Expression of a prometastatic splicevariant of osteopontin, OPNC, inhuman pancreatic ductaladenocarcinomaJennifer Sullivan, MD,a Laurel Blair, MD,a Amer Alnajar, BS,a Tamer Aziz, MD,a Chee Yuan Ng, MD,a

Galina Chipitsyna, PhD,a Qiaoke Gong, MD,a Agnes Witkiewicz, MD,b Georg F. Weber, MD,c

David T. Denhardt, PhD,d Charles J. Yeo, MD,a and Hwyda A. Arafat, MD, PhD,a,b Philadelphia, PA,Cincinnati, OH, and New Brunswick, NJ

Background. Osteopontin (OPN) is a secreted phosphoprotein that confers on cancer cells a migratoryphenotype. We demonstrated recently that nicotine, a major risk factor in pancreatic ductal adenocar-cinoma (PDA), increases OPN expression in PDA cells. An OPN splice variant, OPNc, supportsanchorage independence and maybe the most potent OPN isoform to convey metastatic behavior. In thisstudy, we tested the effect of nicotine on OPNc expression and analyzed the correlation between totalOPN/OPNc levels and patients’ smoking history.Methods. Real-time polymerase chain reaction and ultraviolet light illumination of ethidium-bromidestaining were used to examine the mRNA expression in tissue and in PDA cells treated with or withoutnicotine (3--300 nmol/L). OPN and OPNc were localized by immunohistochemistry, and an enzyme-linked immunosorbent assay was used to analyze OPN serum levels.Results. Nicotine treatment of PDA cells selectively induced de novo expression of OPNc. OPNc wasfound in 87% of invasive PDA lesions, of which 73% were found in smokers. The levels of OPNccorrelated well with higher expression levels of total OPN in the tissue and serum from patients withinvasive PDA.Conclusion. Our data suggest that smoking and nicotine may contribute to PDA metastatic potentialthrough promoting OPNc expression. Although the direct role of OPNc in PDA progression is not defined,OPNc may have value as a diagnostic and prognostic marker, especially in invasive PDA. (Surgery2009;146:232-40.)

From the Department of Surgery, Jefferson Pancreatic, Biliary and Related Cancer Center,a and the Depart-ment of Pathology, Anatomy and Cell Biology,b Thomas Jefferson University, Philadelphia, PA; the College ofPharmacy, University of Cincinnati Medical Center,c Cincinnati, OH; and the Department of Cell Biologyand Neuroscience, Rutgers University,d New Brunswick, NJ

THE INCIDENCE OF PANCREATIC DUCTAL ADENOCARCINOMA

(PDA) was estimated at 37,170 in 2007.1 There isonly a 5% overall 5-year survival rate, and morethan 85% of the tumors are diagnosed after thetumor has infiltrated into adjacent vital organs orwhen distant metastases are present. There is,

Supported by NIH grant 1R21 CA133753-01.

Accepted for publication March 10, 2009.

Reprint requests: Hwyda A. Arafat, MD, PhD, Department ofSurgery, Thomas Jefferson University, 1015 Walnut Street,Suite 618 Curtis, Philadelphia, PA 19107. E-mail: hwyda.ara-fat@jefferson.edu.

0039-6060/$ - see front matter

� 2009 Mosby, Inc. All rights reserved.

doi:10.1016/j.surg.2009.03.036

232 SURGERY

therefore, an urgent need for better understandingof the basic molecular mechanisms that contributeto the aggressive nature of PDA and for the designof more effective therapeutic strategies.

Cigarette smoking is among the most notablerisk factors, lending upwards of a 2-fold increase tothe risk of developing PDA.2 It is estimated thatcigarette smoking may be responsible for 25--30%of all PDA cases.3 Nicotine, a major component oftobacco and cigarette smoke, is an addictive agentand has been characterized as a drug of abuse bythe U.S. Surgeon General.4 Pancreatic cancerhave been linked to nicotine exposure throughcigarette smoking in many human studies.5,6 In ad-dition, animal studies have demonstrated that nic-otine or its metabolites can induce pathologicand functional changes in the pancreas.7 It is

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unknown, however, how these functional or mor-phologic changes could contribute to the progres-sion of pancreatic cancer. A possible mechanism isnicotine-mediated induction of stress responsegenes, which are important in cancer metastasis.8

One such stress response gene is osteopontin(OPN), a secreted noncollagenous, sialic acid-richphosphoprotein, which functions as both anextracellular matrix component and a cytokine.9,10

OPN can support migration and protect againstprogrammed cell death after binding to certainintegrin receptors or a CD44 variant on the cellsurface.11 The biological functions of metastasis-associated gene products are extensively regulatedon the post-transcriptional and post-translationallevels.8 Consistently, OPN secreted from variouscells has diverse structural characteristics12,13 andtumor-derived OPN forms are smaller than OPNsecreted by nontransformed cells.14 Recent studieshave shown that an OPN splice variant (OPNc) isexpressed in invasive, but not in noninvasive,breast and liver tumor cell lines.10,15 It has beensuggested that this isoform may support tumorprogression by conveying anchorage indepen-dence and inducing the expression of oxidoreduc-tases.16 Although high levels of OPN have beenreported in PDA tissue,17 the possible regulationof this OPN metastatic isoform by PDA carcino-gens, such as nicotine, has not been studied.

We have recently shown that nicotine inducesthe expression of OPN in PDA cell lines.18 In thisstudy, we tested the effect of nicotine on OPNcexpression in 3 PDA cell lines. We also correlatedthe patients’ smoking history with the expressionlevels of serum and tissue OPN, and tissue OPNcin premaligant (intrapapillary mucinous neo-plasms [IPMN]) and in invasive PDA.

MATERIALS AND METHODS

Cell culture. The human PDA cell line MiaPacaas purchased from the American Type CultureCollection (Manassas, VA). BxPC-3 and HS766Tcells were kindly provided by Dr Scott Kern (TheJohns Hopkins University School of Medicine,Baltimore, MD). Cells were counted and culturedat 1 3 104 cells to near confluence in 96-well platesand maintained in DMEM supplemented with 10%fetal bovine serum in a humid atmosphere of 5%CO2/95% air. Cells were treated with nicotine(3--300 nmol/L) for 3 and 24 hours, and wereevaluated for the expression of OPN mRNA byreal-time polymerase chain reaction (PCR).OPNc expression was evaluated by ultraviolet lightillumination of ethidium bromide staining of PCRproducts.

RNA extraction and real-time reverse transcrip-tion PCR. Total RNA was isolated from PDA cells orpancreata using Trizol reagent (Life Technologies,Gaithersburg, MD). RNAs were quantified andinput amounts were optimized for each amplicon.OPN and GAPDH (internal control) primers andprobes were designed with the help of PrimerExpress Software (Applied Biosystems, Foster City,CA). cDNA was prepared, diluted, and subjected toreal-time PCR using the TaqMan technology (7500Sequence Detector; Applied Biosystems). The rela-tive mRNA levels were presented as unit values of2^[CT (GAPDH) � CT (OPN)], where CT is the thresh-old cycle value defined as the fractional cycle num-ber at which the target fluorescent signal passes afixed threshold above baseline.

Semiquantitative PCR. RNAs from cells andtissues were quantified, DNase-digested, and cDNAswere prepared using ImProm-II Reverse Transcrip-tion System (Promega, Madison, WI), then sub-jected to semiquantitative PCR using master mix(Promega). The primers used were: OPNc humanforward 59-TCAGGAAAAGCAGAATGCTG-39, re-verse 59-GTCAATGGAGTCCTGGCTGT-39.

Upstream and downstream primers that couldanneal with the 39-untranslated region of humanGAPDH were included in the PCR reaction as aninternal standard forward 59-TGAAGGTCGGAGTCAACGGATTTGGT-39, reverse 59-CATGTGGGCCATGAGGTCCACCAC-39. The linear range of amplifi-cation for each set of primers was determined toensure that we used a number of cycles in the linearrange. PCR products were electrophoresed on 2%agarose gels and band intensities were quantifiedusing Kodak Electrophoresis Documentation andAnalysis System 290 (EDAS 290).

Protein isolation and Western blot analysis. Celllysates were analyzed as described elsewhere. Anti-total OPN and anti--b-actin antibodies werepurchased from Santa Cruz Biotechnology (SantaCruz, CA) and Sigma (St. Louis, MO), respectively.

Human tissue acquisition and analysis. Histo-logically confirmed human invasive PDA (n = 40; 29smokers and 11 nonsmokers), IPMN (n = 6; 2smokers, 4 nonsmokers) were obtained frompatients who underwent operative resection at theThomas Jefferson University Hospital between2005 and 2008. All patients signed an appropriateconsent for tissue acquisition and study. The studywas approved by the Institutional Review Board ofThomas Jefferson University. Tissue samples werestored in RNA Later for RNA analysis. The meanage of the patients at the time of diagnosis was 64years (range, 48--87); 23 were men and 23 werewomen. Patients’ smoking history was extracted

Fig 1. Expression of OPN in PDA cells. (A) Real-time PCR analysis of OPN mRNA transcripts relative to GAPDH in3 PDA cell lines shows that BxPC-3 cells express the highest levels of OPN mRNA transcripts followed by HS766T cells.MiaPaca cells contained minimal levels of OPN. (B) Representative Western immunoblot showing the expression of var-iable levels of OPN protein in PDA cells that is seen at 2 bands at ;65 and ;25 kDa. (C) Representative agarose gel withthe PCR product of PDA cells showing expression of OPNc only in BxPC-3 cells (155- and 109-bp bands correspond withthe amplified OPN and GAPDH, respectively).

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from the clinical notes and correlated with OPN,and OPNc expression levels.

Immunohistochemistry. Paraffin blocks weresectioned at 5 mm stained with a monoclonalantibody against OPN (2A1, Santa Cruz Biotech-nology; 1:100). A vectastain universal elite ABC kitand 3,39-diaminobenzidine tetrahydrochloridechromogenic substrate (Vector Laboratories Inc.,Burlingame, CA) was used to visualize tissue stain-ing. Serial sections were stained with an affinity-purified anti-OPNc chicken IgY (produced byGallus Immunotech from the peptide ac-SEEKQ-NAVSC), at 1:160 dilution, as described previ-ously.16 Antibody specificity was validated withnonimmune isotype serum. Negative control sec-tions, where the primary or secondary antibodieswere omitted, were also prepared.

Enzyme-linked immunosorbent assays. Serumsamples were obtained by venous puncture frominvasive PDA patients who were smokers (n = 29)and nonsmokers (n = 11), and from IPMN patientswho were smokers (n = 2) and nonsmokers (n = 4).OPN protein levels were measured using anenzyme-linked immunosorbent assay kit (R&D,Minneapolis, MN) according to the manufacturer’sinstructions. Spectrophotometric evaluation ofOPN levels was made by Synergy HT multi-detectionMicroplate reader (BioTeck, Winooski, VT).

Statistical analysis. All experiments were per-formed 4--6 times. Data were analyzed for statistical

significance by analysis of variance with a post-hocStudent t test analysis. Data are presented as meanvalues ± standard error of the mean. Continuous,normally distributed variables were analyzed bythe Student t test. The Spearman rank correlationtest was performed to analyze the correlation be-tween OPN, OPNc, and nAchR mRNAs expres-sion. The Fisher exact test or v2 test were alsoused to analyze the distribution of strongly OPN-positive cases and OPNc expression. Analyseswere performed with the assistance of a computerprogram (JMP 5 Software; SAS, Cary, NC). Differ-ences were considered significant at P # .05.

RESULTS

Expression of total OPN and OPNc in PDAcells. Analysis of total OPN levels by real-time PCRrevealed that PDA cells express variable basal levelsof OPN. MiaPaca cells expressed undetectablelevels of OPN mRNA transcripts when comparedwith BxPC-3 cells, which displayed 1.6 times higherlevels of OPN mRNA transcripts than HS766T cells(Fig 1, A). To confirm that the protein expressionlevels match the basal OPN mRNA levels, we ana-lyzed the basal OPN protein expression by westernimmunoblotting. As seen in Figure 1, B, there wasa considerable amount of OPN protein, especiallyin BXpC-3 and HS766T cells. Two bands of OPNprotein at ;65 kDa and ;25 kDa could be

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recognized. Ultraviolet light illumination of ethid-ium-bromide staining of PCR products after aga-rose gel electrophoresis showed a 155-bp bandfor OPNc that was detectable only in BxPC-3 cells(Fig 1, C), but not in HS766T or MiaPaca cells.These data suggest a correlation between high ex-pression levels of OPN and presence of OPNc inPDA cells.

Effect of nicotine on OPN and OPNc expres-sion. Addition of nicotine (3--300 nmol/L) to PDAcells for 3 and 24 hours induced a differentialincrease in total OPN mRNA expression. InMiaPaca cells (Fig 2, A), nicotine induced a dose-dependent significant increase of OPN mRNAat 3 hours. In HS766T cells (Fig 2, B), nicotineinduced a dose- and time-dependent, significantincrease in OPN mRNA. In BxPC-3 cells, nicotinetreatment was associated with a nonsignificanttrend toward increased OPN mRNA expression(Fig 2, C). These data suggest that high basal levelsof OPN mRNA might regulate the synthesis ofadditional OPN in response to nicotine.

Nicotine had no effect on OPNc expression inMiaPaca cells, which lacked the OPNc isoform(data not shown). Interestingly, nicotine (30nmol/L) induced de novo expression of OPNcisoform in Hs667T cells after 24 hours (Fig 3). Nic-otine (3 nmol/L) at 3 and 24 hours increasedOPNc mRNA expression in BxPC-3 cells, whichconstitutively expressed OPNc (Fig 3). These datasuggest that high nicotine concentrationspromote the expression of OPNc isoform in cer-tain PDA cell lines.

Expression of total OPN mRNA in tissue sam-ples of smokers and nonsmokers. Total OPN levelsin IPMN (n = 6; 2 smokers, 4 nonsmokers) andinvasive PDA (n = 40; 29 smokers and 11 non-smokers) were analyzed by real time PCR. RT-PCRrelative quantification values of OPN/GAPDH ofabove 1 indicated high total OPN and was labeled(+++), a value of 0.5 to 1.0 was labeled (++), of 0.1to 0.5 was labeled (+), and of less than 0.1 waslabeled (�; Fig 4, A). In PDA samples taken fromsmokers, more than 70% of the cases expressedhigh (+++) OPN mRNA levels, compared with 35%of the nonsmokers. IPMN lesions, the majority ofwhich were from nonsmokers, expressed minimalamounts of OPN (�). These data suggest that inPDA, the pancreas might be an active source ofOPN and that OPN among the invasive PDA lesionsis expressed significantly more in smokers’ samples(P < .05).

Expression of OPNc mRNA in tissue samples ofsmokers and nonsmokers. OPNc was analyzed byRT-PCR using specific primers and GAPDH as an

internal control. An OPNc band at 155 bp was foundin all (100%) invasive PDA specimens that con-tained high (+++) OPN mRNA (n = 25; 21 smokersand 4 nonsmokers). In the premalignant lesions,OPNc was present in 50% of the smokers; noOPNc could be detected in the nonsmokers. A rep-resentative of these findings is seen in Figure 4, B.OPNc band intensities were labeled (+++) for highintensity, (++) for moderate intensity, (+) for low in-tensity, and (�) for minimal intensity (Fig 4, C).There was a significant (P < .005) correlationbetween OPNc band intensities with total OPNlevels (Fig 4, D). These data suggest that increased

Fig 2. Nicotine induces OPN accumulation in culturedPDA cells. MiaPaca (A) HS766T (B) and BxPC-3 (C) cellswere treated with nicotine (3--300 nmol/L) for 3 and24 hours. Significant induction of OPN mRNA expressionis seen in A and B with the maximum induction after24 hours at 3 and 30 nmol/L of nicotine. BxPC-3 cellsdid not show any significant increase in OPN. Values areexpressed as mean ± standard error of the mean of 3 ex-periments. *P < .05; #P < .005 versus control levels, using1-way repeated analysis of variance with subsequent allpairwise comparison procedure by the Student t test.

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Fig 3. PCR analysis of OPNc mRNA transcripts revealed the presence of no OPN mRNA in control HS766T untreatedcells and small amounts in control untreated BxPC-3 cells. Time response of OPN mRNA in nicotine dose course showsde novo expression of OPNc in HS766T cells at 24 hours at a nicotine concentration of 30 nmol/L. Visible upregulationof OPN mRNA levels in BxPC-3 cells at 3 and 24 hours at nicotine concentration of 3 nmol/L (155- and 109-bp bandscorrespond with the amplified OPN and GAPDH, respectively).

OPN expression in smokers is associated with the ex-pression of OPNc isoform.

Immunohistochemical expression of OPN andOPNc in tissue sections of smokers and non-smokers. As shown in Figure 5, A, in invasive PDAlesions from smokers, intense immunoreactivitiesfor both OPN and OPNc are seen in the malignantducts colocalized to the membrane and cytoplasmof the tumor cells, and in the fibroblasts of thedesmoplastic stroma. In the invasive lesions fromnonsmokers, intense OPN staining still could bedetected in the malignant ducts and surroundingstromal cells. However, evident reduction of OPNcimmunoreactivity could be seen in the invasivelesions from nonsmokers (Fig 5, B). In the IPMNlesions, OPN immunoreactivity was clearly seen inthe transformed ducts (Fig 5, C). No OPNc immu-noreactivity could be detected in IPMN lesionsfrom smokers and nonsmokers (data not shown).These data indicate that an OPN/OPNc generatingsystem is constitutively present in the malignant duc-tal cells as well as in the stroma. The presence of lowexpression levels of OPNc in the invasive lesions ofnonsmokers suggests that it might be regulated bynicotine.

OPN levels in the serum of smokers and non-smokers. To evaluate whether increased tissueOPN and OPNc in invasive PDA from smokers isaccompanied by increased circulating levels ofOPN, an enzyme-linked immunosorbent assay wascarried out in serum samples that matched thetissue we used (Fig 6, A). There was a ;1.5-foldincrease in OPN serum levels in patients with inva-sive PDA when compared with premalignant le-sions from nonsmokers (P < .05). There was nosignificant difference between serum OPN in inva-sive PDA from smokers when compared with

nonsmokers. There was a significant (P < .05) cor-relation between high OPN serum levels (+++) andhigh OPNc tissue levels (+++; Fig 6, B). However,most of the samples that expressed no OPNc(�), especially in the premalignant lesions, still ex-pressed moderate (++) amounts of serum OPN.This suggests that serum OPN does not correlatewith tissue levels of OPN and OPNc. This mightbe due to serum OPN having different sources.Nonetheless, our data suggest that OPN mightnot be an accurate marker for tumor invasiveness.

DISCUSSION

In this study, we investigated the potentialmolecular basis of the role of nicotine as a majorrisk factor in PDA. We show that an OPN isoform,OPNc, which has been shown to support anchor-age independence and metastatic behavior,15,16 isexpressed in high levels in invasive PDA, especiallyin smokers. We demonstrate that the expressionlevels of OPNc correlate significantly with totalOPN levels and the invasive status of PDA.Evidence for the involvement of OPN in tumorprogression, such as growth, metastasis, and angio-genesis, has accumulated in several types of can-cer.19 Until now, however, there have been noreports analyzing the relationship between theexpressions of OPN and OPNc with patient’s smok-ing history as an indication for their regulation bynicotine. Thus, the present study is the first toinvestigate the correlation between OPN andOPNc in invasive and premalignant PDA andpatients’ smoking history.

Our in vitro data show that nicotine increasedOPN mRNA expression, an increase that might beregulated by the basal levels of OPN, becausenicotine could not significantly induce additional

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Fig 5. Representative immunohistochemical staining for OPN and OPNc in invasive PDA and premalignant lesions.Paraffin-embedded pancreatic serial sections were stained with OPN and OPNc antibodies (A) In invasive PDA fromsmokers, OPN and OPNc are colocalized to the membrane and cytoplasm of pancreatic ductal epithelial and stromalcells. (B) In invasive lesions from nonsmokers, less OPNc could be detected in the malignant ducts and surroundingstroma. (C) IPMN lesions from smokers shows OPN immunoreactivity in the transformed ducts. Negative control (-veC) samples where the primary antibody was omitted did not show nonspecific reaction. (Original magnification, 3 200.)

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OPN transcription in BxPC-3 cells (Fig 2, C), whichconstitutively express high levels of OPN. Interest-ingly, nicotine induced de novo expression ofOPNc in HS766T cells (Fig 3). Nicotine also in-creased OPNc expression in BxPC-3 cells. This isthe first report to demonstrate a relationship

between nicotine and the OPNc isoform. Weshowed previously nicotine upregulates OPN pro-moter activity.18 Additional studies are nowrequired to delineate the details of this relation-ship and whether nicotine--OPN promoter activa-tion is related or separated from OPN alternative

Fig 4. (A) Analysis of RT-PCR relative quantification values of OPN/GAPDH of more than 1 indicated high total OPNand was labeled (+++), a value of 0.5 to 1.0 was labeled (++), of 0.1 to 0.5 was labeled (+), and of less than 0.1 was labeled(�). There is significant correlation (P < .05) between high OPN mRNA levels and smoking history in invasive PDA.IPMN lesions from smokers and nonsmokers expressed minimal levels (�) of OPN. (B) Representative of PCR analysisof OPNc mRNA transcripts revealed the presence of considerable amount of OPNc mRNA in invasive PDA, especially insmokers. IPMN lesions from smokers and nonsmokers expressed minimal amounts of OPNc (155- and 109-bp bandscorrespond with the amplified OPNc and GAPDH, respectively). The OPNc mRNA contents are expressed as ODs cor-rected for GAPDH. (C) Representative of PCR analysis of OPNc mRNA. Average densitometry values of the sampleswere multiplied to obtain the arbitrary levels. (+++) = strong band intensities, (++) = moderate band intensities, (+)= mild band intensities, and (�) = no OPNc. (D) Significant correlation (P < .005) between tissue OPN and OPNcmRNAs. High OPNc (+++) was found in 100% of the invasive PDA samples that expressed high OPN (+++). NoOPNc was found in IPMN lesions that expressed very low levels of OPN.

:

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splicing with the resultant expression of OPNc.Furthermore, the effect of the nicotine-mediatedincrease of OPNc on PDA cell behavior and func-tion is the subject of our currently ongoing studiesin the laboratory.

Numerous studies have correlated high levels ofOPN expression with tumor progression and metas-tasis in many cancers, including pancreatic can-cer.9,17 OPN promotes cell survival and facilitatesmetastatic cell behavior through activation of thePI-3 kinase/AKT-nuclear factor-kB pathways20 andmatrix metalloproteinase-2.21 OPN also induces theexpression of vascular endothelial growth factor22

and promotes integrin-mediated endothelial cellmigration.23 In tumor microenvironment macro-phages, OPN downregulates the activity of induciblenitric oxide synthase, leading to protection of tumorcells from the macrophage nitric oxide-mediatedcytotoxicity.24 Alternative splicing has been reportedas one mechanism, by which cancer cells alter thestructure and function of OPN, leading to increased

Fig 6. (A) OPN protein in the serum was measuredusing human-specific enzyme-linked immunosorbentassay kit. Levels of OPN showed significant differenceonly in IPMN lesions from nonsmokers. *P < .05 versusinvasive PDA from nonsmokers. (B) Significant correla-tion (P < .05) between OPNc mRNA and serum OPN.High OPNc (+++) was found in ;70% of the invasivePDA samples that expressed high serum OPN (+++).No OPNc was found in IPMN lesions that expressedmoderate (++) levels of serum OPN.

support of anchorage independence.11 Our analysesreported herein have found that OPN and OPNc areexpressed in the majority (;87%) of invasive PDAcases, out of which 73% were smokers, but not inpremalignant lesions (IPMNs). The levels of OPNccorrelate with smoking history in invasive PDA. Im-munohistochemical analysis of the different lesionsconfirmed our mRNA data (Fig 5). This makesOPNc a candidate marker for the invasive potentialof PDA, which could give rise to novel diagnosticapproaches. It remains to be determined whetherOPN/OPNc levels correlate with pathologic stage,survival, or recurrence. Furthermore, additionalstudies are required to determine whether similarfindings could be obtained from electronic ultra-sound fine needle aspiration samples.

Nevertheless, the presence of OPNc and highOPN levels in the nonsmokers with invasive lesionsopens the door for further questions about thelikelihood that other factors might contribute toelevating OPN/OPNc levels. For example, secondhand smoke exposure could very well have animpact on OPN/OPNc expression levels in thenonsmokers. Additional studies addressing thesepossibilities are currently ongoing in our laboratory.

Several studies have suggested OPN to be acandidate serum marker for PDA. However, morerecent studies have shown that OPN is over ex-pressed in other, nonmalignant conditions.25 Inour studies, although we show a significant correla-tion between serum OPN and tissue OPNc, most ofthe samples that expressed no OPNc expressedmoderate amounts of serum OPN (Fig 6, B).This suggests that there are different sources of se-rum OPN that might affect its value as a diagnos-tic/prognostic marker for invasive PDA.

Our study demonstrates that nicotine elicits aprometastatic response in PDA cells by stimulationof OPNc production. History of cigarette smokingin invasive PDA patients correlated well with in-creased tissue expression levels of OPNc. Althoughthe exact role of OPNc in PDA remains to bedefined, the existence of OPNc as a downstreameffector of nicotine that is capable of mediating itscarcinogenic effects in PDA cells is novel and couldprovide a unique potential target to control pan-creatic cancer aggressiveness, especially in thecigarette smoking population.

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