a novel klf4/ldha signaling pathway regulates aerobic ... · glycolysis. this altered metabolism is...

Biology of Human Tumors

A Novel KLF4/LDHA Signaling Pathway Regulates AerobicGlycolysis in and Progression of Pancreatic Cancer

MinShi1,2, JiujieCui2,3,4, Jiawei Du5, DaoyanWei2, Zhiliang Jia2, JunZhang1, ZhenggangZhu1, YongGao5, andKeping Xie2

AbstractPurpose: Kr€uppel-like factor 4 (KLF4) is a transcription factor and putative tumor suppressor. However,

little is known about its effect on aerobic glycolysis in pancreatic tumors. Therefore, we investigated the

clinical significance, biologic effects, andmechanisms of dysregulatedKLF4 signaling in aerobic glycolysis in

pancreatic cancer cells.

ExperimentalDesign:Expressionof KLF4 and lactate dehydrogenaseA (LDHA) in70primary pancreatic

tumors and 10 normal pancreatic tissue specimens was measured. Also, the underlying mechanisms of

altered KLF4 expression and its impact on aerobic glycolysis in pancreatic cancer cells were investigated.

Results:We found a negative correlation between KLF4 and LDHA expression in pancreatic cancer cells

and tissues and that their expression was associated with clinicopathologic features of pancreatic cancer.

KLF4 underexpression and LDHA overexpression were correlated with disease stage and tumor differen-

tiation. Experimentally, KLF4 overexpression significantly attenuated the aerobic glycolysis in and growth of

pancreatic cancer cells both in vitro and in orthotopic mouse models, whereas knockdown of KLF4

expression had the opposite effect. Enforced KLF4 expression decreased LDHA expression, whereas small

interfering RNA–mediated knockdown of KLF4 expression had the opposite effect. Mechanistically, KLF4

bound directly to the promoter regions of the LDHA gene and negatively regulated its transcription activity.

Conclusions:Dysregulated signaling in this novel KLF4/LDHA pathway significantly impacts aerobic

glycolysis in and development and progression of pancreatic cancer. Clin Cancer Res; 20(16); 4370–80.

�2014 AACR.

IntroductionPancreatic cancer is the fourth leading cause of cancer-

related deaths in industrialized countries, with a 5-yearsurvival rate of 6% in all patients. In 2012, physiciansdiagnosed 43,920 new cases of this cancer, and 37,390patients died of it in the United States (1). Although surgery

offers the best chance for a cure of pancreatic cancer, lessthan 20% of patients are eligible for potentially curativeresection, as most cases have already spread locally or todistant organs at diagnosis precluding resection (2). Thedisappointing survival rates for pancreatic cancer even aftermargin-negative pancreatectomy indicate the aggressivenature of this disease. Therefore, a better understanding ofthe molecular mechanisms underlying pancreatic cancerinitiation and progression is urgently needed (3, 4).

An increase in aerobic glycolysis, which is the continuousconversion of glucose to lactate in the presence of oxygen(Warburg effect), is a distinctive hallmark of solid tumors,including pancreatic cancer (5, 6). Because it is highlyassociated with increased progression and metastasis ofcancer, aerobic glycolysis is considered a metabolic signa-ture for invasive cancer (7, 8). Enzymatically functionallactate dehydrogenase (LDH) consists of 4 subunitsgrouped into 2 types: M (muscle-type, LDHA gene product)andH(heart-type, LDHBgeneproduct). Five LDH isozymeshave different substrate reactivity as a result of the 5 com-binations of the 2 types of LDH subunits: LDH1 (H4),LDH2 (MH3), LDH3 (M2H2), LDH4 (M3H), and LDH5(M4; 9). LDH5 in particular effectively catalyzes the con-version of pyruvate to lactate and is linked with metastaticcancer (10, 11). Therefore, regulation of expression of LDH,especially the LDHA subunit, plays a key role in aerobic

1Department of Surgery, Shanghai Institute of Digestive Surgery, ShanghaiJiaotongUniversity School ofMedicine Affiliated Ruijin Hospital, Shanghai,People's Republic of China. 2Department of Gastroenterology, Hepatologyand Nutrition, The University of Texas MD Anderson Cancer Center,Houston, Texas. 3Shanghai Key Laboratory of Pancreatic DiseasesResearch, Shanghai Jiaotong University Affiliated First People's Hospital,Shanghai, People's Republic of China. 4Department of Oncology, Shang-hai Jiaotong University Affiliated First People's Hospital, Shanghai, Peo-ple's Republic of China. 5Department of Oncology and Tumor Institute,Shanghai East Hospital, Tongji University School of Medicine, Shanghai,People's Republic of China.

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

M. Shi, J. Cui, and J. Du contributed equally to this article.

Corresponding Authors: Keping Xie, Department of Gastroenterology,Hepatology andNutrition, Unit 1466, TheUniversity of TexasMDAndersonCancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone:713-794-5073; Fax: 713-745-3654; E-mail: [email protected]; andYong Gao, [email protected]

doi: 10.1158/1078-0432.CCR-14-0186

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(16) August 15, 20144370

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Published OnlineFirst June 19, 2014; DOI: 10.1158/1078-0432.CCR-14-0186

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glycolysis. This altered metabolism is maintained in cul-tured cells derived from pancreatic tumors when cultivatedunder normoxic conditions. This indicates that aerobicglycolysis is not just transiently produced by the tumormicroenvironment in vivo but also may be constitutivelyupregulated via stable genetic or epigenetic changes (6, 7).Kr€uppel-like factor 4 (KLF4) is a zinc-finger transcription

factor, and KLF4 mRNA expression is found primarily inpostmitotic, terminally differentiated epithelial cells inorgans such as the skin and lungs and those in the gastro-intestinal tract (12, 13). Authors have described accumulat-ing clinical, experimental, and mechanistic evidence thatKLF4 is a potential tumor suppressor in patientswith variouscancers, including pancreatic cancer (14–17). However, theprecise role and underlying signaling cascade of KLF4 inaerobic glycolysis in pancreatic tumors remain unclear.In this study, we sought to determine the roles of regu-

lation of KLF4 expression in aerobic glycolysis in pancreaticcancer cells, the effect of this regulationonaerobic glycolysisin these cells, and the underlying molecular mechanisms.We discovered that the novel KLF4/LDHA signaling path-way critically regulated this aerobic glycolysis.

Materials and MethodsData miningNCBI Gene Omnibus database provides insight into the

biochemical function of dysregulated of KLF4. GSE21768investigated the changes in gene expression of the KLF4-nullmouse embryonic fibroblasts (MEFs). GSE3113 analyzedthe changes in gene expression when RKO colon cancer cellline was treated for up to 24 hours with ponasterone A toinduce expression of KLF4. Processing of gene expressiondata was according to the database’s guideline. InGSE21768 and GSE3113, the values of logFC represent theglycolysis-related genes’mRNA expression in KLF4-inducedcells/KLF4-nullMEFs versus in the control cells. Thepositive

value of logFCmeans upregulation of the gene expression inKLF4-induced cells/KLF4-null MEFs than in control cells.Conversely, the negative value of logFCmeans downregula-tion of the gene expression in KLF4-induced cells/KLF4-nullMEFs in a comparison with that in corresponding controlcells. Statistical significance was set at P < 0.05.

Cell lines and culture conditionsThe human pancreatic adenocarcinoma cell lines PANC-

1, PAU8902, AsPC-1, BxPC-3, and MiaPaCa-2 werepurchased from the American Type Culture Collection. Thepancreatic cancer cell line MDA Panc-28 was a gift fromDr.P.J. Chiao (The University of Texas MD Anderson CancerCenter). The human pancreatic adenocarcinoma cell lineFG was obtained from Michael P. Vezeridis (The WarrenAlpert Medical School of Brown University; ref. 18). All ofthese cell lines weremaintained in plastic flasks as adherentmonolayers in Eagle’s minimal essential medium supple-mented with 10% fetal bovine serum, sodium pyruvate,nonessential amino acids, L-glutamine, and a vitamin solu-tion (Flow Laboratories). The cell lines obtained directlyfrom the American Type Culture Collection, which per-forms cell line characterization or authentication usingshort tandem repeat profiling, and passaged in our labora-tory fewer than 6 months after receipt.

Human tissue specimens and immunohistochemicalanalysis

Expression of KLF4, LDHA, and LDHB was analyzedusing tissue microarrays (TMA; US Biomax) containingboth human pancreatic tumor specimens and normal pan-creatic tissue specimens (Supplementary Table S1). Use ofthe tissue specimens was approved by the MD AndersonInstitutional Review Board. Standard immunohistochemi-cal staining was performed using anti-KLF4 (Santa CruzBiotechnology), anti-LDHA (Epitomics), and anti-LDHB(Epitomics) antibodies. The staining results were scored by2 investigators blinded to the clinical data as describedpreviously (19).

Measurement of intracellular glucose utilization,LDH activity, and lactate concentration in a culturemedium

To estimate the intracellular glucose utilization, LDHactivity, and lactate concentration in pancreatic cancer cells,a Glucose Colorimetric Assay Kit II (BioVision), LactateDehydrogenase Activity Assay Kit (Sigma-Aldrich), andLactate Assay Kit (Sigma-Aldrich) were used according tothe manufacturer’s protocols. The glucose utilization wasestimated using a standard glucose calibration curve pre-pared under the same condition and reported in micro-grams per microliter. The LDH activity was measured bycomparing the initial rate with a calibration curve for aknown concentration of standard LDH created at the sametime. The LDH activity was reported in milliunits permilliliter. The lactate concentration was estimated using astandard lactate calibration curve prepared under the samecondition and reported in nanograms per microliter.

Translational RelevanceWe used a pancreatic tumor tissue microarray, molec-

ular biology, and animal models to evaluate the inacti-vation and function of the Kr€uppel-like factor 4 (KLF4)/lactate dehydrogenase A (LDHA) pathway in pancreaticcancer cells. Our clinical and mechanistic findings indi-cated that LDHA is a direct transcriptional target of KLF4and that dysregulation of KLF4 expression, which occursfrequently, leads to aberrant LDHA expression. More-over, KLF4 negatively regulated aerobic glycolysis in andgrowth of pancreatic cancer cells, suggesting a novelmolecular basis for the critical role of KLF4 inactivationin pancreatic tumor metabolism. It also suggests thatdysregulated KLF4/LDHA signaling is a promising newmolecular target for designing novel preventive and/ortherapeutic strategies to control this malignancy. There-fore, our findings may have a major effect on clinicalmanagement of pancreatic cancer.

Regulation of Pancreatic Cancer Metabolism by KLF4

www.aacrjournals.org Clin Cancer Res; 20(16) August 15, 2014 4371




Reverse transcription-polymerase chain reactionTotal RNA extraction from pancreatic tumor cells was

performed using the TRizol reagent (Invitrogen). Next, 2 mgof total RNA was reverse transcribed using a First-StrandcDNA Synthesis Kit (Promega) to synthesize cDNA sam-ples. Subsequently, 2 mL of cDNA products was subjected topolymerase chain reaction (PCR) amplification with TaqDNA polymerase (Qiagen) using a thermal cycler with thefollowing primers: KLF4, sense strand 50-cccacatgaagc-gacttccc-30 and antisense strand 50-caggtccaggagatcgttgaa-30; LDHA, sense strand 50-ttgacctacgtggcttggaag-30 and anti-sense strand 50-ggtaacggaatcgggctgaat-30; and a-tubulin,sense strand50-gcagccaacaactatgcccg-30 and antisense strand50-cttccgtatgcggtccagca-30. The PCR products were loadedonto 2% agarose gels and visualized using ethidium bro-mide staining under ultraviolet light.

Western blot analysisStandardWesternblottingwas carriedout usingwhole-cell

protein lysates and primary antibodies against KLF4 (SantaCruz Biotechnology) and LDHA (Epitomics) and a second-ary antibody (anti-rabbit IgG; Santa Cruz Biotechnology).Equal protein sample loading was monitored using an anti-a-tubulin antibody (Santa Cruz Biotechnology).

Transient transfection of pancreatic cancer cellsFor overexpression of KLF4 in PANC-1 cells, they were

transduced with adenoviral KLF4 (Ad-KLF4) or adenoviralenhanced green fluorescent protein (EFGP) as describedpreviously (14). To inhibit KLF4 expression in BxPC-3 cells,they were transfected with a pool of KLF4 small interferingRNA (siRNA) oligonucleotides or control siRNA oligonu-cleotides (Santa Cruz Biotechnology; 50 nmol/L). For over-expressionof KLF4 andLDHA inPANC-1 cells, the cells weretransduced with Ad-KLF4 and pLDHA expression vector(OriGene). LDH inhibitor oxamate sodium (Oxa) with aconcentrationof 20mmol/Lwas added into the BxPC-3 cellstransfected with KLF4 siRNA for further rescue experiments.Cells treated with oligofectamine (Life Technologies) alonewere included as mock transfection controls.

Construction of LDHA promoter reporter plasmidsand mutagenesis

A 1.48-kb fragment containing LDHA 50 sequences from�1330 to þ150 bp relative to the transcription initiationsite was subcloned into the pGL3-basic vector (Promega).The resulting full-length reporter plasmid, which containedmultiple KLF4-binding sites, was designated pLDHA1480.Deletion mutation reporters pLDHA1031 and pLDHA493for this plasmidwere then generated from the pLDHA1480.All constructs were verified by sequencing the inserts andflanking regions of the plasmids.

Measurement of promoter reporter activity by usingdual luciferase assay

Pancreatic cancer cells were transfectedwith the indicatedLDHA promoter reporters, siRNAs, or specific gene expres-sion plasmids. The LDHA promoter activity in these cells

was normalized by cotransfecting a b-actin/Renilla lucifer-ase reporter containing a full-length Renilla luciferase gene(20). The luciferase activity in the cells was quantified usinga dual luciferase assay system (Promega) 24 hours aftertransfection (21).

Chromatin immunoprecipitation assayPancreatic tumor cells (2 � 106) were prepared for a

chromatin immunoprecipitation (ChIP) assay using a ChIPAssay Kit (Millipore) according to the manufacturer’s pro-tocol. The resulting precipitated DNA samples were ana-lyzed using PCR to amplify a 207-bp region of the LDHApromoter with the primers 50-caagccactgacagttcttg-30

(sense) and 50-acctaagtcgagtgacctcc-30 (antisense) and a306-bp region of the LDHA promoter with the primers50-gtgctattttggagctgaggtt-30 (sense) and 50-agcccttgagtatgc-caaaat-30 (antisense). The PCR products were resolved elec-trophoretically on a 2% agarose gel and visualized usingethidium bromide staining.

In vitro cell growth assayPANC-1 cells were transduced with Ad-KLF4 or with Ad-

KLF4 plus pLDHA, and BxPC-3 cells were treatedwith siKLF4orwith siKLF4 plus 20mmol/L oxamate for 48 hours. A totalof 5,000 cells/well in 100 mL medium from each treatmentwere plated in 96-well plates. After 24 hours incubation,viable cells were determined by standard MTT assay.

Animal experimentsFemale pathogen-free athymicnudemicewere purchased

from theNational Cancer Institute. The animals weremain-tained in facilities approved by the Association for Assess-ment and Accreditation of Laboratory Animal Care Inter-national in accordance with the current regulations andstandards of the U.S. Department of Agriculture and U.S.Department of Health and Human Services. Pancreatictumor cells (1 � 106) in 0.1 mL of Hank’s balanced saltsolution were injected subcutaneously into the right scap-ular region of each mouse. The tumor-bearing mice werekilled when they became moribund or on day 35 afterinoculation, and their tumors were removed and weighed.

Statistical analysisThe significance of the patient specimen data was deter-

mined using the Pearson correlation coefficient. The signif-icance of the in vitro and in vivo data was determinedusing the Student t test (2-tailed), Mann–Whitney U test(2-tailed), or one-way analysis of variance.P values less than0.05 were considered significant. The SPSS software pro-gram (version 17.0; IBM Corporation) was used for statis-tical analysis.

ResultsDysregulated KLF4 impacts the expression of multipleglycolysis-related genes

To investigate whether dysregulated KLF4 could impactthe expression of glycolysis-related genes, we analyzed the

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Clin Cancer Res; 20(16) August 15, 2014 Clinical Cancer Research4372




glycolysis-related gene expression data that were depositedin the NCBI Gene Omnibus database (KLF4 null MEFs:GSE21768; KLF4 induced: GSE3113). The expression levelsof many glycolysis-related genes were altered upon thealterations of KLF4 expression. Among all the expressionalterations of glycolysis-related genes in these 2 databases,PFKP, PKM2, and LDHA were upregulated in KLF4-nullMEFs cells whereas downregulated in KLF4-induced RKOcells (Supplementary Tables S2 and S3). This initial datamining study indicated that KLF4 play an important role inglycolysis. Given that LDHA was the most altered genesupon the regulation of KLF4, we chose LDHA as the targetgene and try to investigate whether KLF4–LDHA signalingpathway could impact glycolysis in pancreatic cancer.

Direct association of LDHA but not LDHBoverexpression with clinicopathologic features ofpancreatic cancerWe first investigated the expression of LDHA and LDHB

in the 70 primary pancreatic tumor and 10 normal pancre-atic tissue specimens in the TMA. We observed LDHA-positive staining in the cytoplasm of the tumor cells butLDHA-negative or weakly LDHA-positive staining in thecytoplasm of normal pancreatic cells. In contrast, weobservedLDHB-negative staining in the cytoplasmof tumorcells but weakly LDHB-positive or LDHB-positive stainingin the cytoplasmof normal pancreatic cells (Supplementary

Fig. S1). LDHA expression was positively correlated withdisease stage, indicating that LDHA expression was upre-gulated in late-stage pancreatic tumors. This associationbetween stage I and II versus stage III and IV tumors wassignificant (P < 0.001; Fig. 1A and Supplementary Table S4).Moreover, increased LDHA expression correlated withdecreased tumor differentiation (P ¼ 0.008; Fig. 1B andSupplementary Table S4). Nevertheless, LDHB expressionwas not related to clinicopathologic features of pancreaticcancer (Supplementary Figs. S2 and Table S5). These find-ings indicated that LDHA but not LDHB overexpressionplays a critical role in pancreatic cancer development andprogression.

Decreased KLF4 expression and its direct associationwith clinicopathologic features of pancreatic cancer

We further evaluated the expression of KLF4 protein inthe TMA specimens. Typically, we observed KLF4-negativeor weakly KLF4-positive staining in the tumor cells andKLF4-positive staining in the normal pancreatic cells (Sup-plementary Fig. S1). Furthermore, we analyzed the relation-ship between clinicopathologic features and KLF4 expres-sion in the pancreatic cancer cases. We found that KLF4expression was negatively correlated with disease stage,demonstrating that KLF4 expression was downregulated inlate-stage tumors. This association between stage I and IIversus stage III and IV tumors was significant (P < 0.001;

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Figure 1. LDHA expression in pancreatic tumor specimens and its association with clinicopathologic features of patients with pancreatic cancer. A, positivecorrelation of LDHA expression with disease stage. Representative images of stage I and IV tumors are shown (�200magnification). B, positive correlation ofLDHA expression with tumor differentiation. Representative images of grade 1 and 3 tumors are shown (�200 magnification).






Supplementary Figs. S3 and Table S6). Moreover, decreasedKLF4 expression correlated with decreased tumor differen-tiation (P ¼ 0.004; Supplementary Fig. S3 and Table S6).These findings strongly indicated that loss of KLF4 expres-sion plays a critical role in pancreatic cancer developmentandprogression and is a valuable biomarker for this disease.

Association of decreased KLF4 expression with LDHAoverexpression in pancreatic cancer cells

Given that KLF4 expression loss and LDHA overexpres-sion are closely related to the clinicopathologic features ofpatients with pancreatic cancer, we sought to evaluate therelationship between KLF4 expression and LDHA expres-sion in pancreatic cancer cells. First, we analyzed the expres-sion of KLF4 and LDHA in the TMA specimens (Fig. 2A).KLF4 expression was negatively correlated with LDHAexpression at a statistically significant level (r ¼ �0.557,P < 0.001; Fig. 2B). Consistently, expression of KLF4 wasnegatively correlatedwith expression of LDHA in pancreaticcancer cell lines (Fig. 2C). These data were clinical evidencethat KLF4 inactivation is associated with increased LDHAexpression.

Effects of alteredKLF4 expression on aerobic glycolysisand growth in pancreatic cancer cells in vitro

To determine the effect of altered KLF4 expression onaerobic glycolysis in and growth of pancreatic cancer cells,we transfected PANC-1 and BxPC-3 cells with Ad-KLF4 orwith Ad-KLF4 plus pLDHA expression vector; or treatedwith KLF4 siRNAorwith KLF4 siRNAplus oxamate sodium,

respectively, for 48 hours. We calculated the glucose utili-zation, LDH activity, lactate concentrations, and MTT assayof the treated cells. Restored KLF4 expression stronglydecreased the glucose utilization, LDH activity, lactate con-centrations in, and growth of PANC-1 cells, whereas thisinhibitory effect was attenuated by overexpression of LDHA(Fig. 3A and Supplementary Fig. S4). Downregulation ofKLF4 expression increased the glucose utilization, LDHactivity, lactate concentrations in, and growth of BxPC-3cells. Furthermore, when we added oxamate sodium to theBxPC-3 cells transfected with KLF4-siRNA, oxamate sodiumcould rescue these effects which caused by knockdown ofKLF4 (Fig. 3A and Supplementary Fig. S4). We confirmedthese results using reverse transcription-PCR and Westernblot analysis, which demonstrated that the levels of LDHAmRNA and protein expression in Ad-KLF4–transfectedPANC-1 cells were significantly lower than those in controlcells, whereas LDHA mRNA and protein expression wasupregulated in KLF4 siRNA–transfected BxPC-3 cells(Fig. 3B). Thus, our data clearly established that KLF4 couldregulate aerobic glycolysis in and the growth of pancreaticcancer by impacting LDHA expression.

Altered KLF4 expression inhibits the tumorigenicity ofpancreatic cancer cells in vivoby downregulating LDHAexpression

To determine whether KLF4 regulates LDHA expressionin pancreatic cancer cells in vivo, we injected KLF4-over-expressing PANC-1 cells andKLF4 siRNA–transfectedBxPC-3 cells into the pancreas of nude mice. Overexpression of

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Figure 2. Negative association ofKLF4 expression with LDHAexpression in pancreatic cancercells. A, immunohistochemicalstaining of pancreatic tumorspecimens for KLF4 and LDHA.Shown are representativephotographs (�200 magnification)of KLF4 and LDHA staining in 2pancreatic cancer cases. B,negative correlation of LDHAexpression with KLF4 expression(n ¼ 70; Pearson correlation test:r ¼ �0.557; P < 0.001). C, Westernblot analysis of KLF4 and LDHAexpression in pancreatic cancercell lines (left), and quantitativeWestern blot analysis resultsobtained using densitometricanalysis and standardizedaccording to a-tubulin, andnegative correlation between KLF4expression and LDHA expressionin pancreatic cancer cell lines(right, Pearson correlation test:r ¼ �0.560, P < 0.001).

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Figure 3. The effect of KLF4 expression on aerobic glycolysis in pancreatic cancer cells. A, PANC-1 cells were transfectedwith Ad-KLF4 or with Ad-KLF4 pluspLDHA. BxPC-3 cells were transfected with KLF4 siRNA or KLF4 siRNA plus 20 mmol/L oxamate sodium (Oxa) for 48 hours, respectively. The glucoseutilization, LDH activity, and lactate concentrations were determined as described in Materials and Methods. The data represent the mean � standarddeviation results of experiments performed in triplicate. �, P < 0.05 in a comparison between Ad-KLF4– or KLF4 siRNA–transfected cells and mock orcontrol cells. ��,P < 0.05 in a comparison between cells transfectedwith Ad-KLF4 and pLDHA and cells transfectedwith Ad-KLF4; or between groups treatedwith KLF4 siRNA plus oxamate sodium and groups treated with KLF4 siRNA. B, reverse transcription-PCR (mRNA) and Western blot (Protein) analysisof KLF4 and LDHA expression in PANC-1 and BxPC-3 cells transfected with Ad-KLF4 and KLF4 siRNA for 48 hours, respectively. Total RNA and proteinlysates were harvested for measurement of the expression (left). Quantitative results also were shown (right).






KLF4 significantly inhibited tumor growth (P < 0.001),whereas knockdown KLF4 promoted tumor growth (P ¼0.011; Supplementary Fig. S5). Immunohistochemicalstaining of tumor xenografts indicated that LDHA expres-sion was inhibited by KLF4 overexpression (Fig. 4A). Con-versely, we observed LDHA overexpression in xenograftsafter downregulation of KLF4 expression (Fig. 4B). Theseresults were consistent with the effect of altered KLF4expression on glucose utilization, LDH activity, lactateconcentrations in, and the growth of pancreatic cancercells in vitro as described above, and suggested that KLF4inhibited pancreatic cancer by downregulation of LDHAexpression.

Transcriptional inhibitionof LDHAexpressionbyKLF4in pancreatic cancer cells

Our study demonstrated a negative correlation betweenKLF4 and LDHA expression in pancreatic cancer cell lines.To further explore the molecular mechanisms of regulationof LDHA expression by KLF4, we first analyzed the LDHA

promoter sequence for the presence of potential KLF4-binding elements by using the KLF4 consensus sequence50-CACCC-30. We identified 2 putative KLF4-binding ele-ments (referred to as DNA sequences #1 and #2) in theLDHA promoter region. According to these 2 elements, wegenerated an LDHA promoter (pLDHA1480) and 2 dele-tion mutation LDHA promoters (pLDHA1031 andpLDHA493). The pLDHA1480 contains potential KLF4-binding sites #1 and #2, pLDHA1031 contains potentialKLF4-binding site #1 only, and pLDHA493 contains nopotential KLF4-binding sites (Fig. 5A). Transfection of pan-creatic cancer cells with KLF4 greatly decreased the LDHApromoter activity (pLDHA1480 andpLDHA1031),whereasknockdown of KLF4 expression in the cells increased thisactivity. Altered KLF4 expression had no effect on LDHApromoter activity (pLDHA493). These results suggested thatthe KLF4-binding sites were negative regulatory elements inthe LDHA promoter (Fig. 5B).

In addition, to determine whether KLF4 interacts directlywith the LDHA promoter in pancreatic cancer cells, we

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Figure 4. Influence of KLF4expression on pancreatic tumorgrowth. A, overexpression of KLF4by gene transduction usingAd-KLF4 and B, knockdown ofKLF4 expression by using siRNAagainst KLF4 (siKLF4). Presentedare representative photographs(�200 magnification) of KLF4 andLDHA staining inmouse pancreatictumor xenografts.

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conducted ChIP assays using chromatins prepared fromPANC-1 and BxPC-3 cells and 2 primer sets flanking the207-bp (�471 to �265 bp; #1 site) and 306-bp (�1427 to�1122 bp; #2 site). We amplified both of these DNAfragments from PANC-1 and BxPC-3 cell precipitates usinganti-KLF4 antibodies but not control IgG, suggesting thatendogenous KLF4 bound to the regions of the LDHApromoter from �471 to �265 bp and from �1427 to�1122 bp in these pancreatic cancer cells. We confirmedthese results by engineering overexpression of KLF4, whichled to increased KLF4 recruitment to the LDHA promoter,and by knocking down KLF4 expression, which led todecreased KLF4 recruitment to the LDH promoter. Thealtered KLF4 recruitment was consistent with changes inLDHA promoter activity (Fig. 5C). Collectively, these find-

ings demonstrated that KLF4 bound to the LDHA promoterprimarily at positions �371 to �367 bp and �1310 to�1306 bp and negatively regulated LDHA transcription inpancreatic cancer cells.

DiscussionIn this study, we determined the critical roles of KLF4 and

LDHA expression in aerobic glycolysis in pancreatic cancercells and the underlying mechanism. We found that KLF4transcriptionally repressed the expression of the LDHAgene, constituting a novel signaling pathway that directlyimpacted aerobic glycolysis, and that alterations informedthe clinicopathologic features of patients with pancreaticcancer.

–1,330A

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Figure 5. Transcriptionalinactivation of LDHA expression byKLF4 in pancreatic cancer cells.A, sequences and positions ofputativeKLF4-binding elements onthe LDHA promoter #1 and #2.B, relative LDHA promoter activityin PANC-1 and BxPC-3 cellstransfected with the LDHApromoter and with Ad-KLF4 andKLF4 siRNA, respectively, intriplicate. The relative LDHApromoter activity wasmeasured 24hours after transfection. Statisticalsignificance: �, P < 0.05; ��, P <0.01. C, ChIP assay. Chromatinswere isolated from PANC-1 andBxPC-3 cells, and binding of KLF4to the LDHApromoter in these cellswasexaminedusing a specificanti-KLF4 antibody andoligonucleotides flanking theLDHApromoter regions containingputative KLF4-binding sites asdescribed in the Materials andMethods. Similar ChIP assayswereconducted using chromatinsisolated from PANC-1, Ad-KLF4–transfected PANC-1, BxPC-3, andKLF4 siRNA–transfected BxPC-3cells.






Aberrant expression and activity of metabolic enzymesare among the hallmarks of cancer. Advances in under-standing the biology of tumor progression and metastasishave clearly highlighted the importance of aberrant tumormetabolism, which supports not only tumor cells’ energyrequirements but also their enormous biosynthetic needs.Such metabolic alterations modulate glucose, amino acid,and fatty acid–dependent metabolite biosynthesis andenergy production. Researchers are only just beginning tounderstand thesemetabolic alterations in pancreatic cancercells, which have vast implications for diagnosis of andtherapy for this disease (22).

In the 1920s, Warburg (5) made the striking discoverythat even in thepresenceof ampleoxygen, cancer cells preferto metabolize glucose via glycolysis. In contrast, normalcells produce ATPmainly viamitochondrial oxidative phos-phorylation. LDH catalyzes the final step of glycolysis, inwhich pyruvate is directly converted to lactate or vice versa.An isozyme shift toward increased LDH5 expressionpotentiates lactate production (23, 24). In pancreatic can-cer, it has been reported that the expression of LDHA iselevated in both clinical samples and cell lines, and forcedexpression of LDHA promoted the growth and tumorige-nicity of pancreatic cancer cells (25). However, the mech-anism underlying LDHA overexpression in pancreatic can-cer remains unclear.

Aerobic glycolysis in cancer cells has long been regardedas a phenotype acquired in response to environmentalconstraints such as intermittent hypoxia in premalignantlesions (6, 26). However, this metabolic change is currentlythought of as not simply an adaptation to a hypoxic envi-ronment but rather an active cellular strategy that confers asignificant proliferative and malignancy advantage to can-cer cells (27). From a metabolic standpoint, cancer-relatedgene signaling networks may alter the energy profiles ofcancer cells and enhance the production of biosyntheticintermediates, which are required for generation of thebuilding blocks necessary for sustaining the growth andsurvival in unfavorable microenvironments (22). Thus,investigation of the molecular mechanisms leading to thisphenotype and their contributions to pancreatic cancerdevelopment is urgently needed (28).

KLF4, also known as gut-enriched Kr€uppel-like factor, is amember of the KLF family of zinc-finger transcriptionfactors. Investigators have observed inactivation or silenc-ing of KLF4 in cases of a number of human cancers,including gastric, colorectal, pancreatic, esophageal, lung,prostate, and hepatocellular cancer (14, 16, 17, 29–33).Deletion of KLF4 in mouse models have led to the forma-tion, abnormal differentiation, and increased proliferationof intestinal adenomas in the colon and gastric epithelia(34–36). Also, upregulation of expression of KLF4 inhibitscell proliferation, colony formation, and cancer cell migra-tion and invasion; promotes cell-cycle arrest; and inducesapoptosis in vitro and suppresses carcinogenesis and metas-tasis in vivo (16, 37). Moreover, many lines of clinicalevidence demonstrate that KLF4 functions as a tumor sup-pressor and has potential prognostic value for lymph node

metastasis (14, 32). These observations are evidence thatKLF4 has a putative tumor suppressor function for a varietyof malignancies. Furthermore, KLF4 expression is increasedin primary breast ductal carcinoma and oral and dermalsquamous cell carcinoma cells, and functioned as an onco-genic transcription factor (38–40). All of these results sug-gest that KLF4 expression plays a pivotal role in tumordevelopment and progression. Recently, a study shows thatKLF4 activates the transcription of PFKP gene and plays arole in the maintenance of high glycolytic metabolism inbreast cancer cells (41). Inpancreatic cancer, KLF4 functionsas a tumor suppressor, and seems to be oncogenic in breastcancer. Therefore, the regulatory roles and mechanisms ofKLF4 in aerobic glycolysis in pancreatic cancer cells needsfurther study.

Herein we provide evidence that KLF4 expression affectsaerobic glycolysis in pancreatic cancer cells. We assessed theglucose utilization, LDH activity, and lactate concentra-tions, which reflected aerobic glycolysis, in PANC-1 andBxPC-3 cells transfected with Ad-KLF4 and KLF4 siRNA,respectively. Restoration of KLF4 expression strongly atten-uated the LDH activity and lactate concentrations in PANC-1 cells, whereas downregulation of KLF4 expression pro-moted them in BxPC-3 cells.

Given the important role of KLF4 in aerobic glycolysis inpancreatic tumors, we further examined the underlyingmechanisms responsible for this role. We found that KLF4regulated aerobic glycolysis in pancreatic cancer cells viatranscriptional regulation of LDHA. Specifically, (i) KLF4staining loss and LDHA overexpression in primary pancre-atic tumors were highly correlated with clinicopathologicfeatures of the patients, and their negative correlation wasstatistically significant and consistent in pancreatic cancercells; (ii) overexpressionof KLF4 led todecreased expressionof LDHA, whereas knockdown of expression of KLF4 hadthe opposite effect both in vitro and in vivo; and (iii) KLF4bound directly to the promoter regions of the LDHA geneand inactivated LDHA gene transcription. Therefore, ourfindings provided clinical and mechanistic evidence sup-porting the existence of a novel KLF4/LDHA signalingpathway and its critical contribution to aerobic glycolysisin pancreatic cancer cells.

In summary, this study provided both clinical andmech-anistic evidence supporting that KLF4 regulates LDHAexpression and that the KLF4/LDHA signaling pathway hasa critical role in aerobic glycolysis in pancreatic cancer cells.Our study not only identified a novelmolecularmechanismof this aerobic glycolysis but also identified aberrant KLF4/LDHA signaling as a promising newmolecular target for thedesign of novel therapeuticmodalities to control pancreaticcancer development and progression.

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

Authors' ContributionsConception and design: M. Shi, J. Cui, K. XieDevelopment of methodology:M. Shi, J. Cui, J. Du, D. Wei, Z. Jia, Y. Gao,K. Xie

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Acquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M. Shi, J. Cui, J. Du, D. Wei, Z. Jia, Y. GaoAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):M. Shi, J. Cui, J. Du, D.Wei, Z. Jia, Y. Gao,K. XieWriting, review, and or revision of the manuscript: J. Cui, K. XieAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. XieStudy supervision: J. Zhang, Z. Zhu, Y. Gao, K. Xie

AcknowledgmentsThe authors thank Don Norwood for editorial comments.

Grant SupportThis work was supported by the NCI, NIH (grants R01-CA129956, R01-

CA148954, R01-CA152309, and R01-CA172233; to K. Xie) and theNationalNatural Science Foundation of China (grants 81272917 and 81172022; toY. Gao).

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 January 23, 2014; revised May 14, 2014; accepted May 30, 2014;published OnlineFirst June 19, 2014.

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2014;20:4370-4380. Published OnlineFirst June 19, 2014.Clin Cancer Res Min Shi, Jiujie Cui, Jiawei Du, et al. Glycolysis in and Progression of Pancreatic CancerA Novel KLF4/LDHA Signaling Pathway Regulates Aerobic

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