Inhibition of dUTPase Induces Synthetic Lethality with...

Therapeutic Discovery

Inhibition of dUTPase Induces Synthetic Lethality withThymidylate Synthase–Targeted Therapies inNon–Small Cell Lung Cancer

Peter M. Wilson1, Melissa J. LaBonte3, Heinz-Josef Lenz2, Philip C. Mack4, and Robert D. Ladner1

AbstractChemotherapies that target thymidylate synthase (TS) continue to see considerable clinical expansion in

non–small cell lung cancer (NSCLC).Onedrawback to TS-targeted therapies is drug resistance and subsequent

treatment failure. Novel therapeutic and biomarker-driven strategies are urgently needed. The enzyme

deoxyuridine triphosphate nucleotidohydrolase (dUTPase) is reported to protect tumor cells from aberrant

misincorporation of uracil during TS inhibition. The goal of this study was to investigate the expression and

significance of dUTPase inmediating response to TS-targeted agents inNSCLC. The expression of dUTPase in

NSCLC cell lines and clinical specimens was measured by quantitative real-time reverse transcriptase PCR

and immunohistochemistry. Using a validated RNA interference approach, dUTPase was effectively silenced

in a panel of NSCLC cell lines and response to the fluoropyrimidine fluorodeoxyuridine (FUdR) and the

antifolate pemetrexed was analyzed using growth inhibition and clonogenic assays. Apoptosis was analyzed

by flow cytometry. Significant variation in the quantity and cellular expression of dUTPase was observed,

including clear evidence of overexpression inNSCLC cell linemodels and tumor specimens at themRNA and

protein level. RNA interference–mediated silencing of dUTPase significantly sensitizedNSCLCcells to growth

inhibition inducedbyFUdRandpemetrexed. This sensitizationwas accompanied by a significant expansion of

intracellular dUTP pools and significant decreases in NSCLC cell viability evaluated by clonogenicity and

apoptotic analyses. Together, these results strongly suggest that uracil misincorporation is a potent determi-

nant of cytotoxicity toTS inhibition inNSCLCand that inhibition ofdUTPase is amechanism-based therapeutic

approach to significantly enhance the efficacy of TS-targeted chemotherapeutic agents.Mol Cancer Ther; 11(3);

616–28. �2011 AACR.

IntroductionIn2010,more than222,500patientswerediagnosedwith

lung cancer and more than 157,000 lung cancer patientssuccumbed to their disease, making this the deadliestmalignancy in the United States (1). Non–small cell lungcancer (NSCLC) accounts for 85% of cases, and mostpatients are diagnosed at an advanced stage and requirechemotherapy to control their disease (2). However, noveltherapeutics in NSCLC has resulted in only minor

improvements in outcomes for the majority of patientsand novel therapeutic strategies are urgently needed.

For over 50 years, chemotherapeutic agents that targetthymidylate metabolism have seen widespread clinicalimplementation in a wide range of neoplasias. The pro-duction of dTMP from dUMP is catalyzed by the enzymethymidylate synthase (TS), encoded by the TYMS gene,and represents the sole source of de novo cellular thymi-dylate for DNA synthesis (3). Inhibition of TS depletesthymidylate pools, inducing a thymineless state andgrowth arrest (4–6). Two classes of therapeutic agentsthat target thymidylate metabolism via TS inhibitioninclude the fluoropyrimidines and antifolates. The fluor-opyrimidines 5-fluorouracil (5-FU) and fluorodeoxyuri-dine (FUdR) were the first TS inhibitors to show clinicalefficacy (7, 8). The antifolate class includes agents such asraltitrexed (9) and pemetrexed (10) that also functionprimarily through TS inhibition (11).

Recently a large phase III randomized clinical trialshowed superior overall survival for cisplatin/peme-trexed over cisplatin/gemcitabine in NSCLC patientswith adenocarcinoma and large cell histologic subtypes(12). Pemetrexed has since become established asan efficacious therapeutic in multiple lines of NSCLC

Authors' Affiliations: 1Department of Pathology, 2Division of MedicalOncology, Norris Comprehensive Cancer Center, University of SouthernCalifornia; 3Department ofBiology andChemistry, AzusaPacificUniversity,Los Angeles; and 4Division of Hematology/Oncology, University of Cali-fornia Davis Cancer Center, Sacramento, California

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

Corresponding Author: Robert D. Ladner, Room 5322, Norris Compre-hensive Cancer Center, 1441 Eastlake Avenue, University of SouthernCalifornia, Los Angeles, CA 90089. Phone: 323-865-3116; Fax: 323-442-3049; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-0781

�2011 American Association for Cancer Research.

MolecularCancer

Therapeutics

Mol Cancer Ther; 11(3) March 2012616

on July 2, 2019. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst December 15, 2011; DOI: 10.1158/1535-7163.MCT-11-0781

http://mct.aacrjournals.org/

treatment (13–15) and is undergoing significant clinicalexpansion. Although fluoropyrimidine-based regimensare not widely implemented in NSCLC in the UnitedStates, this class is seeing expanding application in Japanand Europe. Tegafur–uracil (UFT) and S-1 are oral 5-FUderivatives containing inhibitors of the 5-FU catabolicenzyme dihydropyrimidine dehydrogenase that is oftenoverexpressed in NSCLC.One drawback to TS-directed therapies is the presence

of intrinsic or acquired resistance. Because therapeuticresponse is difficult to predict using clinical and patho-logic factors, considerable effort has been directed towardunderstanding mechanisms of drug action and identify-ing biomarkers to predict response to therapy.An increas-ing number of studies have shown that elevated TSexpression is associated with resistance to fluoropyrimi-dine-based therapy in colorectal cancer (16–20) and S-1(21) and UFT (22) in NSCLC. Accumulating evidencealso indicates that TS overexpression is a determinant ofsensitivity to pemetrexed (23–29). However, benefits fromTS-targeted chemotherapy in NSCLC seems to have pla-teaued, and their continued clinical success may dependon our ability to correctly administer these agents follow-ing biomarker-driven patient selection (30).A less appreciated component of cell death induced by

TS inhibition is aberrant uracil incorporation into DNAresulting from accumulation of the cytotoxic nucleotideintermediate dUTP.When dUTP pools accumulate, DNApolymerases use dUTP in place of TTP during DNAsynthesis, activating iterative rounds of uracil repair andreincorporationdue to continuedTTPdepletion, resultingin extensive DNA damage and cell death. Although thedepletion of dTMP and the accumulation of dUMP iscommon in cancer cell lines undergoing TS inhibition(31, 32), the ability to accumulate dUTP varies dramati-cally. The enzyme deoxyuridine triphosphate nucleotido-hydrolase (dUTPase), encoded by the DUT gene, is thesole regulator of cellular dUTP pools, hydrolyzing dUTPto dUMP, thereby eliminating dUTP from the DNA bio-synthetic pathway. We previously reported that over-expression of dUTPase could protect colorectal and breastcancer cells from cytotoxicity induced from dUTP poolexpansion and subsequent uracil misincorporation intoDNA induced during TS inhibition (33, 34). In addition,dUTPase overexpression in tumor specimens is associat-ed with resistance to 5-FU in colorectal cancer (34, 35).Numerous solid tumors overexpress dUTPase, abrogat-ing the potential for dUTP pool expansion and cytotox-icity induced by the uracil–DNA misincorporation path-way. However, if dUTPase activity could be impaired inthe presence of TS inhibition, the combined cytotoxicityfrom concomitant TTP pool depletion and uracil misin-corporation would result in enhanced DNA damage andcell death (Supplementary Fig. S1).As TS-targeted therapies are seeing considerable clin-

ical expansion in NSCLC, the goal of this study was toevaluate the expression of dUTPase in NSCLC tumor celllines and clinical specimens and to use in vitro models to

evaluate the role of dUTPase in determining sensitivity to2 class-specific TS-targeted therapies.

Materials and MethodsAdditional details can be found in the Supplementary

Methods.

Compounds and reagentsFUdR and propidium iodide were purchased from

Sigma. Pemetrexedwas purchased from LC Laboratories.CellTiter96 Aqueous MTS was purchased from Promega.Halt protease/phosphatase inhibitorwaspurchased fromThermo Scientific.

Cell cultureThe NSCLC cell lines H1299 (metastatic lung adeno-

carcinoma), H358 (bronchioalveolar carcinoma), andH460 (large cell carcinoma), A549, H1563, H2228, H522,and H23 (adenocarcinoma) were purchased directlyfromAmericanTypeCultureCollection andwere authen-ticated before receipt. Cells were maintained in folate-depleted RPMI supplemented with 25 nmol/L 5-formyl-tetrahydrofolate (Invitrogen), 10% FBS (Gemini) withpenicillin/streptomycin, and L-glutamine (Invitrogen) ina humidified incubator (Forma) at 37�C with 5% CO2.

Growth inhibition assayCells were seeded in 96-well plates at a density of 1,500

cells per well with continuous exposure to increasingconcentrations of drug for 72 hours and quantified by theCell Titer96AQueousMTS (Promega) assay as previouslydescribed (36).

Colony formation assayThe colony formation assay was carried out as previ-

ously described (37). Briefly, cellswere seeded at a densityof approximately 75 cells per well in a 24-well plate andfollowing a 24-hour drug exposure, media was removedand replacedwith drug-freemedium and cells allowed togrow for 7 to 14 days. Colonies were fixed, stained withcrystal violet, scanned, and colonies with more than 100cells counted. Drug-treated samples were compareddirectly with vehicle-treated controls.

Flow cytometric/Sub-G1 analysisApoptosis was determined by flow cytometric analysis

of DNA content using propidium iodide as previouslydescribed (38). Cells were harvested at the indicated time,fixed, stained, and analyzed using an EPICS ELITE flowcytometer with cell populations quantified using Expo32software (Beckman Coulter). Cells with DNA content lessthan 1 were considered apoptotic.

ImmunoblottingImmunoblotting was done as previously described (38).

Cell lysates were prepared using radioimmunoprecipita-tion assay bufferwith Protease and Phosphatase Inhibitors

dUTPase Mediates TS Inhibitor Sensitivity in NSCLC

www.aacrjournals.org Mol Cancer Ther; 11(3) March 2012 617




(Thermo). Western blots were probed overnight at 4�Cwith affinity purified anti-dUTPase and anti-TS generatedin our laboratory (1:500) and 2 hours with goat–antirabbithorseradish peroxidase. Blots were reprobed for b-actin orb-tubulin (Sigma) to control for loading and quantifiedusing ImageJ64 (NIH).

RNA interferenceRNA interference was done as previously described

using the siDUT/230 (si-DUT) small interfering RNA(siRNA) oligos (sense; 5-CGGACAUUCAGAUAGCGCUdTT; and antisense; 5-AGCGCUUAUCUGAAUGUCCGd TT (33, 37). Cells were treated with 25 nmol/Lsi-DUT complexed with Lipofectamine RNAiMAX (Invi-trogen). A nontargeted scrambled control siRNA (si-SCR)was used at equimolar concentration. Transfection com-plexes were removed after 12 hours and dUTPase silenc-ing was confirmed by quantitative (q) real-time reversetranscriptase PCR (RT-PCR) and Western blotting. Nosignificant toxicity or reduction in plating efficiency wasobserved with either the si-SCR or si-DUT transfectionsin any of the cell lines.

Quantitative real-time RT-PCRRNA was isolated using TRizol according to manufac-

turer’s instructions (Invitrogen). For clinical specimens,tumor specimens were fresh frozen at the time of surgeryand stored at �80�C until RNA extraction. cDNA wasreverse transcribed and analyzed using PerfeCTa SYBRGreen (Quanta Biosciences Inc.) on an Applied Biosys-tems 7500 PCR System (Applied Biosystems). For in vitroanalyses, DUT and TYMS were normalized to GAPDHor ACTB (b-actin) and quantified using comparative CT

methodology (39). For clinical specimens, parallel deter-minationswere done forTYMS andDUT.ACTBwas usedfor normalization and the denominator for the ratio usedto determine relative gene expression (40).

Nucleotide pool determinationsCellsweretreatedwithFUdR,orpemetrexedfor24hours,

harvested, and 2 � 105 cells were analyzed for nucleotidepool content using a 96-well fluorescence-based assaydeveloped in our laboratory and as described previously(41). The assay measures the Taq polymerase–mediatedincorporation of a limiting dNTP (TTP) into newly synthe-sized DNA and was modified to distinguish between TTPand dUTP by parallel reactions with and without a pre-incubation step including 10 ng of recombinant dUTPase(41). Fluorescence generated in the presence of dUTPaserepresented the TTP pool, whereas undigested extractsrepresented the combineddUTP and TTPpools. dUTPwasquantified by subtracting the results of extracts treatedwithdUTPase from untreated extracts and presented as pmolTTP/106 cells as determined by standard curve.

Patients and specimensDeidentified clinical specimens were obtained from

35 histopathologically confirmed stage II NSCLC

patients at the University of California Davis CancerCenter. All 35 patients had formalin-fixed, paraffin-embedded (FFPE) tissue available for immunohis-tochemistry (IHC) and 32 had corresponding RNAextracted from microdissected primary tumor biopsymaterial. Of the 32 patients with both FFPE and RNAavailable, 21% had squamous cell carcinoma (SCC),50% had adenocarcinoma, 6% had large cell carcinoma,and 6% had bronchi alveolar carcinoma. The remaining17% of patients had neuroendocrine large-cell type,carcinoid, and large cell carcinoma undifferentiated. Inthese 32 patients, the median age was 68 and 81% had ahistory of smoking. Data and tissue collection were inaccordance with the regulations of the local ethics com-mittee and Institutional Review Board. In addition,6 normal lung tissue FFPE specimens were available.

ImmunohistochemistryIHC was conducted at the University of Southern Cali-

fornia Immunohistochemistry Clinical Laboratory usingtheDUT415 (42) dUTPasemonoclonal antibody (2mg/mL;ref. 43) and the TS polyclonal antibody (1 mg/mL) usingmethods previously described (34). Slides were reviewedby 2 independent observers at the University of SouthernCalifornia Norris Comprehensive Cancer Center (RDLand PMW). The scoring field was �100, and the whole ofeach tissue was scored. Staining intensity was graded as0 (no staining), 1 (weak), 2 (moderate), and 3 (strong).Staining extent was graded by mean percentage of cellsstaining positive. The mean tumor expression index wascalculated by multiplying the percent positive and inten-sity scores for each slide (semiquantitative system; range:0–300).

Statistical analysesQuantitative data are presented as mean � SEM and

analyzed by Student 2-tailed t test in which a P valueof 0.05 or less was considered statistically significant and�, P � 0.05; ��, P � 0.01; ��, P � 0.001 (GraphPad).

ResultsdUTPase and TS show wide-ranging expression inNSCLC cell lines

The expression of dUTPase protein and mRNA wasanalyzed in 8 NSCLC cell lines during log-phase growth.The NSCLC cells show an 38-fold range in nucleardUTPase expression and a 18-fold range in mitochond-rial dUTPase protein expression. Subsequent measure-ment of DUT mRNA by qRT-PCR revealed a similarpattern of mRNA expression to the protein expressionbut with a reduced range of 6.5-fold. TS expressionshowed a similar range in protein expression as observedwith dUTPase of approximately 16-fold. TS protein alsocorrelated with TYMS mRNA although the range ofmRNA expression observed was less than the proteinat 10-fold. TYMS and DUT mRNA expression exhibiteda correlation in these 8 NSCLC cell lines (R2 ¼ 0.6;P ¼ 0.027; Fig. 1).

Wilson et al.

Mol Cancer Ther; 11(3) March 2012 Molecular Cancer Therapeutics618




Aberrant dUTPase expression in NSCLC clinicalspecimensPrimary tumor specimens were obtained from 35

histopathologically confirmed NSCLC patients (stageII) and included the primary histologic subtypes ofadenocarcinoma and SCC. To investigate the expressionand intracellular localization of dUTPase in NSCLC,IHC on FFPE tumor tissue and qRT-PCR on tumor-extracted RNA was done. Thirty-five specimens wereavailable for IHC with 32 of these having matched RNAavailable for qRT-PCR. Expression of dUTPase proteinwas detected in 89% of the tumor specimens and 0% ofthe normal lung specimens. dUTPase expression variedsignificantly in the quantity of expression and intracel-lular localization. Of the specimens that stained positivefor dUTPase, 51.4% exhibited both nuclear and cyto-plasmic expression, 8.6% were nuclear-only and 28.6%were cytoplasmic staining only. Figure 2A shows atypical dUTPase staining pattern for replicating cells

in human palatine tonsil. In contrast, Fig. 2B shows alack of dUTPase staining in a representative normallung specimen. In Fig. 2C, dUTPase is highly abundantin both the NSCLC cell cytoplasm and nucleus, whereasthe surrounding stromal cells exhibit no dUTPaseexpression. In contrast, in Fig. 2D predominantly cyto-plasmic dUTPase expression is observed. Fig. 2E and Fillustrate NSCLC specimens with weak staining andintense staining for dUTPase with IHC scores of 5 and270, respectively. Overall, there was clear evidence ofelevated dUTPase expression at both the nuclear andmitochondrial level in the tumor tissues compared withthe normal and surrounding nontumor tissue. Themedian dUTPase IHC score was 100 (range: 0–300).Seventy-seven percent of specimens (27 of 35) exhibiteda dUTPase IHC score higher than 10. Although thenumber of clinical specimens is limited, the analysis ofdUTPase expression in NSCLC histologic subtypesrevealed a significant difference in expression between

Figure 1. dUTPase and TS proteinand mRNA expression in a panel ofNSCLC cell lines. A, dUTPasenuclear (Nuc) and mitochondrial(Mito) isoforms, TS and b-actinexpression were analyzed byWestern blotting with densitometricanalysis of the representativeWestern blot conducted using ScionImageJ64 normalized to b-actin. B,themRNAs encoded by theDUT andTYMS genes were measured usingqRT-PCR normalized to GAPDH.Histogram represents mean� SEM.

β-Actin

TS

A549

H1299

H358

H460

H522

H1563

H2228

H23

dUTPaseMitochondrialNuclear

A549 H1299 H1563 H2228 H23 H358 H460 H522

Rel

mR

NA

A

B

Protein

Nuc dUTPase Mito dUTPase TS

DUT total

TYMS

A549 H1299 H1563 H2228 H23 H358 H460 H5220.0

0.5

1.0

1.5

2.0

2.5

Rel

pro

tein

0.0

0.1

0.2

0.3

0.4mRNA






SCC versus adeoncarcinoma. dUTPase IHC score abovethe median was found in 57% of SCC as comparedwith 25% of adenocarcinoma (P ¼ 0.01), although theoverall range of expression in adenocarcinoma andSCC were similar (0–300 and 0–280, respectively).

The mRNA encoding dUTPase is aberrantlyexpressed in clinical specimens of NSCLC

Following microdissection, RNA was extracted andprocessed from the 32 tumor specimens, and DUT geneexpression relative to ACTB (b-actin) was determinedby qRT-PCR alongside TYMS (Fig. 2G). A range in DUTmRNA expression of 7-fold was observed across all 32NSCLC specimens, similar to the range observed in thecell line panel. Subgroup analysis of the histologic sub-types revealed a narrow 2-fold range in DUT expressionin SCC compared with the wider 5-fold variationobserved in adenocarcinoma (Fig. 2H). Importantly,DUT

mRNAcorrelatedwith the protein expression detected byIHC (R2 ¼ 0.51; P ¼ 0.027).

Expression of dUTPase does not correlate with TS inNSCLC

The pattern of TS expression in the NSCLC specimenswas also analyzed by IHC and qRT-PCR. Themedian IHCscore for TS was 100 (range 0–300) with 94% of tumorspecimens staining positive (Supplementary Fig. S2).TYMS mRNA expression correlated with TS IHC (R2 ¼0.4; P ¼ 0.035). When all histologic subtypes were com-bined, there was no correlation between DUT and TYMSmRNA expression (R2 ¼ 0.01; P ¼ 0.56; n ¼ 32; Fig. 2G)or protein expression measured by IHC (R2¼ 0.1; P¼ 0.1;n ¼ 35), suggesting that these genes are not coregulatedin NSCLC. The median IHC score for TS was higher inSCC than adenocarcinoma with scores of 200 and 100,respectively, with TS IHC score above the median found

dUTPase

R2 = 0.01; P = 0.56

DUT (ΔΔCT)

TY

MS

(ΔΔ

CT)

G H I

0.000

0.025

0.050

0.075

0.100

0.125

0.150

0.175

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0.09

AllAC

SCC

Other

non

-SCC

0.00

0.02

0.04

0.06

0.08

0.10

AllAC

SCC

Other n

on-S

CC

0.00

0.05

0.10

0.15

0.20

DU

T (

ΔΔC

T)

TY

MS

(ΔΔ

CT)

A B

ED

C

F

Figure 2. Representativeimmunohistochemical staining ofdUTPase with the DUT415antibody. Specimens wereprocessed as described inMaterials and Methods. A,dUTPase expression in replicatingcells of the human palatine tonsilgerminal center (�40). B, dUTPaseimmunostaining in normal lungtissue: pneumocytes in alveolartissue stained negative (�100). C,dUTPase overexpression with highnuclear positivity in NSCLC withnearby stromal cells stainingnegative (�200). D, NSCLCspecimen showing elevatedcytoplasmic dUTPase staining(�200). E, NSCLC specimenshowing very low dUTPaseexpression (�100; IHC score: 5).F, NSCLC specimen showingoverexpression of dUTPase in boththe cytoplasm and nucleus (�100;IHC score: 270). G, scatter plotillustrating the relationship betweenTYMS and DUT gene expressionmeasured by qRT-PCR (R2¼ 0.01;P ¼ 0.56). H, whisker plot showingthemedian and rangeof expressionfor DUT in NSCLC histologicsubtypes (medianAC¼0.044;SCC¼ 0.067; other non-SCC ¼ 0.069).I, whisker plot showing the medianand range of TYMS expression inNSCLC histologic subtypes(median AC ¼ 0.0075; SCC ¼0.015; other non-SCC ¼ 0.013).AC, adenocarcinoma.

Wilson et al.





in 71% of SCC as compared with 31% of adenocarcinoma.Analysis of TYMS mRNA also showed a higher medianexpression in SCCwhen compared with adenocarcinomaconsistent with previous observations (27). However, aswas observed with DUT, the range of expression wassignificantly greater in adenocarcinoma at 80-fold com-pared with just 2.8-fold in SCC (P ¼ 0.006; Fig. 2I).

TS, but not dUTPase, protein expression is induced inresponse to fluoropyrimidine and antifolatetreatmentThe acute induction of TS protein following treatment

with TS-inhibiting agents has been reported in a variety ofneoplasias and proposed as a mechanism of resistance totreatment. However, the expression of dUTPase inresponse to TS inhibition in NSCLC cells has not been

evaluated. FourNSCLCcell lineswere exposed to increas-ing concentrations of FUdR and pemetrexed and proteinexpression of TS and dUTPase was measured. FUdRtreatment was characterized by the appearance of theincreased molecular weight ternary TS complex. Howev-er, with the exception of the A549 cells (low TS expres-sing), all cell lines showed detectable expression of free(uninhibited) TS enzyme as well as the TS-inhibited com-plex. These cells also showed induction of TS proteinranging from 2- to 5-fold higher than vehicle controlsafter FUdR treatment. Similarly, treatment with peme-trexed resulted in an increase in TS protein expressionbetween 2- and 10-fold higher than vehicle controls andwas observed in all 4 cell lines. No evidence of increaseddUTPase expressionwas observed in response to FUdRorpemetrexed treatment (Fig. 3, left).

Figure 3. Evaluation of dUTPaseexpression in response to TSinhibitor treatment and validation ofsiRNA–mediated silencing. Leftcolumn: dUTPase and TS proteinexpression was evaluated inresponse to FUdR and pemetrexedtreatment. Western blot bands werequantified by ImageJ64 andnormalized to their respectiveb-tubulin and compared withvehicle-treated controls. Middlecolumn, 4 NSCLC cell lines weretransfected with either dUTPase-targeted siRNA (si-DUT), scrambledcontrol siRNA (si-SCR), or TS-targeted siRNA at 25 nmol/L.dUTPase and TS expression wasmeasured by Western blotting andbands were quantified by ImageJ64and normalized to b-tubulin andcompared with si-SCR–transfectedcontrol. Numbers below bandscorrespond to the fold changecompared with the si-SCR–transfected control. Right column,DUT and TYMS mRNA expressionwas analyzed by qRT-PCR,normalized to GAPDH, andexpressed as a percentagecompared with si-SCR–transfectedcontrol. ��, P � 0.01;��, P � 0.001.

si-SCR

si-TS

si-DUT

si-SCR

si-TS

si-DUT

0

50

100

150

200

si-TS

si-SCR

si-DUT

TS

β-Tubulin

dUTPase

si-TS

si-SCR

si-DUT

TS

β-Tubulin

dUTPase

% C

on

tro

l

si-TS

si-SCR

si-DUT

TS

β-Tubulin

dUTPase

s-iT

Ssi-

SCR

si-DUT

TS

β-Tubulin

dUTPase

1 0.1 0.7

1 1.1 0.07

1 0.05 1.3

1 0.93 0.19

1 0.04 1.1

1 1.04 0.04

1 0.12 0.85

1 0.83 0.06

% C

on

tro

l%

Co

ntr

ol

% C

on

tro

l

mRNA

mRNA

mRNA

mRNA

TYMS

DUT

TYMS

DUT

TYMS

DUT

TYMS DUT

TS

β-Tubulin

dUTPase

TS

β-Tubulin

dUTPase

1 2.7 3.5 3 3.3

1 0.9 1.1 1.4 1.1

1 1.2 1.2 1.4 2 2.3

1 0.6 0.5 0.5 0.8 1.3

TS

β-Tubulin

dUTPase

1 5.6 5.6 6.3 9.9

1 0.8 0.7 0.8 1.1

TS

β-Tubulin

dUTPase

1 1.1 1.1 1.2 1.4 1.3

1 2 3.2 3.3 3.4 2.9

A549

H358

H1299

H460

0

50

100

150

200

si-SCR

si-TS

si-DUT

si-SCR

si-TS

si-DUT

0

50

100

150

si-SCR

si-TS

si-DUT

si-SCR

si-TS

si-DUT

0

50

100

150

200

si-SCR

si-TS

si-DUT

si-SCR

si-TS

si-DUT

******

*** **

*****

*** ***

PTX nmol/LFUdR nmol/L

500 1,000125 250 500-

- - - -- -


500 1,000250 500-

- - -- -


500 1,000250 500-

- - -- -


500 1,000125 250 500-

- - - -- -






Efficient dUTPase gene silencing in NSCLC cellsHaving confirmed aberrant dUTPase overexpres-

sion in NSCLC, the influence of dUTPase expressionon response to TS-targeted chemotherapy was ana-lyzed. Using a previously validated siRNA targetingdUTPase (si-DUT; ref. 37), 4 NSCLC cell lines weretreated with 25 nmol/L of si-DUT. The specificity ofthe siRNA was confirmed by parallel transfectionswith both 25 nmol/L of a nontargeted scrambled con-trol siRNA (si-SCR) and an additional TS-directedcontrol siRNA (si-TS). In all 4 NSCLC cell lines, effi-cient and specific dUTPase silencing with si-DUT wasobserved with more than 80% reduction in expressionat the protein and mRNA levels measured by qRT-PCRand Western blotting at 24 and 48 hours, respectively(Fig. 3, middle and right). Importantly, siRNA–medi-ated dUTPase silencing had no effect on TS mRNA orprotein expression and the transfection methodologyhad no detectable effect on plating efficiency or sub-sequent cell viability.

Inhibition of dUTPase in NSCLC cells inducessynthetic lethality in combination withfluoropyrimidine and antifolate-based TS inhibitors

The growth inhibitory and cytotoxic effects of FUdRand pemetrexed in NSCLC cells were analyzed in thepresence and absence of dUTPase silencing. Three ofthe 4 NSCLC cell lines were inherently resistant to thegrowth-inhibitory effects of FUdR, with the H460 cellline being the exception. When dUTPase expressionwas suppressed in si-DUT–transfected cells, a signifi-cant increase in growth inhibition was observed in theA549, H1299, and H358 cells in response to increasingFUdR concentrations compared with the si-SCR–trans-fected cells. In H460 cells, sensitivity to FUdR was onlyenhanced at elevated concentrations in which thesi-SCR–transfected cells seemed to recover, possiblydue to salvage of nucleotides from apoptotic cells(Fig. 4). Interestingly, NSCLC cells were more sensitiveto the growth inhibitory effects of pemetrexed, withdose-dependent increases in growth inhibition observ-ed. However, although the concentration at whichgrowth inhibition becomes apparent was similar forsi-DUT- and si-SCR–transfected cells, silencing of dUT-Pase showed a significant overall increase in growthinhibition at all subsequent concentrations analyzed(Fig. 4). Calcein–AM cell viability analysis was alsodone and confirmed the reduction in cell proliferationassociated with dUTPase silencing following treatmentwith pemetrexed. The inclusion of 10 mmol/L thymi-dine in culture media rescued the growth inhibitoryeffects of pemetrexed in both si-SCR- and si-DUT–transfected cells to that of vehicle-treated controls(Supplementary Fig. S3), indicating that the growthinhibitory effects of pemetrexed in these NSCLC cellsis mediated primarily through TS inhibition and thatthe sensitization observed with dUTPase silencing isdependent on TS inhibition.

dUTPase silencing suppresses the ability of NSCLCcells to survive and recover from transient exposureto FUdR or pemetrexed

Colony-forming assays were carried out to assess theability of 4 NSCLC cell lines to recover following a tran-sient 24-hour exposure to increasing concentrations ofFUdR or pemetrexed. A dose-dependent decrease in thecolony formation capacity was observed in NSCLC cellstreated with increasing concentrations of FUdR. Howev-er, suppression of dUTPase induced highly significantreductions in colonies formed at all concentrations ofFUdR. Similarly, treatment with increasing concentra-tions of pemetrexed reduced colony-forming capacity tovarying degrees in NSCLC cells, ranging from 30% up to75% at the highest concentration of 2.5 mmol/L analyzed.However, in all cell lines, suppression of dUTPase con-ferred a highly significant reduction in colony-formingcapacity in NSCLC cells following pemetrexed treatmentat virtually all concentrations (Fig. 5). These results indi-cated that inhibition of dUTPase greatly diminishes theability of NSCLC cells to survive and recover from tran-sient exposure to a TS-targeted therapy.

dUTPase silencing enhances the expansion of dUTPpools during TS inhibition

A novel fluorescence-based assay was used to mea-sure the concentration of dUTP in NSCLC cells treatedwith 0.5 mmol/L FUdR and 0.25 mmol/L pemetrexed. Insi-SCR–transfected A549 cells, treatment with FUdRand pemetrexed significantly reduced TTP pools from19 pmol/106 in vehicle-treated cells to 6.5 pmol and lessthan 1 pmol/106 cells, respectively. FUdR treatment alsoinduced expansion of the dUTP pool to 23.5 pmol/106 cellswith pemetrexed increasing dUTP pools to 4 pmol/106

cells. However, in si-DUT–transfected cells treated withFUdR, TTP was reduced to less than 0.1 pmol/106 cellsand dUTP pools were increased to 39 pmol/106 cells. Fol-lowing pemetrexed treatment, TTP pools were also reduc-ed to less than 0.1pmol/106 cells anddUTPpools increasedto 21 pmol/106 cells (Fig. 6A). In H460 si-SCR–transfectedcells, treatment with FUdR or pemetrexed depleted TTPfrom 24 pmol/106 in vehicle-treated cells to less than1 pmol/106 cells with less than 2 pmol/106 cells of dUTPdetected. However, in vehicle-treated si-DUT–trans-fected cells, approximately 20 and 5 pmol/106 cells ofTTP and dUTP, respectively, could be detected. In si-DUT–transfected cells treated with FUdR and peme-trexed, the dUTP pool expanded significantly to morethan 15 pmol/106 cells, whereas the TTP pool was signi-ficantly reduced from 24 to less than 5 pmol/106 cells(Fig. 6A). These data confirmed that silencing dUTPaseactivity results in significant increases in dUTP poolexpansion during TS inhibition in NSCLC.

dUTPase silencing induces apoptosis in NSCLC cellsfollowing exposure to FUdR and pemetrexed

Flow cytometry was used to assess whether silencingdUTPase was effective at inducing apoptosis with FUdR

Wilson et al.





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Figure 4. dUTPase silencing enhances NSCLC sensitivity to FUdR and pemetrexed. Growth inhibition was determined by MTS assay for 72 hours. FourNSCLC cell lines were transfected with either si-SCR or si-DUT for 24 hours and subsequently exposed to increasing doses of FUdR (left column) orpemetrexed (right column). Data points representmeanþSEMpercent growth inhibition (n¼3 experiments) comparedwith vehicle-treated controls at 100%.�, P � 0.05; ��, P � 0.01; ��, P � 0.001.






or pemetrexed treatment. Two NSCLC cell lines weretransfected with si-DUT or si-SCR and then treated withincreasing concentrations of FUdR or pemetrexed. Boththe A549 and H358 cell lines transfected with si-DUT

showed significant increases in the induction of apoptosisfollowing treatment with FUdR or pemetrexed acrossthe concentration ranges tested. Specifically, increases inapoptosis of approximately 3- to 5-fold were observed in

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Figure 5. dUTPase silencing reduces the colony-forming capacity of NSCLC cells following transient exposure to FUdR and pemetrexed. Four NSCLC celllineswere treatedwith increasing concentrations of FUdR (left column) or pemetrexed (right column) for 24 hours andmedia subsequently replacedwith drug-free media for 12 to 15 days. Data are presented as meanþ SEM percentage colony formation compared with vehicle-treated control (n¼ 3). Representativeimages at selected concentrations illustrate key differences in colony-forming capacity between si-DUT and si-SCR–transfected cells following treatment.�, P � 0.05; ��, P � 0.01; ��, P � 0.001.

Wilson et al.





si-DUT–transfected cells treated with FUdR comparedwith si-SCR–transfected cells. Similarly, following treat-mentwith pemetrexed, increases of approximately 2- to 4-fold in apoptosis were noted with si-DUT compared withsi-SCR–transfected cells (Fig. 6B). Similar data wereobtained for additional cell lines analyzed (Supplemen-tary Fig. S4).

DiscussionInhibitors of thymidylate metabolism represent an

important class of antineoplastic agents used for thetreatment of head and neck, breast, gastrointestinal, andlung cancers. Elevated intratumoral expression of TS isreported to limit the severity of TTP pool depletion lead-ing to resistance to TS inhibitors (29, 44). However,although TTP depletion can be lethal, a prolonged period

of continued TS inhibition is typically required for this tooccur, and cancer cells can retain viability in a growth-arrested state following 120 hours of continuous exposureto TS inhibitors (4, 31). What is unclear is whether currenttherapeutic regimens can achieve the sufficient and per-sistent inhibition of TS necessary to induce tumor celldeath, particularly in tumors that overexpress TS. Asecond, less-understood mechanism of TS inhibitor activ-ity occurs due to the induction of dramatically increasedratios of dUTP toTTP, resulting in themisincorporation ofaberrant uracil metabolites into DNA. However, elevatedexpression of dUTPasewill prevent dUTPpool expansionand subsequent DNA damage from uracil–DNA misin-corporation and repair, thus contributing to tumor resis-tance to TS-targeted therapy (32, 45, 46). The results of thisstudy clearly show the presence of dUTPase overexpres-sion that can protect NSCLC cells from uracil–DNA

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Figure 6. dUTPase silencing enhances dUTPpool expansion andapoptosis inNSCLC treatedwith FUdRandpemetrexed. A, nucleotide pool analysis of FUdRor pemetrexed-treated cells. Both TTP anddUTPpool levelswere determined inA549 (A) andH460 (B) cells following 24-hour exposure to 0.5mmol/L FUdRor0.25 mmol/L pemetrexed in cells previously transfected with si-SCR or si-DUT. Bars represent mean� SD. B, 2 NSCLC cells were transfected with si-SCR orsi-DUTand treated for 72hourswith increasing concentrations of FUdR (left) or pemetrexed (right). Apoptosiswasevaluatedbyflowcytometry. Bars representmean � SEM. �, P � 0.05; ��, P � 0.01; ��, P � 0.001.






misincorporation. In addition, this study shows thatNSCLC cell lines have an inherent and robust ability tosurvive transient exposure to both fluoropyrimidine andantifolate-based TS inhibitors. Although transient expo-sure to a TS inhibitor did induce growth arrest, the level ofapoptosis induced in response to treatment was minimaland the majority of NSCLC cells retained viability andresumed proliferation upon drug removal. However,when dUTPase expression was suppressed, cells exhib-ited an expansion of the dUTP pool and highly significantincreases in growth arrest, potent suppression of colonyformation, and the induction of cell death. Previous stud-ies in colorectal cancer cells reported a significant increasein DNA damage and a more rapid loss of viability asso-ciatedwith dUTP pool expansion during TS inhibition, anobservation that was reversed by forced expression ofdUTPase (47, 48). The data presented herewould stronglysuggest that NSCLC cells are resistant to cytotoxicityinduced by transient TS inhibition; however, this resis-tance can be effectively overcome by a synthetic lethalapproach targeting both TS and dUTPase simultaneouslyand resulting in activation of the uracil–DNA misincor-poration pathway.

Tailored treatments strategies based on histopathologyand biomarker expression for NSCLC are receivingincreased acceptance based on accumulating clinical evi-dence (27, 49). As pemetrexed-based therapies are seeingrapid clinical expansion, several studies are reporting thatpatients with non-SCC derive greater clinical benefit frompemetrexed-based therapy (12).Conversely, patientswithSCC seem to benefit more from gemcitabine-based ther-apy, correlatingwith the increased TS expression recentlyreported in SCC as a mechanism of resistance to peme-trexed (27, 50). We identified an aberrant pattern of dUT-Pase overexpression that varies dramatically in NSCLCtumor specimens, both in magnitude and intracellularlocalization. Despite the limited number of availablespecimens in this study, our data support previousreports in which TS expression is elevated in SCC com-pared with adeoncarcinoma. In addition, our data alsosuggest that SCCmay have elevated dUTPase expressionthat may contribute to the lack of clinical benefit observedwith pemetrexed-based therapy in SCC. Although therewas no significant correlation between the expression ofdUTPase and TS in the clinical specimens, there werenotable examples with concomitant overexpression of TSand dUTPase at both the mRNA and protein levels. Suchpatients are likely to be nonresponders to pemetrexed-based therapy. Thewidevariation indUTPase expression,observed particularly in adenocarcinoma, suggests that asubset of NSCLC patients treated with a TS-directedtherapy will not benefit from the cytotoxic effects ofuracil–DNA misincorporation as a result of elevateddUTPase. These preliminary observations warrant eval-uation in larger NSCLC patient populations, includingthose who received pemetrexed-based therapy to evalu-ate the relationship between dUTPase expression, treat-ment outcome, and patient prognosis.

Our increasing knowledge of tumor biology, especi-ally in aggressive and treatment-refractory tumors, suchas NSCLC, is providing clear evidence that multiplecellular targets need to be neutralized to induce tumorcell death and elicit lasting clinical efficacy. AlthoughTS may be an important determinant of sensitivity toTS-directed therapy, this study strongly suggests thatdUTPase overexpression in NSCLC induces resistanceto the parallel mechanism of cytotoxicity induced byuracil misincorporation into DNA (37, 46). TargetingdUTPase and thus exploiting this pathway during TSinhibition represents a mechanism-based syntheticlethal combination approach, with the potential to signi-ficantly improve the clinical efficacy of this class ofchemotherapies. However, it is also recognized thatchemotherapeutic agents that inhibit multiple targetswithin a specific DNA biosynthetic pathway or DNAprocessing have the potential to increase treatment-relat-ed toxicities, and this remains a consideration withthe concomitant targeting of TS and dUTPase. However,despite the continued development of technologies toevaluate potential toxicities with novel therapeuticsin silico, it remains difficult to accurately predict howsuch toxicities will manifest themselves until such timesas an agent is evaluated in appropriate combinationanalyses and schedules in vivo. One promising observa-tion to date is that therapeutic combinations that targetmultiple aspects of DNA synthesis and processing suchas those using combinations of platinum agents withfluoropyrimidines or antifolates (5-FU þ oxaliplatin orpemetrexed þ cisplatin) and fluoropyrimidines withtopoisomerase 1 inhibitors (5-FU þ irinotecan) and,indeed, combinations of 3 cytotoxic DNA-damagingagents such as FOLFIRINOX (5-FU, irinotecan, andoxaliplatin) can be successfully implemented in patientswith good performance status with manageable toxi-cities and acceptable patient quality of life, while show-ing additive and synergistic efficacy (12, 51–53). Thesecombination strategies are not only manageable inthe majority of patients, they also continue to serve asthe most effective foundational regimens in some of themost difficult-to-treat solid tumors, including colorectal,lung, and pancreatic cancers. The continued develop-ment of technologies that assist in targeting drug deliv-ery to tumor cells such as those using prodrug, nano-materials, or immunoconjugates also have the potentialto reduce exposure to normal tissues and representsone strategy to maximize antitumor activity while min-imizing the increased risk of adverse events (54). Thedata presented herein strongly support continued effortsto develop inhibitors of dUTPase as a novel therapeuticstrategy in NSCLC and other solid tumors that routinelyuse TS-targeted therapies. The potential broad-reachingclinical benefit of improving these foundational classesof anticancer therapies is worth the inherent challenges.

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

Wilson et al.





Grant SupportThe work was funded by NIH awards P30CA014089 and R21

5R21CA104796-3; The Margaret E. Early Research Trust and the RobertE. Burns Memorial Fund.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 30, 2011; revised November 30, 2011; acceptedDecember 2, 2011; published OnlineFirst December 15, 2011.

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2012;11:616-628. Published OnlineFirst December 15, 2011.Mol Cancer Ther Peter M. Wilson, Melissa J. LaBonte, Heinz-Josef Lenz, et al.

Small Cell Lung Cancer−Targeted Therapies in Non−SynthaseInhibition of dUTPase Induces Synthetic Lethality with Thymidylate

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