de novo synthesis of deoxythymidine monopho-sphate (dTMP; ref. 1). TS utilizes 5,10-methylenetetrahydrofolate(5,10-CH2THF) as the methyl-group donor and catalyzes thereductive methylation of deoxyuridine monophosphate (dUMP)to dTMP (1). dTMP is subsequently phosphorylated to deoxythy-midine triphosphate (dTTP), a critical precursor for DNA repli-cation and repair. As TS contains binding sites for the substratenucleotide (dUMP) and the cofactor folate (5,10-CH2THF), twostructurally different classes of inhibitors, nucleotide, or folateanalogs block the activity of TS (2). The class of fluoropyrimidinesincluding 5-fluorouracil (5-FU) and floxuridine (5-FdU) targetthe nucleotide-binding site, whereas the antifolates such as peme-trexed target the folate-binding site of TS.

Fluoropyrimidines are widely used in the treatment of varioustypes of malignancies for their broad antitumor activity. Oncetaken into cells, fluoropyrimidines can be metabolized into fluor-odeoxyuridine monophosphate (FdUMP) and fluorodeoxyuri-dine triphosphate (FdUTP; refs. 2–5). The metabolite FdUMPinhibits TS by forming a stable ternary complex with TS andCH2THF (6–8), which ultimately leads to the depletion of dTTPand accumulation of deoxyuridine triphosphate (dUTP). Theresulting imbalance of deoxynucleotide pools favors the utiliza-tion of dUTP and FdUTP during DNA replication and leads to theaccumulationofbothuraciland5-FUinDNA(2–5).Multitargetedantifolates such as pemetrexed have been approved as compo-nents of first-line therapy in combination with cisplatin for thetreatment of advancednon–small cell lung cancer (9). Pemetrexedinhibits several folate-dependent enzymes; however, TS is itspredominant target (10–13). Administration of pemetrexed leadsto a global reduction in nucleotide synthesis as well as accumu-lation of dUTP (14). As a result, dUTP is used in DNA synthesis inplace of dTTP, generating uracil misincorporation into DNA (15).

Misincorporated uracil and 5-FU are both primarily recognizedand repaired by the uracil DNA glycosylase (UDG)–initiated baseexcision repair pathway (16). Although incorporation of uraciland 5-FU into DNA is well documented as a consequence ofexposure to TS inhibitors (15), the impact of the downstreamrepair pathway directed by UDG on cell survival is not consistent.It has been hypothesized that thymine-less futile cycles of uracilmisincorporation, excision byUDG, and further dUTP reinsertionresult in DNA strand breaks and cell death (17). If thymine-lesscell death was dependent on UDG-mediated removal of uracil

1Department of Pharmacology, Case Western Reserve University, Cleveland,Ohio. 2Case Comprehensive Cancer Center, Division of General MedicalSciences-Oncology, Case Western Reserve University, Cleveland, Ohio.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

CorrespondingAuthor: Stanton L. Gerson, Case Comprehensive Cancer Center,Case Western Reserve University, 11100 Euclid Avenue, Cleveland, OH 44106.Phone: 216-844-8562; Fax: 216-844-4975; E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-17-0215

�2017 American Association for Cancer Research.

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and 5-FU, onewould expect a correlation between the cytotoxicityof TS inhibitors and UDG expression. However, the majority ofstudies reported that neither overexpression, nor inhibition, ofUDG affected the sensitivity to TS inhibitors in human, mouse, orchickenDT40 cells (16, 18–23). In contrast, recently both our andthe Karnitz group observed that loss of UDG highly potentiatedthe cytotoxicity of 5-FdU in several cancer cell lines, indicatingthat uracil and 5-FU incorporation played a key role in cell killing(24, 25).

As themediators of cell killing due to persistent uracil and 5-FUlesions in DNA are not clear, we assessed the likely pathways andnoted that one of themajor differences in these disparate findingsis that cancer cells bearing p53 mutations were used in our andKarnitz's experimental system, whereas nontransformed or p53(wild-type) WT cancer cells were used in the majorities of others(16, 18–20, 22).MutationofTP53 is themost frequently observedgene alteration in cancers (26). Mutations in p53 have beenshown to influence cellular response to chemotherapeutic agentssuch as cisplatin, etoposide, and 5-FU (27, 28). Notably, sub-stantial evidence reveals that loss of p53, or p53 mutations, islinked to resistance to 5-FU due to inability to activate apoptosispathway. For example, a study using isogenic cell systems dem-onstrated that deletion of p53 from a p53 WT colon cancer cellline (HCT116) rendered cells remarkably resistant to apoptosisinduced by 5-FU (29). In addition, 5-FU resistance was alsodescribed in a variety of p53-mutated cancer cells, includingcolon, bladder, pancreatic, and gastric cancer (30–33). However,few studies have reported on the link of p53 status with theresponse to other TS inhibitors such as 5-FdU.

Given the divergent cell models with different p53 status usedin our and other studies, the following questions remain unan-swered: (1) does loss ormutation of p53 render cells resistant to 5-FdU, and (2) is the potentiated cytotoxicity of 5-FdU after UDGdepletion reliant upon p53 status? To gain insight into thesequestions, we tested the impact of UDG depletion on 5-FdUcytotoxicity in a number of cancer cell lines with differing p53status. We found that, in general, loss or mutation of p53 remark-ably reduced the sensitivity to 5-FdU, and depletion of UDGselectively resensitized p53-deficient or -mutated cancer cells to 5-FdU. In order to understand the underlying mechanism thatcontributes to the distinct response after UDG depletion, weutilized paired HCT116 cell lines with, or without, deletion ofthe TP53 gene and observed that loss of UDG selectively resensi-tized HCT116 cells with p53 deletion. This resensitization wasalso observed with pemetrexed, but to a lesser extent with 5-FU,whichmainly causes damage in RNA (21, 34–37). In the presenceof WT p53, 5-FdU treatment induced activation of the apoptosispathway in both UDG competent, or UDG-depleted cells atcomparable levels. However, in the absence ofWT p53, apoptosisactivation was compromised in UDG-expressing cells and dra-matically elevated in UDG-depleted cells. Collectively, thesefindings suggest that loss, or mutation, of p53 is associated with5-FdU resistance, and UDG depletion can significantly restoresensitivity, indicating that UDG may serve as a therapeutic targetto improve the clinical effectiveness of 5-FdU.

Materials and MethodsCell lines and drugs

HCT116 p53 knockout (KO) cells were a gift from Dr. Guang-bin Luo (Department of Genetics, Case Western Reserve Univer-

sity, Cleveland, OH). Other cancer cell lines were purchased fromthe American Type Culture Collection. Details of the cell linesused in this study are listed in Table 1. All cells weremaintained inDMEM (Corning 15-017-CV) supplemented with 10% dialyzedFBS, 2 mmol/L L-glutamine, 1% MEM NEAA, 100 U/mL penicil-lin, and 100 mg/mL streptomycin. Cells were incubated at 37�C ina humidified atmosphere of 95% air and 5% CO2. 5-FdU and 5-FU were purchased from Sigma-Aldrich, dissolved respectively inMilli-Q water and DMSO, and stored as a 10 mmol/L stock at�80�C. Pemetrexed was purchased from LC laboratories andprepared fresh for each experiment by dissolving inMilli-Qwater.

Lentiviral shRNA knockdownp53 or UDG knockdown (KD) was achieved via shRNA trans-

duction. Lentiviral vectors LV-THM-shp53 (which also expresses aGFP reporter) or LV-Bleo-shp53 to perform p53 KD in WTHCT116 cells were obtained from Dr. Mark Jackson's laboratoryat Case Western Reserve University, Cleveland, OH (38). Lenti-viral vector targeting GFP (sh-GFP) was used as control. UDGshRNA vectors (shUDG: NM_003362.2-656s21c1, shUDG-2:NM_003362.2-758s21c1, and shUDG-3: NM_003362.2-893s21c1) were purchased from Sigma, and a scramble targetingshRNA vector (Sigma) was used as paired control. The lentiviralproduction and infection were performed as previously described(24). Cells stably infected with LV-THM-p53 were isolated by cellsorting on the basis of their GFP expression. Cells stably infectedwith LV-Bleo-p53 were selected with zeocin (Sigma). Selection ofpositive UDG KD cells was assessed with puromycin (Sigma).

Clonogenic survival assayAs described previously (24), cancer cells (200–300 cells/well)

were seeded in 6-well culture dishes and allowed to adhereovernight. For 5-FdU, cells were treated for 24 hours, then gentlywashedwith PBS once, and incubatedwith freshmedia for at least10 days to allow individual colonies to form. For 5-FU orpemetrexed, cells were treated continuously for at least 10 daysto form colonies. After 10 to 18 days, the plates were stained withmethylene blue. Colonies containing�50 cells were counted. Thepercentage of survival was determined relative to untreated con-trol averaged over three independent experiments.

Western blots and qPCRWestern blots were performed as previously described (39).

Twentymicrogram of protein was loaded on SDS-polyacrylamidegel. The following antibodies were used to detect proteins onthe membrane: a-Tubulin (Calbiochem); GAPDH (Santa CruzBiotechnology); UDG (FL-313; Santa Cruz Biotechnology);cleaved PARP (Asp214)(19F4; Cell Signaling Technology);cleaved caspase 3 (Cell Signaling Technology); p53 (FL-393; Santa

Table 1. Cell lines and strains used in this work

Cell line Origin p53 status

A375 Melanoma wtLoVo Colon cancer wtRKO Colon cancer wtA2780 Ovarian cancer wtH460 Large cell lung cancer wtH1299 Non–small cell lung cancer nullOVCAR8 Ovarian cancer del 126-132DLD1 Colon cancer S241FHEC1A Endometrial cancer R248Q

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Cruz Biotechnology); and p21 (Santa Cruz Biotechnology). ForqRT-PCR, total RNA from cells was extracted by using the RNeasyPlus Mini Kit (Qiagen). cDNA synthesis was performed by usingthe SuperScript III First Strand Kit (Life Technologies). qPCR wasachieved with validated TaqMAN MGB FAMTM dye–labeledprobes (Applied Biosystems) for nuclear UDG on an ABI 7500Fast Real-time PCR System (Applied Biosystems). b-Actin wasused as an endogenous control, and relative gene expression wascalculated as 2�DDCt.

Flow cytometric assay of apoptosisCells were seeded in 6-well tissue culture plates (1.5� 105 cells/

well) and allowed to attachovernight. Cellswere then treatedwith25 nmol/L 5-FdU for 24 hours, washed twice with PBS, andreplenished with drug-free medium at 48, 72, and 96 hours. Afterrecovery, the cells floating in the medium were collected. Theadherent cells were trypsinized, pelleted, washed in ice-cold PBS,and resuspended in 1X binding buffer according to the manu-facturer's instructions (FITC Annexin V Apoptosis Detection Kit;BD Pharmingen). Cells were then stained with FITC–Annexin Vandpropidium iodide (PI) for 15minutes at room temperature inthe dark. Annexin V–FITC detects translocation of phosphatidy-linositol from the inner to the out of cell membrane during earlyapoptosis, and PI can enter the cells in late apoptosis or necrosis.Untreated cells were used as control for the double staining. Thecells were analyzed immediately after staining using an AttuneNXT instrument and FlowJo software. For each measurement, atleast 20,000 cells were counted.

Statistical analysisStatistical significance between two treatment groups was ana-

lyzed using unpaired two-tailed Student t test. Significance wasassigned for a P value < 0.05. Standard software GraphPad Prismand Excel 2013 (Microsoft Corp.) were used for all statisticalanalysis.

Resultsp53 mutation or deficiency affords 5-FdU resistance amongdifferent types of cancer cells

Given that p53 mutations or deficiencies are frequentlyobserved in cancers, studies have demonstrated that mutationsof p53 reduce 5-FU cytotoxicity (29–33). To understandwhether these mutations also alter the response to 5-FdU, apanel of human cancer cell lines from colon, lung, ovarian,skin, and endometrium with intrinsically differing p53 statuswas utilized in this study. The p53 status of each cell line islisted in Table 1. To determine p53 protein functionality inp53 WT and p53-mutant (Mut) or -deficient cancer cell lines,we assessed p53 levels and expression of p21, a widely accept-ed initiator of p53-activated signaling (40), 24 hours afteradministration of 8 Gy gamma irradiation. All the p53 WTcancer cell lines used in this work induced p21 expression afterirradiation, indicating functional p53 in these cell lines (Sup-plementary Fig. S1). In order to establish the relationshipbetween p53 status and 5-FdU sensitivity, we evaluated thecytotoxicity of 5-FdU in these cell lines by clonogenic survivalassay. As shown in Fig. 1A, the cell lines tested displayed aspectrum of 5-FdU sensitivities with IC50 values ranging from1.32 � 0.33 to 269.55 � 0.73 nmol/L for A2780 and H1299lines, respectively. Importantly, we observed that, in general,cell lines with p53 mutation or deficiency (Fig. 1A, solid lines)were significantly more resistant to 5-FdU than p53 WTcells (Fig. 1A, dashed lines), with the exception of A375 whichhas WT p53 but an IC50 of 110.81 � 1.80 nmol/L. In addition,except for A375, the IC50 values for the p53 WT cancer linesclustered together at a lower dose range (<10 nmol/L), whereasp53-mutant or -deficient lines clustered at a higher range(>100 nmol/L; Fig. 1B). These observations are consistent withthe hypothesis that p53 mutation or deficiency is associatedwith resistance to 5-FdU.

Figure 1.

5-FdU resistance in different types of cancer cellswithp53 mutation or deficiency. A, Clonogenic survivalassay in cancer cell lines shown in Table 1 in responseto 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 independentexperiments that were done in duplicate each time.B, IC50 values of 5-FdU for cancer cell lines listedin Table 1 with WT p53 or deficient (or mutant) p53status, respectively.

UDG Depletion Resensitizes p53-Mutant Cells to 5-FdU

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UDG depletion sensitizes cancer cells with p53 mutation ordeficiency to 5-FdU exposure

Previously, the discordant findings on sensitization to 5-FdUfollowing UDG depletion were reported using cell models withdiffering p53 status (16, 18–25). To understand whether thedivergent responses could be attributed to p53 status, we exploredwhether UDG depletion could sensitize p53-mutant or -deficientcancer cells to 5-FdUdifferentially. For these experiments, we usedshRNA to deplete UDG in various cancer cell lines with differingp53 status, as listed in Table 1. UDG stable KD was evaluated byWestern blot (Fig. 2A and B, insert). Based on a clonogenicsurvival assay, we observed that UDG depletion selectively sen-sitized cells with p53 mutation or deficiency to 5-FdU exposure(Fig. 2A). However, in p53 WT cell lines, UDG depletion did notalter the cytotoxicity of 5-FdU (Fig. 2B). Collectively, these resultsdemonstrate that UDG depletion resensitizes p53-mutant or-deficient cancer cells, providing a novel therapeutic target forpatients with p53-mutant tumors.

5-FdU resistance in p53 KO or KD cells is reversed by UDGdepletion

Because many studies have identified gain of various functionsfor specific p53-mutated proteins (41, 42), we next askedwhether

loss ofWTp53 protein expression can alter the response to 5-FdU.To address this, we utilized paired HCT116 colon cancer cell lineswith or without genetic TP53 deletion and tested their sensitivityto 5-FdU, and the loss of p53 expressionwas evaluated byWesternblot (Fig. 3A).Using a clonogenic survival assay, we demonstratedthat p53 KO cells were more resistant to 5-FdU than p53WT cells(Fig. 3B). KD of p53 by shRNA recapitulates the resistanceobserved in p53 KO cells (Fig. 3B), indicating that p53 status isa key mediator of the response of HCT116 cells to 5-FdU.

To understand whether loss of p53 protein will affect theresponse to 5-FdU after UDG depletion, we knocked down UDGby shRNA in both HCT116 p53 WT and p53 KO cells. UDG KDlevels were shown to be greater than 90% as evaluated byWesternblot and qPCR (Fig. 3C and D). In agreement with our data usingp53-mutant cells, UDGdepletion greatly enhanced cytotoxicity of5-FdU in p53 KO cells but did not significantly affect p53WT cells(Fig. 3E and F), indicating that p53 is involved in regulating theresponse to 5-FdU following UDG depletion. To exclude the off-target effect of a single shRNA, we also utilized two other shRNAsthat target UDG in HCT116 p53 WT and p53 KO cells andobserved similar effect (Supplementary Fig. S2). In addition,depletion of UDG also potentiated 5-FdU cytotoxicity in twoHCT116 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 indicatemean values � S.E.M. The results represent three independent experiments that were done in duplicate each time (� , P < 0.01).

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(Fig. 4A–E). Collectively, these results confirm that loss of p53protein renders cells resistant to 5-FdU, and UDG depletionselectively resensitizes p53 KO and KD cells to 5-FdU.

UDG depletion selectively sensitizes p53 KO cancer cells topemetrexed and 5-FU

Although all TS inhibitors have the ability to block TS, disrupt-ing DNA replication and leading to uracil incorporation intoDNA, differences among distinct TS inhibitors have been reportedin terms of their other metabolism-mediated cytotoxic pathways(2). For example, pemetrexed polyglutamate derivatives alsodemonstrate inhibitory activity for other folate-dependentenzymes such as glycinamide ribonucleotide, but to a lesser extent(10–13). Moreover, unlike 5-FdU, which mainly exerts its cyto-toxicity due to effects at the DNA level (24), studies have revealedthat the cytotoxicity of 5-FU is primarily RNA-mediated, as 5-FU ismetabolized to fluorouridine triphosphate which affects multipleRNAprocesses following its incorporation intoRNAs (21, 34–37).In order to address the question of whether p53 status is respon-sible for the differences in sensitivity to other TS inhibitors,including pemetrexed and 5-FU, in UDG-depleted cells, we eval-uated cell viability following drug exposure inUDG-depleted p53WT and p53 KO cancer cells. Similar to our observations with5-FdU, no significant survival differences were found between

UDG-expressing and UDG-depleted cells in the presence of p53after pemetrexed or 5-FU treatment (Fig. 5A and B). However, inthe absence of p53, UDG depletion sensitized cells to pemetrexed(Fig. 5C), whereas loss of UDGonlymoderately sensitized cells to5-FU at high concentrations (Fig. 5D), reaffirming that the pri-mary cytotoxic effect of 5-FU depends on RNA incoporation.Together, these results indicate that UDGdepletion also sensitizescells without p53 to other TS inhibitors, mainly through gener-ation of DNA damage.

5-FdU activates cell death in p53 KO cancer cells with depletedUDG

To understand whether 5-FdU resistance observed in p53 KOcells is due to a failure to activate cell death pathways, wemonitored cell death progression by Annexin V and PI staining.Cells were exposed to 5-FdU for 24 hours, washed with PBS,and then allowed to recover in drug-free medium for a total of48, 72, and 96 hours (Fig. 6A). In cells with WT p53, 5-FdUcaused significant cell death (Annexin V and PI positive) at48 hours which was retained at 72 and 96 hours in bothUDG-expressing and UDG-depleted cells (Fig. 6B and C).However, in the absence of p53, cell death caused by 5-FdUwas significantly lower in UDG-expressing cells, whereas inUDG-depleted cells, cell death was detected at 24 hours and

Figure 3.

5-FdU resistance due to loss of p53 isreversed by UDG depletion. A, p53expression levels were analyzed byWestern 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 forincreasing doses of 5-FdU in HCT116p53WT, p53KO, shGFP-infected, andshp53-infected cells. HCT116 p53WT andp53KO cells stably infected withnontargeted scramble control shRNA(shSCR) or UDG-directed shRNA(shUDG)were analyzed byWestern blot(C) and qPCR (D) to examine UDGlevels. Clonogenic survival assay forincreasing doses of 5-FdU in HCT116 (E)p53WT cells, with shSCR, orwith shUDG;and (F) p53KO cells, with shSCR, or withshUDG. Viable colonies (>50 cells)stained with methylene blue after 10days of culture were counted. Theresults represent three independentexperiments that were done in duplicateeach time (� , P < 0.01).






significantly elevated at 48 to 96 hours (Fig. 6B and C). Thesedata suggest that 5-FdU–induced cell death is dependent uponp53, supporting the observation that drug resistance can beobserved as a result of abrogation of the p53-mediated celldeath pathway. Importantly, UDG depletion significantlypotentiates death of cells lacking WT p53 activity through ap53-independent pathway.

To further elucidate whether the cell death caused by 5-FdU isdue to apoptosis, we examined expression of proteins involved inthe activation of the apoptotic pathway. In WT p53 cells, weobserved that p53 expression was induced at 24 hours, and theinduction remained for 96 hours in both shSCR and shUDG cellsfollowing 5-FdU exposure (Fig. 6D). The expression of cleavedPARP, a hallmark of apoptotic cell death, was induced at 48 hoursand persisted through 72 and 96 hours in p53WT cells regardlessof whether UDGwas present or not (Fig. 6D). In addition, cleavedcaspase 3 was also detected in both UDG-expressing or -depletedp53WT cells (Fig. 6D). In the absence of p53, induction of cleavedPARP or caspase 3 was not readily detected in cells expressingUDG after 5-FdU exposure (Fig. 6E), whereas both were robustlyinduced from 48 to 96 hours in cells depleted of UDG (Fig. 6E).Taken together, our results suggest that 5-FdU–induced apoptosisis mediated through p53, and the lack of apoptosis activation due

to loss of p53 is responsible for the enhanced cell survivalobserved in p53 KO cells. However, in p53 KO cell with coinci-dent UDG depletion, 5-FdU selectively activates a p53-indepen-dent apoptotic pathway through a mechanism which needsfurther investigation.

DiscussionIn this study, we utilized multiple cancer cells bearing differing

p53 statuswith orwithoutUDGexpression.Weobserved that lossof UDG selectively resensitized cancer cells with p53 mutation ordeficiency to 5-FdU, but did not alter the response of p53WT cells.These results demonstrate that UDG, through its function ofremoving uracil or 5-FU, plays a major role in the effect of 5-FdUon the response of cells lacking WT p53 activity. Our findingsresolve the unexplained discrepancy observed in a number ofprior studies regarding the role of UDG in sensitivity to TSinhibitors. Prior studies revealed that either loss of UDGenhanced the cytotoxicity of 5-FdU or pemetrexed in cancer cells(24, 25), or overexpression or inhibition of UDG had no effecton the sensitivity of human or mouse cells to TS inhibition(16, 18–20, 22). The difference, we propose, is dependent onp53 status.

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 wereanalyzed by the Western blot to examine p53 KD levels (� , nonspecific bands). B, HCT116 cells expressing shGFP or shp53 (shp53-THM or shp53-Bleo) werefurther 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 thatwere done in duplicate each time (� , P < 0.01).

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p53 plays a key role in determining the sensitivity of cells to5-FU. A number of studies reported that enhanced 5-FU resistancehas been observed in cells bearing TP53 deletion or mutations(29–33). However, unlike other TS inhibitors, 5-FU exposurecaused only slightly potentiated cytotoxicity at higher doses inUDG-depleted, p53 KO cell lines. Recently, several studies haveobserved that the cytotoxicity of 5-FU is more dependent on itsincorporation into RNA than its inhibition of TS, diminishing itseffect on DNA (21, 34–37). In addition, activation of p53 fol-lowing 5-FU exposure has been identified as working throughRNA mechanisms (43, 44). Because UDG recognizes only DNAlesions, it is not surprising that depletion of UDG does notsignificantly alter cellular responses to agents that primarily affectRNA function. Together, this suggests that the increased cytotox-icity of 5-FdU and pemetrexed observed in UDG-depleted cells isprimarily due to uracil and 5-FU incorporation into DNA.

The present results illustrate that cells with p53 mutation ordeficiency are significantly resistant to 5-FdU in comparison withp53 WT cells. It is clear that many different mutant p53s alsoacquire oncogenic functions that are distinct from the activities ofWT p53 (41, 42). Some p53mutants provide enhanced resistanceto apoptosis induced by a variety of treatments, including certainchemotherapeutic drugs (27, 45). In particular, one study iden-tified that p53mutants activate expressionof dUTPase (46),whichhas been related to the resistance to TS inhibitors (47–49). Ourresults on a select groupof p53mutants aswell as p53KOcell linesrevealed resistance to 5-FdU treatment. However, the p53 KO cellline is much less resistant to 5-FdU than other p53-mutant celllines, suggesting the potential for enhanced resistance due togained functions for certain p53 mutants.

Our results demonstrated that inhibition of UDG selectivelysensitized p53-mutant and -deficient cancer cells to 5-FdU, butdid not alter the response in p53 WT cells. Importantly, we haveobserved that apoptosis following 5-FdU is efficiently induced inthe presence of p53 but highly compromised in cells lacking p53,indicating that the activation of the 5-FdU–induced cell deathpathway is dependent on p53. Further studies with different p53WT cell lines also revealed cells highly sensitive to 5-FdUwith IC50

values lower than 10 nmol/L. One exception we observed was inthe A375 melanoma cells line, which has a WT TP53 gene. A375was relatively insensitive to 5-FdU and had an IC50 of 110.81 �1.80 nmol/L. Clearly, more knowledge is needed regarding thep53-mediated cell death pathway and how 5-FdU, with or with-out UDG, causes damage and triggers cell death. In response to5-FdU, cells lacking WT p53, combined with UDG depletion,activate cell death in a p53-independent manner, which reverseschemoresistance and selectively resensitizes these cancer cells to5-FdU.

The current findings of this article focus on the role of p53 inapoptosis and show that in the presence of p53, both UDG WT–and UDG-depleted cells activate apoptosis at similar levels; how-ever, in the absence of p53, apoptosis induction is compromisedin the UDGWT cells but significantly increased in UDG-depletedcells. These data explain that p53WT cells have 5-FdU IC50 valuesless than 10 nmol/L, whereas p53-mutant or -deficient cells haveIC50 values higher than 100 nmol/L. The result is consistent with aprevious publication from Janet Houghton's group (30) that cellswithWTp53displayed acute apoptosis,whereas cellswithmutantp53 showed delayed or compromised apoptosis following fluor-opyrimidine treatment. Based on these results, we propose the

Figure 5.

UDG depletion selectively sensitizes p53KO cells to pemetrexed and 5-FU.Clonogenic survival assay in HCT116 p53WT cells (shSCR and shUDG) treated withincreasing doses of (A) pemetrexed and(B) 5-FU. Clonogenic survival assay inHCT116 p53 KO cells (shSCR and shUDG)treated with increasing doses of (C)pemetrexed and (D) 5-FU. Viable colonies(>50 cells) stained with methylene blueafter 10 days of culture were counted. Theresults represent three independentexperiments that were done in duplicate(� , P < 0.01).






mechanism of cell death that: first, DNA damage due to loss ofUDG enhanced the apoptosis in p53-mutant or -deficient cancercells, but did not change the apoptosis in p53 WT cells which has

already been activated in the presence of WT p53 following5-FdU–induced stress. Second, loss of UDG increases levels ofpersistent uracil and 5-FU incorporation at the replication forks

Figure 6.

UDG depletion induces cell deathcaused by 5-FdU in p53 KO cancercells. A, Schematic diagram of thetreatment for HCT116 p53WT (shSCRand shUDG) and p53KO (shSCR andshUDG) cells with 25 nmol/L 5-FdUfor 24 hours, washed, and replenishedwith drug-free medium at indicatedtime points. B, Untreated (Unt) ortreated cells were subjected to FITC–Annexin V and PI staining andanalyzed by flow cytometry.Representative flow plots of threeindependent experiments are shown.C, Cell death is expressed as thepercentage of Annexin V-positivecells. Values indicate mean values �SD. All experiments were performedindependently for 3 times (� ,P <0.01).Protein expression involved inregulation of apoptotic cell death inresponse to 5-FdU was detected inHCT116 (D) p53 WT (shSCR andshUDG) and (E) p53 KO (shSCR andshUDG) cells (� , nonspecific bands).

Yan et al.





that without p53 results in early S phase arrest and disintegrationof the replication fork progression, as we have shown previously(24). Further support for this mechanism comes from an articlepublished recently by Swati Palit Deb's lab (50), which has shownthat lack of WT p53 increases DNA origin firing compared withp53WT cells. In our system,moreDNA origin firings would resultin both more 5-FU and uracil incorporation and further disrup-tion of these replication forks that improve the killing effect inp53-mutant or -deficient cancer cells. Taken together, these resultsprovide an explanation for the discordant findings in previouspublished data regarding the role of UDG in mediating thecytotoxicity of TS inhibitors and suggest that UDG is an attractivetherapeutic target in cancer cells with p53mutation or deficiency,to enhance their response to TS inhibitors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: Y. Yan, Y. Qing, S.L. GersonDevelopment of methodology: S.L. GersonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Y. YanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Y. Yan, Y. Qing, J.J. Pink, S.L. GersonWriting, review, and/or revision of the manuscript: Y. Yan, Y. Qing, J.J. Pink,S.L. GersonStudy supervision: S.L. Gerson

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 21, 2017; revised August 2, 2017; accepted October 26, 2017;published OnlineFirst November 8, 2017.

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Yan et al.




Published OnlineFirst November 8, 2017.Mol Cancer Res Yan Yan, Yulan Qing, John J. Pink, et al. p53-Mutant and -Deficient Cells to 5-FdULoss of Uracil DNA Glycosylase Selectively Resensitizes

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