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Imaging, Diagnosis, Prognosis

Reduced p21WAF1/CIP1 via Alteration of p53-DDX3 Pathway IsAssociated with Poor Relapse-Free Survival in Early-StageHuman Papillomavirus–Associated Lung Cancer

De-Wei Wu1, Wen-Shan Liu2, John Wang3, Chih-Yi Chen4, Ya-Wen Cheng5, and Huei Lee1,5,6,7

AbstractPurpose: DDX3 alteration has been shown to participate in hepatocellular tumorigenesis via

p21WAF1/CIP1 (p21) deregulation. We observed that DDX3 and p21 expression in lung tumors was

negatively associated with E6 expression. Therefore, the aim of this study was to clarify whether

deregulation of p21 by DDX3 via an E6-inactivated p53 pathway would enhance tumor progression in

HPV-associated lung cancers.

Experimental Design: Real-time PCR, luciferase assays, immunoprecipitation, and chromatin immu-

noprecipitation (ChIP) were performed to determine whether DDX3 was regulated by p53 to synergis-

tically enhance p21 transcriptional activity. Cell proliferation was examined by cell counting and colony

formation assays. DDX3 and p21 expression were evaluated in 138 lung tumors by real-time PCR and

immunohistochemistry. The prognostic value of p21 expression on relapse-free survival (RFS) was

analyzed by Kaplan–Meier analysis.

Results: Real-time PCR, luciferase assays, and ChIP assays indicated that three putative p53 binding

sites, located at �1,080/�1,070, �695/�685, and �283/�273 on the DDX3 promoter, were required for

DDX3 transcription. DDX3 deregulation by the E6-inactivated p53 pathway could promote cell prolifera-

tion and the ability to form colonies via reduced Sp1 binding activity on the p21 promoter. Among tumors,

p21 expression was positively associated with DDX3 expression and negatively related with E6 expression,

particularly in early-stage (Iþ II) tumors. Interestingly, low p21 expression was associated with a poor RFS

in early-stage lung cancer.

Conclusion: The reduction of p21 by the alteration of the p53-DDX3 pathway plays an essential role in

early-stage HPV-associated lung tumorigenesis and is correlated with poor RFS of lung cancer patients. Clin

Cancer Res; 17(7); 1895–905. �2011 AACR.

Introduction

Lung cancer in persons who have never smoked (never-smokers) is now becoming increasingly apparent asapproximately 25% of lung cancer cases worldwide arenot attributable to cigarette smoking (1). In East Asia,

where few women are smokers, lung cancer incidence ratesare higher andmore variable than in other geographic areasthat have low numbers of female smokers (2). For example,more than 90% of Taiwanese women are lifetime never-smokers (3), but lung cancer has been the leading cause oftheir cancer deaths for the past 3 decades (4). Humanpapillomavirus (HPV) 16/18 infection has been shownto be associated with the development of lung cancer infemale Taiwanese never-smokers (5). Therefore, identifica-tion of molecular markers for this disease is urgentlyneeded to improve therapeutic strategies for never-smokerswith lung cancer.

The cyclin-dependent kinase (CDK) inhibitorp21WAF1/CIP1 (p21) is the target of the HPV E6 oncoprotein(6, 7). Induction of p21 by p53-dependent (8) or p53-independent pathways (9, 10) results in the inhibition ofcyclin/CDK complexes that regulate cellular proliferation(11, 12). In humans, loss of p21 expression correlates withlung cancer that has poor prognosis (13–16). In mousemodel studies, p21�/�mice showed an increased incidenceof spontaneous lung tumors compared with p21-sufficientmice (17). Therefore, p21 is considered to act as atumor suppressor in lung tumorigenesis. In addition,

Authors' Affiliations: 1Institute of Medical and Molecular Toxicology,2Department of Radiation Oncology, Chung Shan Medical UniversityHospital; 3Department of Pathology, Taichung Veterans General Hospital;4Department of Surgery, China Medical University Hospital; 5Institute ofMedicine, Chung Shan Medical University; 6Department of MedicalResearch, Chung Shan Medical University Hospital, Taichung, Taiwan,ROC; and 7Division of Environmental Health and Occupational Medicine,National Health Research Institutes, Miaoli, Taiwan, ROC

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

Y-W. Cheng and H. Lee contributed equally to this work.

Corresponding Author: Huei Lee, Institute of Medicine, Chung ShanMedical University, No. 110, Sec. 1, Chien-Kuo Rd., 402 Taichung, Taiwan,ROC. Phone: 886-4-2475-9400; Fax: 886-4-2472-0407; E-mail: [email protected] or [email protected]

doi: 10.1158/1078-0432.CCR-10-2316

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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real-time reverse-transcriptase (RT) PCR data have revealedthat p21 expression decreases significantly in HPV 16/18E6–positive lung tumors compared with E6-negativetumors (18). Thus, p21 appears to be a relevant target ofE6 in HPV-associated lung tumors. However, the role ofp21 in tumor progression andmetastasis is unclear in HPV-associated lung tumorigenesis.

The human DEAD-box RNA helicase DDX3 may play arole in the regulation of gene expression via RNA metabo-lism including transcription, splicing, mRNA export, andtranslation (19). Recently, DEAD-box RNA helicases havebeen shown to participate in the development of certainviral-associated cancers. For example, inactivation of DDX3by HBx and HCV core proteins may promote tumor growthvia suppression of p21 transcription through the p53-independent pathway (20, 21). In addition, inactivationof DDX3 by phosphorylation at Thr204 by the cyclin B/cdc2 complex halts HeLa cells in the S phase throughreduced cyclin A expression (22). However, the role ofDDX3 in HPV-associated tumorigenesis remains to beelucidated, especially with respect to lung tumorigenesis.

In the present study, we identified HPV E6 regulation ofDDX3 transcription via p53 inactivation. We constructeddifferent lengths of the DDX3 promoter by 50-deletionmutation for use in luciferase reporter assays in HPV 16E6–positive TL-1 and E6-negative H1299 lung cancer cells.Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis were performed to identify directbinding of p53 onto the putative p53 binding sites ofthe DDX3 promoter. We also used E6, p53, and p21 smallRNA interference (RNAi) to confirm that DDX3 transcrip-tion is predominantly regulated by p53 and that p21transcription was synergistically suppressed by the altera-tion of the p53-DDX3 pathway via E6. In addition, weobserved that cell proliferation and colony formation weredependent on p21 expression and consistent findings werealso observed in lung tumors. More importantly, reductionof p21 expression by the alteration of the p53-DDX3pathway in lung tumors was associated with poorrelapse-free survival (RFS) in lung cancer patients.

Materials and Methods

Study subjects and cell linesLung tumor specimens were collected from 138 patients

with primary lung cancer in the Department of ThoracicSurgery, Taichung Veterans General Hospital between 1998and 2004 and patients were asked to submit a writteninformed consent approved by the Institutional ReviewBoard. The tumor type and stage of each collected specimenwere histologically determined according to the WHO clas-sification system. Cancer relapse data were obtained by chartreview and confirmed by thoracic surgeons. The A549,H1299, Ch27, and H460 lung cancer cell lines were main-tained in DMEM (Dulbecco’s modified Eagle’s medium).The H1355, H441, H520, TL-1, TL-2, and TL-4 lung cancercells and SiHa cervical cancer cells weremaintained in RPMI-1640. C33A cervical cancer cells were maintained in MEM.The medium contained 10% FBS supplemented with peni-cillin (100 U/mL) and streptomycin (100 mg/mL). Cellswere grown at 37�C in a humidified incubator at 5% CO2.

Plasmid construction and transfection reactionThe wild-type (WT) p53, p21-Luc, and HPV 16 E6 were

kindly provided by Drs. J.L. Ko and J.H. Chang Tsai fromthe Institute ofMedical andMolecular Toxicology at ChungShan Medical University. The DDX3 overexpression plas-mid was purchased from OriGene (OriGene Technolo-gies). The p21 overexpression plasmid was purchasedfrom Addgene (Addgene Technologies). The DDX3-Lucplasmid was constructed by inserting a 2,060 bps Hin-dIII/KpnI fragment (spanning the promoter region�2,060/þ1 and 50-deleted�1,242/þ1,�734/þ1,�309/þ1 relatedto the translation start site of the humanDDX3 gene) into aHindIII/KpnI-treated pGL3 vector (Promega Corp.). DDX3promoter–driven luciferase reporters containing multiplepoint mutations of the p53 sites (Mut1) DDX3-Luc, (Mut2/3) DDX3-Luc, and (Mut1/2/3) DDX3-Luc were generatedusing the QuickChange site-directed mutagenesis system(Stratagene). RNAi was performed by expression of smallhairpin RNA (shRNA) to target p53, DDX3, and p21mRNAin lung cancer cell lines. The shRNA template was con-structed from 2 oligonucleotides with a complementarysequence in the loop region (Supplementary Table 1; ref.23). The different concentrations of expression plasmidswere transiently transfected into lung cancer cells (1� 106)using the Transfast reagent. After 48 hours, cells wereharvested and whole-cell extracts were assayed in the fol-lowing experiments.

Silencing of endogenous HPV-16 E6 expression byRNA interference

The RNAi target sequences for HPV 16 E6 siRNA (E6si)have been previously verified (24, 25). The procedures andmethods were as described previously (18).

RNA isolation and real-time PCRTotal RNA was extracted by homogenization in 1 mL

TRIzol reagent, followed by chloroform extraction and

Translational Relevance

Human papillomavirus (HPV) 16/18 infection hasbeen shown to be a possible etiologic factor of lungcancer. However, the pathogenesis and therapeutic strat-egy of lung cancer with HPV infection are largely uni-dentified. Here, we provide evidence to show that areduction in p21 in response to E6, mediated via thep53-DDX3 pathway, synergistically enhances tumorgrowth. In addition, reduction in p21 expression inlung tumors was correlated with poor relapse-free sur-vival in lung cancer patients, particularly those withearly-stage lung cancer. Therefore, we suggest that p21may be a potential target for therapeutics in HPV-asso-ciated early-stage lung cancers.

Wu et al.

Clin Cancer Res; 17(7) April 1, 2011 Clinical Cancer Research1896




isopropanol precipitation. A 3-mg sample of total RNA fromlung tumor tissues was reverse transcribed using Super-Script II Reverse Transcriptase (Invitrogen Life Technolo-gies) and oligo(dT)15 primer. Primers used for real-timePCR analysis are listed in Supplementary Table 1. Reactionswere performed as previously described (26). Since DDX3is located on chromosome X and escapes X-inactivation inthe female (27), the expression levels of DDX3 in male andfemale genders might be different. When at least a 2-foldreduction existed in the mRNA level of the tumor tissuecompared with the normal tissue, DDX3 mRNA wasdefined as "low". Conversely, the situation was definedas "high" (20).

Luciferase reporter assayFor the luciferase reporter assay, appropriate numbers of

cells were transfected with sufficient reporter plasmid, p21-Luc, DDX3-Luc or its derivatives, and either the controlvector or the p53 and DDX3 expression plasmid. Fornormalization of transfection efficiency, b-gal was alsocotransfected. Transfected cells were harvested at 48 hoursposttransfection and a luciferase assay was performedaccording to the manufacturer’s instructions. The luciferaseactivity was measured with an AutoLumat LB953 lumin-ometer (Berthold) and normalized with the cotransfectedb-gal activity.

ChIP assayChIP analysis was performed as described in a previous

report (28) with the following modifications: Immunopre-cipitated DNA was precipitated with ethanol and resus-pended in 20 mL ddH2O (double distilled water). Sampleswere resuspended in 100 mL ddH2O and diluted 1:100before PCR analysis. PCR amplification of immunopreci-pitated DNA was carried out with diluted aliquots, usingthe primers consisting of the oligonucleotides that encom-pass the promoter region of DDX3 and p21 (Supplemen-tary Table 1). PCR products were separated on 2% agarosegels and analyzed using ethidium bromide staining. AllChIP assays were performed at least twice with similarresults.

In vivo immunoprecipitation assayFor the immunoprecipitation experiments, cells trans-

fected with plasmids were harvested and cell lysates wereprepared using immunoprecipitation lysis buffer [20mmol/L Tris-Cl (pH 7.5), 150mmol/L NaCl, 10% glycerol,and 1% Triton X-100]. Cell extracts (1.5 mg) were incu-bated with 40 mL of anti-Sp1-agarose affinity gel (Milli-pore). After extensive washing with immunoprecipitationlysis buffer, the immunoprecipitated proteins were ana-lyzed by immunoblotting using specific antibodies againstDDX3, Sp1, and p53 antibody (Dako).

Cell proliferation and colony formation assaysCell counting and colony formation assays were per-

formed to assess cell proliferation rates as previouslydescribed (18).

Flow cytometric analysisFlow cytometric analysis was performed for cell knock-

down by DDX3si or overexpression by DDX3 overexpres-sion plasmid as described previously (18).

Invasion assayThese assays were performed according to a previously

report (29).

ImmunohistochemistryThe immunohistochemical procedures for the tissue

array were similar to those described in a previous report(18). The anti-rabbit DDX3 antibody was kindly providedby Professor Yan-Hwa Wu Lee (National Yang-Ming Uni-versity; ref. 20) and the monoclonal anti-p21 and anti-Ki-67 antibody was purchased from Zymed Laboratories andDako. Immunostaining results of HPV 16/18 E6 expressionin lung tumors were obtained from a previous report (18,26). Negative immunostaining of HPV 16/18 E6, p21, andKi-67 was defined as less than 10% of tumor cells showingimmunoreactivity in the nuclei, whereas a value greaterthan 10% was defined as positive immunostaining (18).The scoring of DDX3 immunostaining was performed asdescribed previously (20).

Statistical analysisStatistical analysis was performed using the SPSS statis-

tical software program (Version 15.0 SPSS Inc.). The c2 test(2-tailed) was applied for statistical analysis. The associa-tions between HPV 16/18 E6, DDX3, p21, and Ki-67protein expression were analyzed by a c2 test. Survivalplots were generated using the Kaplan–Meier methodand differences between patient groups were determinedby a log-rank test.

Results

DDX3 expression in lung cancer is dependenton p53 status

Our data indicated that DDX3 mRNA expression wassignificantly reduced in 73 of 138 (53%) of the tumorsamples compared with their normal counterpart tissues(Table 1). However, the actual factor involved in DDX3transcription remained unclear. Therefore, we used real-time PCR to study 10 lung cancer cell lines and 2 cervicalcancer cell lines known to harbor p53 WT, p53 mutation,p53-null, or HPV 16 E6, to understand whether DDX3mRNA expression could be related to p53 status. Interest-ingly, DDX3mRNA levels in p53WT lung cancer cells werehigher than in E6-positive and p53-mutant or p53-nullcancer cells, except for H1355 cells (Fig. 1A). Expression ofHPV 16/18 E6 protein has been demonstrated in lungtumors and appears to be related to p53 inactivation(18). We next investigated whether the deregulation ofDDX3 by E6 is mediated through p53-dependent pathway,E6 knocked down in TL-1 cells, and E6 overexpressed inA549 cells were performed. E6 knockdown graduallyincreased the p53 protein level, whereas forced expressionof E6 gradually decreased the p53 level (Fig. 1B). The

p21 Reduction Promotes HPV-Associated Tumorigenesis

www.aacrjournals.org Clin Cancer Res; 17(7) April 1, 2011 1897




elevation and reduction of DDX3 expression was depen-dent on the restoration and degradation of p53 by E6knockdown and E6 overexpression (Fig. 1B). This phenom-enon was also observed in HPV 16 E6–positive TL-2 andHPV 16 E6–negative TL-4 cells (Supplementary Fig. 1).Decreased and increased DDX3 expression levels occurredin a dose-dependent manner in p53-knockdown or p53-overexpression cells (Fig. 1B). These results suggest that p53may regulate DDX3 transcription.

DDX3 transcription is regulated by p53The DDX3 promoter region located at �1,100 to �700

has been shown predominantly to activate DDX3 transcrip-tion in HeLa cells (30). In this study, 3 putative p53binding sites in the promoter region were predicted bythe software from the web site: http://motif.genome.jp/(Fig. 1C). This region contained a sequence between

�1,080/�1,070 (50-GCGCGTGTCT-30), �695/�685 (50-TGGCCTGCCG-30), and �283/�273 (50-AGGCAGGACT-30) and was homologous to the consensus p53-binding site50-NGRCWTGYCY-30, where R is a purine and Y is apyrimidine base. Four lengths of the DDX3 promotersequence (�2,060 to þ1, �1,242 to þ1, �734 to þ1,and �309 to þ1) were constructed by PCR and deletionmutation (Fig. 1C) and were transfected into different cellsfor luciferase reporter activity analysis. As shown in Figure1C, the reporter activity of the 4 lengths of the DDX3promoter increased markedly in E6-knockdown TL-1 cellsand in H1299 cells with p53 overexpression (Fig. 1C). Thereporter activity of DDX3 (�1,242/þ1)-Luc was similar tothat of DDX3 (�2,060/þ1)-Luc, revealing that the�1,242/þ1 promoter region may be sufficient to regulate DDX3transcription in E6-knockdown TL-1 cells. To delineatewhich of the 3 putative p53 binding sites were responsible

Table 1. The association of DDX3 expression with HPV 16/18 E6 and p53 mutation status in lung tumors(n ¼ 138)

DDX3 mRNA DDX3 proteina

No. Low High P Low High P

All casesHPV 16/18 E6

Negative 95 42 530.002

36 590.003

Positive 43 31 12 28 15p53 mutation

Negative 92 44 480.091

37 550.040

Positive 46 29 17 27 19E6/p53 mutationþ/þ, þ/�, �/þ 76 50 26

0.00145 31

0.001�/� 62 23 39 19 43Early stageHPV 16/18 E6

Negative 52 19 330.002

16 36<0.001


Negative 52 23 290.262

19 330.036


0.00328 16

<0.001�/� 34 10 24 7 27Late stageHPV 16/18 E6

Negative 43 23 200.226

20 230.653


Negative 40 21 190.195

18 220.465


0.08017 15

0.427�/� 28 13 15 12 16

aP value for the correlation between DDX3 mRNA and protein was 0.001.

Wu et al.





for the DDX3 reporter activity, DDX3 promoter–directedreporter plasmids harboring the mutations of the p53binding site at �1,080/�1,070 (Mut1), the other 2 p53binding sites at �695/�685 and �283/�273 (Mut2/3),and all 3 p53 binding sites (Mut1/2/3; SupplementaryFig. 2) were introduced into E6-positive or E6-knockdownTL-1 cells and p53-positive or p53-negative H1299 cells forluciferase reporter assay. The reporter activity of Mut1,Mut2/3, and Mut1/2/3 reporter plasmids in E6-positiveTL-1 cells was similar to that of the WT reporter plasmid.However, the reporter activity of Mut1 andMut2/3 reporterplasmids in E6-knockdown TL-1 cells decreased signifi-cantly compared with that of the WT reporter plasmid

(Fig. 1D). The reporter activity of Mut1/2/3 in E6-knock-down TL-1 cells was almost completely abolished and wassimilar to that of the WT reporter plasmid (Fig. 1D). ChIPanalysis further showed that p53 binding ability on p53binding sites was significantly modulated by E6 and p53status (Fig. 1D). These results clearly indicate that DDX3transcription is directly regulated by p53.

DDX3 synergistically enhances p53-activated p21transcription via increased Sp1 binding affinity ontothe p21 promoter

The transcription of p21 is predominantly transactivatedby p53 via increased Sp1 binding affinity (31). A recent

Figure 1. DDX3 transcription is dependent on E6 and p53 status. A, differential expression of DDX3 mRNA revealed by real-time PCR among10 lung and 2 cervical cancer cell lines. B, left, HPV 16 E6 was knocked down by 2 E6 small interfering RNA (E6si1 and 2) in TL-1 cells. HPV 16 E6 wasoverexpressed using various doses of E6 overexpression plasmid in A549 cells. The DDX3mRNA level was determined by real-time PCR and the levels of HPV16 E6, p53, DDX3, and b-actin protein were evaluated by Western blotting. b-Actin was used as a protein loading control Right, p53 of A549 wastransiently knocked down by p53-knockdown plasmid for this experiment; p53 was overexpressed in H1299 cells by the p53 WT overexpression plasmid(p53WT). DDX3 mRNA was determined by real-time PCR and the levels of p53, DDX3, and b-actin protein were evaluated by Western blotting. b-Actinwas used as a protein loading control. In all experiments, the relative mRNA level in the NC and vector controls (VC) was arbitrarily assigned as one. C,schematic diagram of DDX3 promoter–driven luciferase reporters: DDX3 (�2,060/þ1)-Luc, DDX3 (�1,242/þ1)-Luc, DDX3 (�734/þ1)-Luc, and DDX3(�309/þ1)-Luc. These 4 DDX3 constructs and E6 small interfering RNA 2 (E6si) and p53-overexpressed plasmid (p53WT) were cotransfected into theindicated cell types. Luciferase activity was measured at 48 hours posttransfection. In all experiments, the relative luciferase activity shown is indicated asfold-activation relative to that of DDX3 (�2,060/þ1)-Luc. D, left, the E6 knockdown and p53 overexpression-mediated transactivation of the DDX3promoter driven by WT [DDX3 (�1,242/þ1)-Luc] or mutant constructs of the p53 binding site (Mut1) at �1,080/�1,070, (Mut2/3) at �695/�685, and�283/�273, and (Mut1/2/3) at �1,080/�1,070, �695/�685 and �283/�273 in indicated cells was measured as described in (C). Right, binding activityof p53 on the DDX3 promoter evaluated by ChIP in TL-1 and H1299 cells with or without these 2 constructs (E6si and p53). Chromatin was isolated andimmunoprecipitated with an antibody specific for p53.






report indicated that p21 transcription was upregulated byDDX3 via the p53-independent pathway (21). In the pre-sent study, we consistently observed that DDX3 overexpres-sion in TL-1 and H1299 cells significantly increased p21reporter activity (Fig. 2A). Because DDX3 transcription isdirectly regulated by p53 in lung cells, we studied the roleof DDX3 in p53 transactivation of the p21 promoter byconducting a series of transient transfection experiments inTL-1 and A549 cells. E6 knockdown in TL-1 cells resulted ina significant increase in p21 promoter activity (3.5-fold),whereas cotransfection with DDX3 knockdown decreasedthe reporter activity to 1.31-fold (Fig. 2B). When p53 wassilenced in E6-knockdown TL-1 cells, the p21 reporteractivity was significantly reduced to 0.33-fold of that ofTL-1 NC (nonspecific RNAi control) cells. These resultsclearly indicate that DDX3 may play an important role inp21 transcription via a p53-dependent mechanism. Con-versely, forced expression of DDX3 increased p21 reporteractivity 5-fold in A549 cells, but p21 reporter activity onlyincreased 2-fold in DDX3þp53si A549 cells, as comparedwith p53si A549 cells (Fig. 2B). These results clearly indi-cate that p21 activated by p53 is synergistically enhancedby DDX3.

We next examined whether the binding activity of Sp1 onthe p21 promoter could be increased by DDX3, perhaps viaan increase in interaction between Sp1 and p53. Immu-

noprecipitation and ChIP analysis indicated that the inter-action between p53 and Sp1 and the binding affinity of Sp1onto the p21 promoter was markedly increased in E6-knockdown TL-1 cells compared with TL-1 NC cells butchanged slightly in DDX3-knockdown TL-1 cells when E6was further silenced (Fig. 2C and D). Conversely, forcedexpression of DDX3 in A549 cells increased the interactionbetween p53 and Sp1 and the ability of Sp1 to bind ontothe p21 promoter, but these factors were increased onlyslightly in p53-knockdown A549 cells (Fig. 2C and D).Thus, DDX3 appeared to enhance p53-activated p21 tran-scription via increased Sp1 binding onto the p21 promoter.

Because DDX3 synergistically enhances p53-activatedp21 transcription, we next examined whether this situationcould alter cell proliferation, colony formation, and cellinvasion capability. Cell growth and colony formationwere significantly inhibited by E6 knockdown in TL-1 cells,but no effect was seen in response to E6si in DDX3si, p53si,or p21si cells compared with NC cells (Fig. 3A and B).DDX3 significantly reduced cell proliferation and colonyformation in A549 cells but only slightly retarded theseprocesses in p53-knockdown A549 cells (Fig. 3C, left). Inaddition, p21 protein expression in H1299 cells evaluatedby Western blotting was significantly increased by transfec-tion of p53, DDX3, and p53 þ DDX3 expression vectors.Among these, p53 þ DDX3 transfection had the most

Figure 2. DDX3 enhances p53-activated p21 transcription via an increase in interaction between p53 and Sp1. A, p21 promoter–driven luciferasereporter (p21-Luc; 1 mg) and an increasing amount (0.5–2 mg) of DDX3 expression plasmid were transfected into TL-1 and H1299 cell lines as indicated. Thetotal amount of transfected DNA was kept constant by adding the control vector. Luciferase activity was measured at 48 hours posttransfection. In allexperiments, the relative luciferase activity was shown as fold-activation relative to that of the control cells. B, TL-1 cells were transfected with E6-, DDX3-, andp53-knockdown plasmid, and p21-Luc reporter as indicated. A549 cells were transfected with p53-knockdown plasmid, DDX3-overexpressionplasmid, and p21-Luc reporter as indicated. The total amount of plasmid DNA and siRNA was kept constant by the addition of the empty vector and thenegative control in each transfection. Luciferase activity was measured at 48 hours posttransfection. In all experiments, the relative luciferase activity ispresented as fold-activation relative to that of the control cells. C, TL-1 cells were transfected with E6- or DDX3-knockdown plasmid. A549 cells weretransfected with p53-knockdown plasmid or DDX3-overexpression plasmid and were immunoprecipitated with anti-Sp1–conjugated beads. Theimmunoprecipitates were analyzed by SDS-PAGE, followed by immunoblotting with anti-p53 antibody or anti-DDX3 antibody. The input control was 30% ofthe cell extract without any treatment. D, binding activity of Sp1 onto the p21 promoter was evaluated by ChIP in various conditions as described in (C).Chromatin was isolated and immunoprecipitated with an antibody specific for Sp1.

Wu et al.





inhibition on cell proliferation and colony formationfollowed by p53 or DDX3 transfection (SupplementaryFig. 3). Cell invasion ability was significantly inhibitedby E6 knockdown in TL-1 cells; however, there was nodifference by p21-knockdown þ E6-knockdown in TL-1cells as compared with E6-knockdown TL-1 cells (Fig. 3C,left). Similar observations were also seen in A549 cells(Fig. 3C, right). These results suggest that the alterationof DDX3 could predominantly inhibit p53-dependent cellgrowth arrest via the p21 pathway.

DDX3 expression is negatively associated with E6 andis positively related with p21 expression in lungtumorsTo understand whether DDX3 transcription could be

influenced by E6 and p53 mutations in lung tumors, 138lung tumors were examined by real-time PCR to determine

their DDX3 mRNA level. Low DDX3 mRNA expressionlevels were more prevalent in E6-positive tumors than inE6-negative tumors (72% vs. 44%, P¼ 0.002; Table 1). Thisphenomenon was observed particularly in early-stagetumors (I þ II; 73% vs. 37%, P ¼ 0.002; Table 1) butnot in late-stage tumors. However, the association betweenthe DDX3 mRNA level and p53 mutation was marginal inall studied tumor groups, except in early- or late-stagetumors (Table 1). Low levels of DDX3 mRNA expressionweremore prevalent in E6-positive or p53mutation tumorsthan in E6-negative and p53WT tumors (66% vs. 37%, P¼0.001; Table 1), especially in early-stage tumors (64% vs.29%, P ¼ 0.003; Table 1). Expression of DDX3 mRNA inlung tumors was also consistent with DDX3 immunostain-ing results (P ¼ 0.001; Table 1).

We next examinedwhether p21 expression could be affect-ed by DDX3 in lung tumors by immunohistochemistry.

Figure 3. Cell proliferation andcolony formation efficiency areattenuated by p21. A, TL-1 cellswere transfected with E6-, DDX3-,p21-, and p53-knockdownplasmids as indicated. A549 cellswere transfected with p53-knockdown plasmid and DDX3-overexpression plasmid asindicated. The levels of p21 andb-actin protein were evaluated byWestern blotting (A). b-Actin wasused as a protein loading control.These cells were used to evaluatethe cell proliferation rate (B),colony formation efficacy andinvasion ability (C) and comparedwith that of the vector controlcells. In all experiments, therelative colony number of vectorcontrol cells was arbitrarilyassigned as one.






Representative immunostaining results for DDX3 andp21 expression in lung tumors are shown in Figure 4A.DDX3 expression correlated positively with p21 expressionin early-stage tumors (P ¼ 0.001; Table 2). A high mitoticand Ki-67 proliferative index has been shown to be corre-lated with reduction of cell-cycle regulators (32–34) andthe prognostic value of Ki-67 has been shown in non–small

cell lung cancer (NSCLC; refs. 35–37). Ki-67 expression wasalso examined by immunohistochemistry and a represen-tative immunostaining of Ki-67 in lung tumors was shownin Figure 4A. Due to sample availability, Ki-67 analysis wasperformed on samples from 78 patients. As shown inTable 2, p21 expression in lung tumors correlated nega-tively with Ki-67 expression (P ¼ 0.001), particularly in

Figure 4. The representativeimmunostaining results of DDX3,p21, and Ki-67 expression andp21 reduction by E6-p53-DDX3regulation was associated with apoor RFS in lung cancer patients,especially those with early-stagetumors. A, lung tumors with "high"and "low" expression levels ofDDX3, p21, and Ki-67 weredistinguished byimmunohistochemistry. B, theRFS curves in all patients withearly- and late-stage tumors withhigh or low p21 expression. C,RFS curves by p53/E6/DDX3/p21status in all patients with early-and late-stage tumors.

Table 2. The association of p21 expression with DDX3 and Ki-67 expressions in lung tumors (n ¼ 138)

All cases, p21 protein Early stage (I, II), p21 protein Late stage (III), p21 protein

No. Low High P No. Low High P No. Low High P

DDX3 proteinNegative 64 36 28

0.00135 21 14

<0.00129 15 14

0.311Positive 74 21 53 43 9 34 31 12 19

Ki-67 proteinNegative 48 15 33

0.00126 4 22

<0.00122 11 11

0.476Positive 30 21 9 18 13 5 12 8 4

NOTE: 78 of 138 tumors were available for Ki-67 immunostaining.

Wu et al.





early-stage tumors (P < 0.001). Notably, these observationsfrom all studied patients were predominately shown inpatients from nonsmokers (Supplementary Tables 2 and3). These results suggest that DDX3 deregulated by E6 maypromote tumor growth via decreased p21.

Low p21 expression is associated with poor RFS inearly-stage lung cancer patientsTo verify whether decreased p21 expression could be

associated with a poor RFS rate in patients with early-stagetumors, Kaplan–Meier analysis was performed. Of the 111patients who were enrolled in this study, over a medianfollow-up period of 32.4 months, 44 patients relapsed (4had local recurrence, 26 had distant metastasis, and 14 hadlocal and distant metastasis) and 30 patients died from thisdisease. None of the patients received adjuvant treatmentbefore surgical therapy. Patients with low p21 expressionhad a shorter median RFS than those with high p21expression (26.2 months vs. 43.8 months, P ¼ 0.029;Fig. 4B). However, the prognostic value of p21 expressionwas not seen in all patients (22.1 months vs. 35.4 months,P ¼ 0.124; Fig. 4B), which suggests that decreased p21expression may partially contribute to early-stage tumorprogression. In addition, patients with p53mutation or E6-positive/low DDX3/low p21 expression had a shortermedian RFS compared with the late-stage group (23.5months vs. 50.0 months, P ¼ 0.001; Fig. 4C). Moreover,p53 mutation or E6-positive/low DDX3/low p21 expres-sion correlated with a shorter median survival than seen inpatients with low p21 expression (23.5 months vs. 26.2months). These results suggest that reduction of p21 byp53-DDX3 pathway may contribute to tumor progressionand a poor RFS in early-stage HPV-associated lung cancer.

Discussion

Lung cancer is characterized by sequential accumulationof specific genetic and morphologic changes in the normalepithelial cells of the lung. The most important pathwayinvolved in tumor malignancy is disruption of the tightlyregulated cell-cycle regulators that control cell prolifera-tion. In the present study, the reduction of p21 levels by thealteration of the p53-DDX3 pathway via E6 appeared toplay a crucial role in early-stage HPV 16/18-associated lungtumorigenesis. Among tumors, DDX3 expression corre-lated negatively with E6 expression, particularly in early-stage tumors (stage Iþ II; Table 1), whereas p21 expressioncorrelated positively with DDX3 expression (P ¼ 0.001;Table 2). The latter finding was consistent with our pre-vious report in which p21 mRNA expression levels in lungtumors were found to correlate negatively with HPV 16/18E6 expression (18). In the present study, patients withearly-stage tumors with low p21 expression had a worseRFS rate compared with patients with high p21 expression.In addition, p21 expression in lung tumors correlatednegatively with the expression of the cell proliferationmarker Ki-67. Therefore, we suggest that reduction ofp21 in response to alteration of p53-DDX3 via E6 may

further enhance tumor growth and recurrence in early-stagelung cancers associated with HPV infection.

p21 is frequently deregulated during human tumorigen-esis and may act as a tumor suppressor that inhibits cell-cycle progression (38). Chao and colleagues (21) showedthat DDX3 may inhibit hepatocellular cell growth viaupregulated p21 expression. This suggestion is consistentwith our present study, the expression of p21 was signifi-cantly altered by DDX3 overexpression in TL-1 cells, evenwhen p53 was inactivated by E6 (Supplementary Fig. 4A).The doubling time, colony formation, and flow cytometricassay further showed that the doubling time of DDX3-knockdown TL-4 and A549 cells were reduced and con-comitantly, the S-phase cell proportion and colony num-bers of both cells was elevated compared with their controlcells. Conversely, an increase of doubling time anddecrease of S-phase population and colony numbers wasobserved in DDX3-overexpressed TL-1 cells (Supplemen-tary Fig. 4A). In addition, DDX3 inhibited lung cancer cellgrowth via the reduction of p21 transcription (Supplemen-tary Fig. 4B). These results suggest that p21 deregulated byp53-DDX3 pathway may increase lung cancer cell growth.Cell proliferation and colony formation were significantlyaltered by p21 expression in TL-1 and A549 cells (Fig. 4).We also observed a dose dependency in the inhibition ofcolony formation by exogenously expressed p21 plasmid inTL-1 cells (Supplementary Fig. 4C). However, cell invasionactivity was not changed by a reduction in p21 expression(Fig. 4). Among lung tumors, p21 expression was corre-lated with expression of the cell proliferation marker Ki-67,further supporting an association of the reduction of p21with tumor growth in early-stage tumors. Therefore, p21deregulation by the p53-DDX3 pathway may predomi-nantly increase the cell proliferation rate in vitro and tumorgrowth in vivo.

The prognostic value of p21 has been examined exten-sively in lung cancer, but results for prognosis based on p21have been inconsistent (13–16, 39). In the present study,patients, and especially early-stage patients, with positivep21 expression had favorable prognosis. This observationwas consistent with most previous reports (13–16) but wascontrary to Dworakowska’s report. Possibly, the few femalecases and the few adenocarcinomas studied by Dwora-kowska and colleagues (2005; ref. 39) may account forthis difference. In the present study, early-stage patientswith negative p21 expression showed a more negative RFSrate when associated with a p53 mutation and/or the E6-positive condition, compared with other combinations(P < 0.001; Fig. 4C). This finding was consistent withprevious reports indicating that lung cancer patients withp21 reduction due to alteration of the p53 pathway mayhave a poor prognosis (13).

In cell experiments, the reduction inDDX3 in response toE6has been shown to result in a synergistic reduction in p21expression and a promotion of cell proliferation (Fig. 4).Pyeon and colleagues (2009) showed that G1, S, G2, andearly M phase cell-cycle inhibitors efficiently inhibited cell-cycle progression and prevented HPV infection (40). Soft






agar assay further showed that the independent anchoragesoft agar colony number in TL-1 cells wasmarkedly reducedby E6si transfection but slightly changed by the combinedtreatment ofDDX3siþ E6si as comparedwih TL-1 cellswithDDX3si transfection (data not shown).Therefore, the altera-tion in the p53-DDX3 pathway may promote the initiationstep of lung tumorigenesis in HPV-infected tissues. How-ever, this conclusion needs to be further investigated in E6transgenic mouse model.

An association between HPV 16/18 and lung cancer innever-smokers has been noted (5, 18). However, the mole-cular pathogenesis of HPV-associated lung cancer remainsunclear. This study is the first report to indicate that DDX3transcription is regulated by p53. Tumor progressionappears to require E6-mediated inactivation of DDX3,degradation of p53, and synergistic suppression of p21transcription. These factors contribute to a poor RFS inearly-stage lung cancer associated with HPV infection.

Therefore, we suggest that DDX3 could represent a promis-ing molecular target for therapeutics of HPV-associatedlung cancer.

Disclosure of Potential Conflicts of Interest

The authors declare no conflict of interest.

Grant Support

This work was jointly supported by grants from the National Health ResearchInstitute (NHRI96-TD-G-111-006; NHRI97-TD-G-111-006), Department ofHealth (DOH94-TD-G-111-017; DOH100-TD-C-111-005) and the NationalScience Council (NSC-96-2628-B-040-002-MY3) of Taiwan, ROC.

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 August 30, 2010; revised November 9, 2010; acceptedNovember 14, 2010; published OnlineFirst February 16, 2011.

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Correction

Correction: Reduced p21WAF1/CIP1 viaAlteration of p53–DDX3 Pathway IsAssociated with Poor Relapse-Free Survivalin Early-Stage Human Papillomavirus–Associated Lung Cancer

In this article (Clin Cancer Res 2011;17:1895–905), whichwas published in the April1, 2011, issue of Clinical Cancer Research (1), the cell lines presented in Fig. 1B,specifically the Western blotting bands used in E6-overexpressing A549 cells, areincorrect. The authors have provided a new version of Fig. 1 (below). The conclusionsset forth in the article remain unchanged. The authors regret this error.

Reference1. Wu D-W, Liu W-S, Wang J, Chen C-Y, Cheng Y-W, Lee H. Reduced p21WAF1/CIP1 via alteration of

p53–DDX3 pathway is associated with poor relapse-free survival in early-stage humanpapillomavirus–associated lung cancer. Clin Cancer Res 2011;17:1895–905.

Published online February 17, 2015.doi: 10.1158/1078-0432.CCR-14-3282�2015 American Association for Cancer Research.

Figure 1.

ClinicalCancerResearch

www.aacrjournals.org 935

2011;17:1895-1905. Published OnlineFirst February 16, 2011.Clin Cancer Res De-Wei Wu, Wen-Shan Liu, John Wang, et al.

Associated Lung Cancer−Papillomavirus Associated with Poor Relapse-Free Survival in Early-Stage Human

via Alteration of p53-DDX3 Pathway Is WAF1/CIP1Reduced p21

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