overexpression of ecdysoneless in pancreatic …...human cancer biology overexpression of...

Human Cancer Biology

Overexpression of Ecdysoneless in Pancreatic Cancer andits Role in Oncogenesis by Regulating Glycolysis

Parama Dey1, Satyanarayana Rachagani1, Subhankar Chakraborty1, Pankaj K. Singh4, Xiangshan Zhao2,Channabasavaiah Basavaraju Gurumurthy2, Judy M. Anderson4, Subodh Lele3, Michael A. Hollingsworth4,Vimla Band2, and Surinder K. Batra1,4

AbstractPurpose: To study the expression and function of a novel cell-cycle regulatory protein, human

ecdysoneless (Ecd), during pancreatic cancer pathogenesis.

Experimental Design: Immunohistochemical expression profiling of Ecd was done in nonneoplastic

normal pancreatic tissues and pancreatic ductal adenocarcinoma lesions (from tissuemicroarray and Rapid

Autopsy program) as well as precancerous PanIN lesions and metastatic organs. To analyze the biological

significance of Ecd in pancreatic cancer progression, Ecd was stably knocked down in pancreatic cancer cell

line followed by in vitro and in vivo functional assays.

Results:Normal pancreatic ducts showed very weak to no Ecd expression compared to significant positive

expression in pancreatic cancer tissues (mean� SE composite score: 0.3� 0.2 and 3.8� 0.2 respectively, P <0.0001) as well as in PanIN precursor lesions with a progressive increase in Ecd expression with increasing

dysplasia (PanIN-1–PanIN-3). Analysis of matched primary tumors and metastases from patients with

pancreatic cancer revealed that Ecd is highly expressed in both primary pancreatic tumor and in distant

metastatic sites. Furthermore, knockdown of Ecd suppressed cell proliferation in vitro and tumorigenicity of

pancreatic cancer cells in mice orthotopic tumors. Microarray study revealed that Ecd regulates expression of

glucose transporter GLUT4 in pancreatic cancer cells and was subsequently shown to modulate glucose

uptake, lactate production, and ATP generation by pancreatic cancer cells. Finally, knockdown of Ecd also

reduced level of pAkt, key signaling molecule known to regulate aerobic glycolysis in cancer cells.

Conclusion: Ecd is a novel tumor-promoting factor that is differentially expressed in pancreatic cancer

and potentially regulates glucose metabolism within cancer cells. Clin Cancer Res; 18(22); 6188–98.�2012

AACR.

IntroductionPancreatic cancer is one of the most lethal malignancies

characterized by late clinical presentation and an extremelypoor prognosis (median survival of patients with pancreaticcancer is about 3–6 months; ref. 1). It is estimated that in2012, about 43,920 individuals will be diagnosed withpancreatic cancer, and nearly 37,390 will die from it (2).Its aggressive nature together with the poor response tochemo and radiotherapy and a tendency for recurrencemakes it one of the few cancers with a nearly 100% post-

diagnosismortality. The identification of biomarkers for theearly detection of pancreatic cancer is thus an urgent clinicalneed (1).

Human ecdysoneless protein (Ecd) is the human ortho-log of the fruit fly (Drosophila melanogaster) ecd protein. Theecd protein is named after its role in regulating the synthesisof the steroid hormone ecdysone, which is required formolting in insects (3, 4). Ecd is required for both develop-ment and oogenesis inDrosophila (5). However, the molec-ular function of its mammalian orthologue (Ecd) is not yetdefined. The human Ecd gene was first identified in acomplementation assay conducted to rescue yeast mutantsdefective in glycolytic gene expression (6). Initial studiesreported role of Ecd in stabilizing p53 and preventing itsturnover by mdm-2 (7). Recently, it was reported that Ecdplays a role in cell-cycle regulation (8). The conditionaldeletion of Ecd (the mouse homologue of Ecd) in murineembryonic fibroblasts led to an arrest of cells in the G1–Sphase of the cell cycle and a marked downregulation ofgenes involved in cell-cycle progression, whereas a wholebody knockout of the protein was embryonically lethal (8).Studies done with Ecd null mouse embryonic fibroblasts(MEF) also showed that Ecd indirectly regulates E2F target

Authors' Affiliations: 1Departments of Biochemistry and Molecular Biol-ogy, 2Genetics, Cell Biology and Anatomy, 3Microbiology and Pathology,and 4Eppley Institute forResearch inCancer andAlliedDiseases,Universityof Nebraska Medical Center, Omaha, Nebraska

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

Corresponding Author: Surinder K. Batra, Department of Biochemistryand Molecular Biology, Eppley Institute for Research in Cancer and AlliedDiseases, University of Nebraska Medical Center, Omaha 68198, NE.Phone: 402-559-5455; Fax: 402-559-6650; E-mail: [email protected].

doi: 10.1158/1078-0432.CCR-12-1789

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(22) November 15, 20126188

on April 14, 2020. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 12, 2012; DOI: 10.1158/1078-0432.CCR-12-1789

http://clincancerres.aacrjournals.org/

gene expression during cell cycle through competitive bind-ing to Rb protein (8). Furthermore, a recent study showsthat even in the absence of a DNA binding domain, Ecdpossesses transactivation property, which is enhanced by itsinteraction with the histone acetyltransferase p300 (9).On the basis of the role of Ecd in cell-cycle regulation,

which is often dysregulated in pancreatic cancer, we inves-tigated the expression and functional significance of Ecd inpancreatic cancer. Here, we report that although the normalpancreatic ducts have little or no Ecd expression, it issignificantly upregulated inpancreatic cancer tissues includ-ing premalignant lesions that are known to precede thedevelopment of invasive ductal carcinoma (termed as pan-creatic intraepithelial neoplasia or PanINs) as well as in themetastatic organs. Functional analysis shows that knock-down of Ecd reduces pancreatic cancer cell growth andtumorigenicity. Ecd depletion also downregulates GLUT4mRNA and protein levels with consequent decrease inglucose uptake, ATP, and lactate levels. In conclusion, ourresults show that Ecd is aberrantly expressed during theprogression of pancreatic cancer and regulates pancreaticcancer cell growth through modulation of glucosemetabolism.

Materials and MethodsTissues, cell lines, and reagentsTissue microarrays (TMA) made from formalin-fixed,

paraffin-embedded (FFPE) tissues were purchased from USBiomax comprising normal and pancreatic cancer tissuesections (catalog number PA481 and PA804). Ecd mono-clonal antibody has been described previously (8). Further-more, matched tissues from primary pancreatic cancer andmetastases were obtained from the University of NebraskaMedical Center (UNMC, Omaha, NE) Pancreatic RapidAutopsy Program (IRB number 091–01). In addition,

FFPE-archived tissue sections comprising adjacent areas ofnormal, PanIN, and pancreatic cancer were provided by ourcollaborator Dr. S. Lele (pathologist). Thirteen pancreaticcancer cell lines were grown in complete medium[Dulbecco’s modified Eagle’s medium (DMEM) supple-mented with 10% FBS and 1% pencillin–streptomycin) at37�C and 5% CO2. Ecd scrambled and knockdown (KD)cells were generated by retroviral transfection of vectorcontrol or 2 different Ecd-specific shRNA vectors (7) intophoenix cells (package cells) and viral supernatant was usedto infect CD18/HPAF, Capan1, and MiaPaCa pancreaticcancer cells followed by selection with puromycin (5 mg/mL). Selected cells were maintained in 10% DMEM con-taining puromycin (3 mg/mL).

Western blotting analysisThe cell lysates preparation and Western blotting was

done as per the standard procedures (10). For all Westernblots, 20 mg of protein lysate was resolved by 10% SDS-PAGE conducted under reducing conditions. Primary anti-bodies used for immunodetection were anti-Ecd mousemonoclonal antibody (Dr. V. Band), GLUT4 (H-61, SantaCruz), p-Akt (Ser-473), and Akt antibodies from Cell Sig-naling Technology Inc. Resolved proteins were transferredon to the polyvinylidene difluoride (PVDF)membrane andimmunoblot assay was conducted.

ImmunohistochemistryThe immunostaining technique was conducted as

described previously (8, 11, 12). All tissue sections wereobserved under a Nikon light microscope and photographsof representative areas taken with the Q-capture Micropub-lisher 5.0 camera (Leeds Precision Instruments) byusing theQ-capture suite software package (QImaging). The intensityof Ecd expression was graded on a scale of 0 to 3 (0: nostaining, 1þ: weakly positive, 2þ: moderately positive, 3þ:strongly positive). The percentage of cells stained for Ecd ina given section was graded on a scale of 1 to 4 (1: <25%; 2:25%–50%; 3: 50%–75%; and 4: 75%–100%). The overallstaining was then represented by a composite score (prod-uct of the above 2 scores, ranging from 0–12).

Statistical analysisThe data was analyzed using the Medcalc software for

Windows version 9�6�4�0 (MedCalc Software). The inten-sities of Ecd expression were considered as continuousvariables, whereas the stage and grade of pancreatic cancerand gender of the patient were considered as categoricalvariables. Continuous variables were compared using theStudent’s 2-tailed t test assuming unequal variance, whereasthe categorical variables were compared using the 2-wayANOVA. A P value of less than 0.05 was consideredsignificant.

RNA isolation from pancreatic tissues, cDNApreparation, and real-time PCR analysis

12pancreatic cancer patient tissue sampleswere obtainedafter approval of the protocol by the IRB (IRB- 491-97) at

Translational RelevancePancreatic cancer is one of the deadliest forms of

cancer owing to its cryptic nature and late clinical man-ifestation. In this study, we analyze the expression andfunction of a novel cell-cycle regulatory protein, calledhuman ecdysoneless (Ecd), in pancreatic cancer. Ourstudy shows that Ecd is overexpressed in preneoplasticpancreatic cancer precursor lesions as well as in primaryand metastatic pancreatic cancer tissues as compared tonormal pancreas. Knockdown of Ecd in pancreatic can-cer cells reduces cancer cell proliferation and tumorige-nicity. Functional analysis shows role of Ecd in regulat-ing pancreatic cancer cell glucose uptake through regu-lation of glucose transporter GLUT4 and thereby affectlactate andATP production. Together, our results suggestthat Ecd, which is overexpressed in pancreatic cancer,plays an important role in pancreatic cancer cell growthandmetabolism and hence represent a novel prognosticbiomarker and therapeutic target for the disease.

Overexpression of Ecdysoneless in Pancreatic Cancer

www.aacrjournals.org Clin Cancer Res; 18(22) November 15, 2012 6189




the University of Nebraska medical Center. RNA was iso-lated from the flash frozen tissues using the mirVana kitfrom Ambion using the manufacturer’s protocol. ThemRNA was converted to cDNA using oligo dT primers. Theprimers used to determine Ecd expression are: Ecd (forwardprimer; FP) 50-ACT TTG AAA CAC ACG AAC CTG GCG-30and Ecd (reverse primer; RP) 50- TGA TGC AGG TGT GTGCTA GTT CCT-30.

Confocal microscopyThe Ecd scrambled and KD cells were seeded at 80%

confluency on sterile coverslips in 12-well plates. Followingovernight serum starvation and subsequent insulin stimu-lation with 100 nmol/L insulin for the mentioned timeperiod, the cells were fixed in ice-cold methanol for 2minutes. The cells were then washed with 1� PBS, blockedwith 10%goat serum, and incubatedwith primary antibodyagainst GLUT4 (Santa Cruz) for 1 hour. After washing, thecells were incubated further with Texas-red–labeled anti-rabbit secondary antibody and then washed and mountedwith Vectashield containing 40 60-diamidino-2-phenylin-dole (DAPI) stain.

Glucose uptake assayFor in vitro glucose uptake assay, cells were seeded at a

density of 50,000 cells per well in a 12-well plate and thenserum starved for 24 hours. Following starvation, cellswere stimulated with 100 nmol/L insulin for half hour.Cells were then washed with 1� PBS and incubated with 1mCi of (3H)-2DG for 15 minutes at 37�C. After washingwith 1� PBS, cells were permeabilized using 1% SDSsolution, and the radioactivity was measured with a liquidscintillation counter. The total radioactive counts werenormalized for the cell count in each well. For in vivoglucose uptake assay, 0.5 � 106 cells of each type (Ecd KDand SCR, 10 mice/group) were injected orthotopicallyinto the pancreas of athymic female mice. 20 days post-implantation, the mice were intraperitonially injectedwith 100 mL of 10 nmol/L IR800 dye coupled 2-deox-yglucose (IRDye 800CW 2DG, from LI-COR Biosciences).The mice were imaged 24 hours after injection by usingPEARL IMPULSE, a near-infrared in vivo imager from LI-COR. The total intensity and mean fluorescence intensitywas measured using Pearl Cam software and comparedbetween the 2 groups.

Lactate assayFor lactate measurement, 50,000 cells were seeded per

well in a 12-well plate. After 24 hours, the supernatant wascollected and lactate production was measured using theLactate Assay Kit (Biovision) following the manufacturer’sprotocol.

ATP measurementThe total ATP content of the cells was determined using a

luminescence-based assay (Cell titer Glo, Promega). 10,000cells were seeded perwell of a 96-well plate (provided by themanufacturer) and after 24 hours, 100 mL of the reagent

mixture was added to each well and the luminescence wasmeasured using luminometer. The luminescence per wellwas normalized with the amount of protein in each well.

Microarray analysisThe scrambled and Ecd KD HPAF/CD18 cells were pro-

cessed for RNA isolation as previously described in thissection. The purity of the RNA was determined by Nano-Drop Spectrophotometer followed by verification withBioanalyzer. The RNA samples in 3 biologic replicates weresubmitted formicroarray using the Phalanx Biotech SpottedMicroarray (HOA_005). The microarray results were ana-lyzed by biostatistician for top differentially expressed genesusing LOWESS Normalization protocol using BRB Array-Tools. The data discussed in this publication have beendeposited inNCBI’sGene ExpressionOmnibus (Edgar et al.,2002) and are accessible through GEO Series accessionnumber GSE39988 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE39988).

ResultsEcd is overexpressed in pancreatic cancer tissues

Given the role of Ecd as a cell-cycle regulator, we wishedto examine if Ecd expression is altered in pancreaticcancers. Commercial TMAs containing pancreatic tissuesfrom 201 patients, representing normal pancreatic tissue,cancer adjacent normal and ductal adenocarcinoma, wereimmunostained using the mouse anti-Ecd monoclonalantibody. In 3 of 25 (12%) normal pancreatic tissues, thenonneoplastic ducts showedweak focal expression of Ecd. Astrong Ecd staining was, however, noted in the islets andweak to moderate staining was seen in the acinar cells ofnormal pancreas tissue sections (Fig. 1A). In comparison,127 of 144 (88%) cases of ductal adenocarcinoma showedsome staining for Ecd protein (i.e., composite score >0). Themean (�SE) composite score for Ecd expression in pancre-atic cancer was significantly higher than that in the non-neoplastic ducts (3.8 � 0.2 and 0.3 � 0.2 respectively, P <0.00001). Among other histologic subtypes of pancreaticcancer, Ecd staining was strongest in pancreatic neuroen-docrine tumors (panNETs) followed by intraductal papil-lary mucinous neoplasm (IPMN), mucinous carcinoma(either mucinous cystadenomacarcinoma derived fromMCNs or noncystic mucinous carcinoma derived fromIPMNs), acinus cell carcinoma, and squamous cell carci-noma. The results are summarized in Supplementary TableS1.

Notably, Ecd expression showed an inverse correlationwith the degree of differentiation being highest in welldifferentiated and lowest in poorly differentiated pancreaticcancer (Fig. 1A). Although the percentage of Ecd-positivecases increased with increasing stage of pancreatic cancer(87%, 92%, 100%, and 100% in stage 1, 2, 3 and 4,respectively), the intensity (measured by the compositescore) was not significantly different between differentstages (Supplementary Table S2)

We further analyzed variation in Ecd expression duringthe initiation andprogressionof pancreatic cancer. In recent

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Clin Cancer Res; 18(22) November 15, 2012 Clinical Cancer Research6190




years, a progression model that describes the transforma-tionof normal ducts into invasive adenocarcinoma througha series of premalignant lesions has been proposed. Theselesions termed as PanINs; ref. 13) are characterized by

specific cytologic, molecular, and genetic changes (1, 8).We analyzed Ecd expression in tissues containing pancreaticcancer, PanIN lesions of various grades, and normal adja-cent pancreatic tissue in terms of the composite score. Therewas a trend towards increase in the percentage of Ecd-positive ducts (composite score >0) with increasing gradeof dysplasia (0%, 55%, 80%, and 74% of nonneoplastic,PanIN-1, PanIN-2, and PanIN-3 lesions were positive,respectively). Furthermore, there was a significant increasein Ecd expression with increasing dysplasia as evidenced bythe increase in composite score. Overall, the immunohis-tochemical staining results indicate that Ecd is overex-pressed during pancreatic cancer initiation and progressionas compared with nonneoplastic normal tissue, therebysuggesting its potential contribution in pancreatic cancerpathogenesis.

Positive correlation of Ecd expression in primarypancreatic cancer and metastatic sites

Many cell-cycle regulatory proteins are known to undergoan alteration in either their level of expression or subcellularlocalization as the malignancy progresses from a localizedto a metastatic stage. Given the fact that Ecd is differentiallyupregulated in the malignant ducts, we next sought toinvestigate whether there was a variation in Ecd expressionduring the metastasis of pancreatic cancer. For this, tissuesamples collected from patients with metastatic pancreaticcancer as part of the rapid autopsy program (at UNMC)were stained for Ecd protein. Samples were obtained from32patients that consisted of primary pancreatic tumors (n¼29 cases) andmetastases occurring in distant organs includ-ing the lungs (n¼ 14 cases), liver (n¼ 18), lymph nodes (n¼ 16), and the omentum (n ¼ 14). In 14 of 29 (48%)patients, expression of Ecd was noticed in primary pancre-atic tumor (composite score¼4.3�1, Fig. 2A; III, IV). Therewas also Ecd expression in metastatic deposits from allorgans at levels similar to that of the primary tumor (Sup-plementary Table S3). An analysis of Ecd positivity at sites ofdistant metastasis revealed that cases where the primarytumor was positive for Ecd expression were more likely tobe also positive for Ecd at the site of metastasis (Supple-mentary Table S4). Specifically, the percentage positivityfor Ecd was higher in the lungs (80% vs. 56%), lymphnodes (100% vs. 44%), and omentum (75% vs. 50%)metastasis when comparing primary tumor specimensthat were positive (top) to those which were negative(bottom) for Ecd expression. The intensity of Ecd expres-sion (as measured by the composite score) was higher inthe omentum and lymph nodes, but not in the lungs forcases where the primary pancreatic cancer was positive forEcd (Fig. 2A and B; III–2XII). Interestingly, primarytumors negative for Ecd also had corresponding meta-static lesions without Ecd expression (Fig. 2B; I–X). Incase of liver metastases, however, 100% of the metastatictumors expressed Ecd irrespective of the positivity of theprimary pancreatic tumor. Overall, the intensity of Ecdexpression was significantly higher in metastatic sites forcases where the primary pancreatic cancer was positive

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Figure 1. Ecd expression in normal pancreas and pancreatic ductaladenocarcinoma. A, expression of Ecd in different grades of PDAC andnonneoplastic pancreas. I, normal pancreatic tissue with non-neoplasticpancreatic ducts (black arrowhead) shows no significant Ecd expression,whereas islet cells (red arrowhead) shows strong and adjacent normalacini (star) shows weak Ecd expression. II–IV, Ecd expression wassignificantly upregulated in invasive ductal adenocarcinoma showingdecreasing expression with advancing grade of pancreatic cancer: II–IVrepresents well, moderate, and poorly differentiated pancreatic cancer,respectively. B, expression of Ecd during initiation and development ofpancreatic cancer. I, Ecd was not expressed in any of the nonneoplasticpancreatic ducts. II, normal pancreatic islet cells on the other handshowed a strong expression of Ecd. III, PanIN-1 lesion showing a weak,predominantly granular cytoplasmic Ecd staining in the dysplasticcolumnar epithelial cells, occasional strong focal staining is noted in theapical portion of the cells (inset). IV, PanIN-2 lesion showing moderatelystrong Ecd staining, predominantly cytoplasmic in distribution. Ecdexpression was significantly stronger towards the apical region of thecells compared with the basal region (inset). V, PanIN-3 lesions alsoshowing strong Ecd expression with cytoplasmic and apical membranestaining similar to that for PanIN-2 (inset). VI, well-differentiatedadenocarcinoma showing moderate Ecd expression. Notably, theexpression was cytoplasmic with an apical membrane accentuation(inset). Original magnification � 100.






(8.6 � 1.1) compared with cases where the primarypancreatic cancer was negative (4.3 � 0.9, P ¼ 0.017).Therefore, the above results show that Ecd expression ismaintained in the metastatic stage of pancreatic ductaladenocarcinoma (PDAC) in different distant organs andis positively correlated to its expression within the pri-mary tumor.

Ecd knockdown inhibits proliferation andtumorigenicity of pancreatic cancer cells

Our previous observation that Ecd is a cell-cycle regulatoroverexpressed in pancreatic ductal adenocarcinoma as com-pared with normal pancreatic ducts prompted us to inves-tigate the functional role of Ecd in pancreatic cancer path-ogenesis. For this, Ecd expression was analyzed in a panel of

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Figure 2. Ecd expression in primary versusmetastatic pancreatic cancer specimens fromRapid Autopsy A.I and II, immunostaining of cancer adjacent normalpancreatic ducts from a patient with metastatic pancreatic cancer shows no Ecd expression (black arrow head). III and IV, moderate to poorly differentiatedprimary pancreatic cancer tissue section from the same patient showing high Ecd expression. V–X, metastatic pancreatic cancer from the same patientreveals strong Ecd expression in the malignant cells at all sites (liver, lung, lymph node, and diaphragm, respectively). Ecd expression was primarilycytoplasmic in both primary andmetastatic cancer cells. III, V, VII, IX, and XI¼ 3,30 -diaminobenzidine (DAB) stained; brown areas indicate Ecd expression. IV,VI, VIII, X, and XII ¼ corresponding hematoxylin and eosin (H&E)–stained sections. Ecd expression in metastatic organs shows strong correlation withthat in the primary tumor. B, immunostaining of Ecd in poorly differentiated pancreatic cancer tissue and corresponding metastatic lesions from liver, lung,lymph, omentum, respectively from the same patient. Primary tumor as well as corresponding metastatic tissues show no Ecd expression. (II, IV, VI, VIII, X)represent H&E staining for each of PDAC, liver, lung, lymph node, and omentum metastasis tissue sections.

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pancreatic cancer cell lines, all ofwhich showedmoderate tohigh levels of Ecd expression both at protein and mRNAlevels (Supplementary Fig. S1). Following this, stableknockdown clones of Ecd were established in CD18/HPAF,Capan1, and MiaPaca pancreatic cancer cells using 2 dif-ferent shRNA (KD1 and KD2) constructs. Growth kineticsassay conducted on the Ecd shRNA (Ecd KD) and scram-bled shRNA-transfected cells (Scr) revealed that downregu-lation of Ecd leads to reduced proliferation of HPAF/CD18(Fig. 3A I) and MiaPaca pancreatic cancer cells. Cell-cycleanalysis of Scr and Ecd KD CD18 pancreatic cancer cellsusing flow cytometry analysis further revealed that decreasein Ecd induces cell-cycle arrest of pancreatic cancer cells atthe G1–S checkpoint (Fig 3A). Ecd KD cells showedincreased cellular population at G1 phase and decreasedcell population at the S phase. This data is in corroborationwith our earlier observation that Ecd depletion causes G1–Sblock in breast cancer cells (7). Annexin V-staining analysison Scr versus EcdKDcells did not showany varitation in celldeath (data not shown), thereby indicating that differencein cell proliferation could be primarily attributed to inhi-bition of cell growth. In addition, there was no significanteffect of Ecd knockdown on cell motility or invasive prop-erties (data not shown) of pancreatic cancer cells. To inves-tigate the effect of Ecd knockdown on pancreatic cancer cell

tumorigenicity, CD18/HPAF Ecd KD or Scr cells wereinjected orthotopically into the pancreas of nude mice andtumor growth was evaluated after 21 days. In vivo tumor-ogenesis data revealed that decrease in Ecd protein levelleads to reduced tumor weight (Fig. 3B; I) as well as lessernumber ofmetastatic foci at distant organs (SupplementaryTable S5A). Immunohistochemical analysis of the tumorsresected from Scr and Ecd KD cell injected mice confirmedthat knockdown of Ecd was maintained within the tumors(Fig. 3B II). Although the exact mechanism of decreasedtumor weight and less metastatic foci upon Ecd deletionneeds further evaluation, these results are consistent withcell-cycle regulatory function of Ecd.

Ecd regulates expression of insulin-dependent glucosetransporter GLUT4

To elucidate themechanism underlying the role of Ecd inpancreatic cancer pathogenesis, microarray analysis wasconducted by using the mRNA isolated from Scr and EcdKD HPAF/CD18 pancreatic cancer cells to identify differ-entially regulated genes (GEO Series accession numberGSE39988). There were 227 genes that showed more than2-fold change in expression due to Ecd downregulation.Clustering of the differentially expressed genes indicatedthatmanyof the geneswere involved in the cholesterol/fatty

Figure 3. Knockdown of Ecd reduces cell growth (in vitro) and tumorigenicity (in vivo) of pancreatic cancer cells. A, Ecd depletion reduces pancreatic cancercell proliferation (I). Ecd KD pancreatic cancer cells exhibit reduced rate of proliferation compared with Ecd scrambled (Scr) cells. (�, P < 0.01). Experimentswere carried out in triplicates (n ¼ 3; II). Bar diagram representing the distribution of Ecd scr and KD cells in different phases of cell cycle analyzed byAnnexin V staining and fluorescence-activated cell sorting (FACS). Ecd KD cells exhibit G1–S block having significantly higher population of cells inthe G1 phase and lower in the S1 phase compared with the control cells; n ¼ 3. B, Ecd knockdown inhibits pancreatic cancer cell tumorigenicity.I, boxed-plot representation of the distribution of tumor weight isolated from mice injected with Ecd KD pancreatic cancer cells versus control cells.Error bars represent SE for n¼ 10 mice. II, immunohistochemistry of Ecd in tumor sections (left) obtained from mice injected with Ecd KD pancreatic cancercells (bottom) and control cells (top) along with corresponding H&E sections (right).






acid synthetic pathway (Supplementary Table S5B), con-sistent with yeast Ecd KD data (14). However, given thepreviously established role of Ecd in regulation of glyco-lytic pathway, we focussed on the insulin-dependentglucose transporter GLUT4 gene, which was one of thedifferentially expressed genes in Ecd KD versus Scr pan-creatic cancer cells. Both quantitative real-time PCR (Fig.4A left) as well as Western blotting confirmed that GLUT4mRNA and protein (Fig. 4A right) were downregulated inpancreatic cancer cells upon Ecd knockdown. Similarchanges in GLUT4 protein and transcript levels wereobserved in MiaPaCa and Capan1 Scr and Ecd KD pan-creatic cancer cells (Supplementary Fig S2A). These find-ings although contradictory to the initial microarray datashowing upregulation of the GLUT4 gene, were validatedby multiple experiments and reanalysis of microarraydata further revealed downregulation of critical GLUT4transcriptional regulator GEF (GLUT4 Enhancer Factor)in Ecd KD cells. These results suggest that human ecdy-soneless protein regulates GLUT4 expression in pancre-atic cancer cells. GLUT4 is a facilitative glucose transport-er, which resides within cytoplasmic vesicles that aretranslocated to the plasma membrane for glucose uptakeupon insulin stimulation. Because we observed an effect

of Ecd downregulation on GLUT4 level, we then inves-tigated if Ecd regulated GLUT4 membrane translocation.Membrane fractionation of MiaPaca pancreatic cancercells showed reduced GLUT4 protein level in the mem-brane fraction of Ecd KD cells as compared with the SCRcells (Supplementary Fig S2B). Further confocal micros-copy of GLUT4 under basal condition and insulin stim-ulation in HPAF/CD18 cells showed that there is delayedtransport of GLUT4-containing vesicles to the membraneupon insulin stimulation in Ecd KD cells comparedwith the scrambled cells (Fig 4B). Overall, these resultsindicate that Ecd regulates expression as well as traffickingof the glucose transporter GLUT4 in pancreatic cancercells.

Ecd depletion leads to reduced glucose uptake in vitroand in vivo

Glucose, the chief energy metabolite of cancer cells, ismainly used by the aerobic glycolytic pathway followingglucose uptake through the transmembrane glucose trans-porters. Because we showed that Ecd regulates expressionand localization of the glucose transporter GLUT4 in pan-creatic cancer cells, we then sought to investigate the effectof Ecd on glucose uptake by pancreatic cancer cells, whichsimultaneously also represents the rate of glycolysis withinthe cells. Because GLUT4 is an insulin-responsive glucosetransporter, radiolabeled glucose was used to study glucoseuptake in HPAF/CD18 Scr and Ecd KD cells either in thepresence or absence of insulin (100 nmol/L) at differenttime intervals. The Ecd KD cells showed decreased level ofglucose uptake compared with the scrambled cells both atbasal level and upon insulin stimulation (Fig. 5A). Theeffect was observed to be more significant on insulin stim-ulation at the initial time points. We extended the study toinvestigate glucose uptake by the tumor mass formed fromHPAF/CD18 scr and Ecd KD pancreatic cancer cells afterorthotopic injection into nude mice. 20 days followingimplantation, IRDye 800CW 2DG was injected intraperi-toneally into each mouse. After 24 hours, the mice wereimaged by using PEARL IMPULSE IR imager, which showedthat the glucose uptake by tumors formed in mice injectedwith Scr pancreatic cancer cells was significantly higher thanthat of the Ecd KD cells (Fig. 5B). Similar differences inglucose uptake were also obtained in mice orthotopictumors developed from Capan1 Scr and Ecd KD pancreaticcancer cells (Supplementary Fig. S3). Comparison of pro-tein level of Ecd and GLUT4 isolated from mice orthotopictumors also show decreased expression in the Ecd KD cellinjected group versus the Scr cells (Fig 5B III). Interestingly,decrease in level of pAkt level was also observed in thetumor lysates from Ecd KD–injected mice. Akt pathway isknown to be a critical regulator of GLUT4 expression andtranslocation (15, 16) as well as the cancer cell glycolyticpathway (17–19). Therefore, our observation suggests acritical role of Ecd in modulation of glucose uptake andconsequently glycolysis by pancreatic cancer cells poten-tially through regulation ofGLUT4protein andmodulationof key signaling pathways.

Figure 4. Ecd regulates expression of insulin-dependent glucosetransporter GLUT4. A, the graph represents quantitative real-timePCR analysis of Ecd and GLUT4 genes in RNA isolated from Ecd scr andKD cells. The Ecd KD cells show a significant decrease in the level of Ecdas well as GLUT4 mRNA compared with the Ecd scrambled cells (left);n ¼ 3. Western blot analysis also shows decrease in the total level ofGLUT4 protein in Ecd-depleted pancreatic cancer cells (right). B,distribution of GLUT4 molecule (red) in Ecd scr versus KD cells, beforeand 10 and 20minutes after insulin stimulation (100 nmol/L) were studiedby confocal microscopy. Ecd KD cells show delayed transport of GLUT4to the plasma membrane on insulin stimulation compared with thescrambled cells.

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Figure 5. Ecd depletion affectsglucose uptake by pancreatic cancercells both in vitro and in vivo. A, thegraph represents the rate ofradiolabeled glucose uptake by Ecdscr and KD pancreatic cancer cellsunder basal condition as well asunder insulin (100 nmol/L)stimulation. Ecd KD cells show asignificant decrease in glucoseuptake rate under insulin stimulationcompared with the control cells;n ¼ 3. B in vivo glucose uptake wasmeasured in mice orthotopic tumorsby intraperitoneal injection of IR800-labeled 2-deoxy glucose; (n ¼ 5mice/group). I, the images obtainedthrough PEARL IR imager reflectglucose uptake in representativeanimals from Ecd scr (top) and EcdKD (bottom) tumor-bearing groups.II, the images show markedlyreduced glucose uptake in tumormasswithEcdKDcomparedwith thecontrol tumors, also reflected by asignificant decrease (P < 0.005) in thecalculated average intensity pergram of tumor. III, The protein lysatesprepared from Ecd scr and KDtumors also show reducedexpression of Ecd and GLUT4 in theEcd KD tumors.






Ecd promotes aerobic glycolysis in pancreatic cancercells

According to the Warburg’s hypothesis, cancer cells useglucose primarily through glycolysis, whereas themitochon-drial oxidative respiration pathway is inhibited. As a result ofthis, the final product of glycolysis, pyruvate, is converted tolactate instead of entering the tricarboxylic acid (TCA) cycle.The results of the current study have shown that Ecd pro-motes pancreatic cancer cell growth as well as glucoseuptake. Hence, we examined whether Ecd favors pancreaticcancer cell aerobic glycolysis by estimating lactate produc-tion. The HPAF/CD18 Ecd KD cells showed decrease inlactate production as compared with the Scr cells (Figure6A). The chief energyquotientof the cells fromanymetabolicpathway including glycolysis is ATP. Quantification of ATPproduction by the CD18 pancreatic cancer cells showed thatEcd depletion leads to reduction in total ATP content in cells(Fig. 6B). Thus, our data shows that Ecd affects the glucosemetabolism pathway in pancreatic cancer cells and that Ecdmay be a key player in the energy production network.

DiscussionWith an incidence rate almost equal to the mortality rate,

pancreatic cancer is the 4th leading cause of cancer-relateddeaths in the United States (1). In this report, we observethat while the nonneoplastic pancreatic ducts had aweak tono expression of Ecd, nearly 88%of ductal carcinomas havehigh expression of this protein. In addition, Ecd expressionwas higher in well andmoderately differentiated pancreaticcancer compared with poorly differentiated pancreatic can-cer indicating that Ecd expression was altered during theprogression of invasive adenocarcinoma. This aberrantexpression pattern of Ecd underlines its potential role inpancreatic cancer pathogenesis. Interestingly, upregulationof Ecd occurs as early as in the PDAC precursor lesions orPanINs, increasing gradually from the low-grade PanINsreaching the highest expression in the high-grade PanIN-3lesions. While PanIN-1 and 2 lesions may be discoveredincidentally and could represent age-related changes,PanIN-3 lesions have been shown to be at the highest riskfor the development of invasive adenocarcinoma (1). Ourobservations suggest that Ecd immunostaining might bepotentially useful for the early detection of pancreaticcancer in high-risk patients and future use of this novelmolecule as an early biomarker for pancreatic cancer.

The increased Ecd expression in pancreatic ductal ade-nocarcinoma as compared with its minimal expression inthe nonneoplastic pancreatic ducts, suggests its potentialrole as a growth promoting factor that facilitates the processof pancreatic cancer pathogenesis. The in vivo studywith EcdKD in pancreatic cancer cells (Fig 4) in our current reportsupports the above hypothesis, as it shows that Ecd knock-down reduces the tumorigenicity of pancreatic cancer cellswhen orthotopically injected into nude mice. Previousstudies showing that knockdown of Ecd in breast cancercells leads to decrease in cell proliferation andG1–S block incell cycle, also corroborate our observations (8). Therefore,role of Ecd in cell cycle might define the underlying

Figure 6. Reduced ATP and Lactate production in Ecd KD pancreaticcancer cells. A, lactate production by Ecd scr and KD pancreatic cancercellswasmeasuredunder basal and insulin-stimulated conditions.Underboth the conditions, Ecd KD cells exhibited significantly reduced lactateproduction compared with the Ecd scr cells. B, graph representsdecrease in ATP production by the Ecd KD pancreatic cancer cells withrespect to the control cells. The ATP content was normalized to theprotein content of the cells. Error bars represent SE for n ¼ 3. C,schematic representation depicting the interconnected network of thepossible role of Ecd in modulating glucose metabolism and cell cyclethrough p53, Rb and Akt pathways.

Dey et al.





mechanism related to its potential role in pancreatic cancerinitiation, development, and metastasis.Otto Warburg had described cancer metabolism early in

the year 1924, mentioning that tumor cells characteristicallythrive on glycolysis rather than oxidative phosphorylationfor their energy source. In this phenomenon defined as"aerobic glycolysis", pyruvate, the end product of glycolysisis converted to lactate along with ATP generation instead ofentering the TCA cycle (20, 21). In this study, we show thatEcd is a novel regulator of pancreatic cancer cell metabolismas depletion of Ecd affects the expression of glucose trans-porter GLUT4 and subsequently glucose uptake by thepancreatic cancer cells. Because glucose is the chief sourceof energy for the cancer cells, hence Ecd might be a criticalplayer in pancreatic cancer cell survival and growth bymodulating the glucose transporters. Furthermore, decreasein lactate and ATP concentration in Ecd knockdown cellsreflects that energy production by the pancreatic cancer cellsis compromised in the absence of Ecd. Also, key signalingpathways, like PI3K/Akt, which regulate glycolysis and cellsurvival (22), are affected by Ecd downregulation, indicatingmultidimensional involvement of this protein in controllingpancreatic cancer cell glucose metabolism. However, themechanism of regulation needs further exploration. In anearlier study, the humanEcd genewas found to complementfor the yeast gene GCR2, which is a transcriptional coacti-vator for glycolytic genes, indicating its role in regulation ofglucose metabolism (6). Hence, it is also known as hSGT1(human suppressor of GCR 2). Another study in yeastillustrated the role of Ecd in modulating carbohydrate andamino acid metabolic pathways (14). Consistent with yeastdata, our current results indicate a conserved function of Ecdfrom yeast to mammals in controlling glucose metabolism,potentially by regulation of genes involved in the metabolicnetwork through its transactivation function. Interestingly,the cell-cycle regulatory function and metabolic function ofEcd seems highly interdependent, placing it at the crossroadof these 2 fundamental cell survival pathways (Figure 6C).Earlier studies with Ecd-null MEFs show that a decrease inEcd levels leads toG1–S blockwith reduced levels of cyclin E.It is important to note that cyclin E is known to serve as acritical "sensor" for any imbalance in cellular ATP/AMPratio, ensuring G1–S block until the cellular energy pool isrestored (23, 24). p53, another binding partner for Ecd, alsohas direct effects on metabolic fate of the cells throughdownstream effectors such as TIGAR (TP53-induced glycol-ysis andapoptosis regulator; ref. 25).AnotherEcd interactingcell cycle regulatory protein Rb (Retinoblastoma) has beenshown to regulate the expression of PDK4 (Pyruvate Dehy-drogenase Kinase), which inhibits PDH (Pyruvate Dehydro-

genase) thereby preventing the entry of pyruvate into theTCA (Tri Carboxylic Acid) cycle, redirecting it towards lactateproduction (26). Therefore, Ecd emerges as a critical mole-cule relaying the signals between cell cycle and metabolicpathways of the cell.

In conclusion, our study suggests that Ecd, a proteinhighlyconserved across species, is differentially expressed duringthe transformation of the normal exocrine pancreatic ductsinto invasive ductal adenocarcinoma. The expression of Ecdin PanIN lesions suggests a potential role for Ecd as a novelearly diagnostic biomarker in pancreatic cancer tissues. Inaddition, role of Ecd in modulating glucose metabolismpathways in pancreatic cancer cellsmight underlie its growthpromoting potential during pancreatic cancer pathogenesis.The current results have identified Ecd as a novel protein,overexpression of which might provide the rapidly prolifer-ating cancer cells with a growth advantage due to increasedpotential for aerobic glycolysis. Future studies should delin-eate the role of Ecd for early detection, prognosis, and/ortherapy target in pancreatic adenocarcinoma.

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

Authors' ContributionsConception and design: P. Dey, S. Chakraborty, P.K. Singh, M.A. Hollings-worth, V. Band, S.K. BatraDevelopment of methodology: P. Dey, P.K. Singh, X. Zhao, V. BandAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): P. Dey, P.K. Singh, J.M. Anderson, S. LeleAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): P. Dey, S. Chakraborty, P.K. Singh, V.Band, S.K. BatraWriting, review, and/or revisionof themanuscript: P.Dey, S. Chakraborty,P.K. Singh, C.B. Gurumurthy, S. Lele, M.A. Hollingsworth, V. Band, S.K. BatraAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): P. Dey, X. Zhao, J.M. AndersonStudy supervision: V. Band, S.K. BatraAnimal experiments like orthotopic implantation, glucose uptake, andtumorigenecity metastasis: S. Rachagani

AcknowledgmentsThe authors thank the invaluable technical support from Erik Moore and

Kavita Mallya. We also thank Janice A. Tayor and James R. Talaska of theconfocal laser scanning microscope core facility at the UNMC, and LynetteSmith from the department of Biostatistics for their support.

Grant SupportThis research is supported by the NIH grant SPORE P50CA127297,

U54163120, and UO1CA11294 (to S.K. Batra and M.A. Hollingsworth);CA96844 and CA144027, and Department of Defense grants W81XWH-07-1-0351 and W81XWH-11-1-0171 (to V. Band).

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 7, 2012; revised August 8, 2012; accepted August 26, 2012;published OnlineFirst September 12, 2012.

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2012;18:6188-6198. Published OnlineFirst September 12, 2012.Clin Cancer Res Parama Dey, Satyanarayana Rachagani, Subhankar Chakraborty, et al. in Oncogenesis by Regulating GlycolysisOverexpression of Ecdysoneless in Pancreatic Cancer and Its Role

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