AGR2 Is a Novel Surface Antigen That ... - Cancer...

Tumor and Stem Cell Biology

AGR2 Is a Novel Surface Antigen That Promotes theDissemination of Pancreatic Cancer Cells throughRegulation of Cathepsins B and D

Laurent Dumartin1, Hannah J. Whiteman1, Mark E. Weeks5, Deepak Hariharan1, Branko Dmitrovic6,Christine A. Iacobuzio-Donahue7, Teresa A. Brentnall8, Mary P. Bronner9, Roger M. Feakins3,John F. Timms4, Caroline Brennan2, Nicholas R. Lemoine1, and Tatjana Crnogorac-Jurcevic1

AbstractPancreatic ductal adenocarcinoma (PDAC) remains one of themost lethal cancers largely due to disseminated

disease at the time of presentation. Here, we investigated the role and mechanism of action of the metastasis-associated protein anterior gradient 2 (AGR2) in the pathogenesis of pancreatic cancer. AGR2 was induced in allsporadic and familial pancreatic intraepithelial precursor lesions (PanIN), PDACs, circulating tumor cells, andmetastases studied. Confocal microscopy and flow cytometric analyses indicated that AGR2 localized to theendoplasmic reticulum (ER) and the external surface of tumor cells. Furthermore, induction of AGR2 in tumorcells regulated the expression of several ER chaperones (PDI, CALU, RCN1), proteins of the ubiquitin-proteasomedegradation pathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4), and lysosomal proteases [cathepsin B (CTSB)and cathepsin D (CTSD)], in addition to promoting the secretion of the precursor form pro-CTSD. Importantly,the invasiveness of pancreatic cancer cells was proportional to the level of AGR2 expression. Functionaldownstream targets of the proinvasive activity of AGR2 included CTSB and CTSD in vitro, and AGR2, CTSB,and CTSD were essential for the dissemination of pancreatic cancer cells in vivo. Taken together, the resultssuggest that AGR2 promotes dissemination of pancreatic cancer and that its cell surface targeting may permitnew strategies for early detection as well as therapeutic management. Cancer Res; 71(22); 7091–102.�2011 AACR.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is almost invari-ably lethal and remains one of the most devastating cancers inman (1). Most deaths from pancreatic cancer are due to thesilent and aggressive nature of this malignant disease. Atpresentation, the disease has, in most cases, already spreadlocally and to distant organs, and patients usually succumbwithin 3 to 6 months. Moreover, even after curative resection,

the vast majority of patients relapse due to undetected dis-seminated tumor cells (DTC) that have spread prior to primarytumor diagnosis. A better understanding of the molecularmechanisms that promote dissemination of cancer cells is,therefore, essential for the development of novel detection andtherapeutic strategies for this, at present, largely incurablemalignant disease.

Anterior gradient 2 (AGR2) protein is upregulated in mul-tiple cancers, including breast (2, 3), lung (4), ovarian (5),esophageal (6), and prostate cancers (7), and is associatedwith a metastatic phenotype and poor prognosis (2). It hasbeen identified previously by gene profiling as a marker forDTC detection (8). In sporadic pancreatic cancer, AGR2 pro-tein is present from the earliest precursor lesions [pancreaticintraepithelial precursor lesions (PanIN1)] to PDACs but is notexpressed in normal pancreas (9). Recent data also suggestedthat silencing of AGR2 in the pancreatic cancer cell lineMPanc-96 results in fewermetastases in an orthotopic pancreas tumormodel (10).

AGR2 has been shown to have structural characteristics ofthe protein disulfide isomerase (PDI) family, including a car-boxy-terminal endoplasmic reticulum (ER) retention signalKTEL and a single thioredoxin-like domain with a CXXS motif(11). PDI proteins catalyze formation, reduction, and isomer-ization of disulfide bonds, thereby facilitating the maturationof proteins in the ER and ensure correct folding and multi-merization of proteins targeted for the secretory pathway (11).

Authors' Affiliations: 1Centre for Molecular Oncology, Barts Cancer Insti-tute, 2School of Biological and Chemical Sciences, Queen Mary Universityof London; 3Department of Histopathology, Royal London Hospital; 4Can-cer Proteomics Laboratory, EGA Institute for Women's Health, UniversityCollege London, London, United Kingdom; 5Proteomics, Metabolomicsand Bioimaging, AHVLA-Weybridge, Addlestone, United Kingdom;6Department of Pathology and Forensic Medicine, KBC Osijek, Osijek,Croatia; 7Department of Pathology, The Sol Goldman Pancreatic CancerResearch Center, Johns Hopkins Medical Institutions, Baltimore, Mary-land; 8Division of Gastroenterology, Department of Medicine, University ofWashington, Seattle, Washington; and 9Department of Anatomic Pathol-ogy, University of Utah, Salt Lake City, Utah

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Tatjana Crnogorac-Jurcevic, Barts Cancer Insti-tute, Centre for Molecular Oncology, Queen Mary University of London,John Vane Science Centre, Charterhouse Square, London EC1M 6BQ,United Kingdom. Phone: 44-0-20-7882-3554; Fax: 44-0-20-7882-3884;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1367

�2011 American Association for Cancer Research.

CancerResearch

www.aacrjournals.org 7091

Research. on October 30, 2020. © 2011 American Association for Cancercancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 26, 2011; DOI: 10.1158/0008-5472.CAN-11-1367

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Park and colleagues have recently shown that AGR2 loca-lizes in the ER of normal intestinal epithelial cells and isessential for in vivo production of protective mucus. Theyshowed that AGR2mediates processing of the intestinal mucinMUC2 through formation ofmixeddisulfidebonds and that theabsence of AGR2 resulted in a dramatic reduction of mucusproduction and secretion and an increased sensitivity to colitisin Agr2�/� mice (12). The precise molecular effects of AGR2 incancers, however, remain largely unknown.

Here, we report that AGR2 is almost universally expressed intumor cells of patients with pancreatic cancer in both sporadicand familial settings and that it localizes both in the ER and atthe external surface of the plasma membrane. We revealproteomic changes resulting from the induction of AGR2expression and show that AGR2 promotes in vitro and in vivodissemination of cancer cells through posttranscriptionalinduction of 2 proteases, cathepsin B (CTSB) and cathepsinD (CTSD).

Materials and Methods

Tissues and cell linesThree tissue arrays comprising 42 normal, 48 PanIN, and

84 PDAC cores from both familial and sporadic PDAC cases(University of Washington, Seattle, Washington); 8 primaryPDACs and matched infiltrated lymph nodes (Department ofPathology, KBC Osijek); and 10 cases of primary PDAC and 9matched liver and 1 lung metastases (GICRMDP, JohnHopkins University) were analyzed. Thirty cases of peri-neural invasion found within the PDAC tissues were alsoexamined. All specimens were obtained with full ethicalapproval from the host institutions.

The human pancreatic ductal epithelial (HPDE) cell line wasobtained from Dr. Ming-Sound Tsao, University of Toronto,Toronto, ON, Canada, and grown as described previously (13).Other cell lines, verified by short tandem repeat profiling(February 2010) were obtained from Cancer Research UK CellServices (Clare Hall, Middlesex, UK) and cultured in Dulbecco'sModified Eagle Medium (DMEM; Invitrogen) supplementedwith 10% heat-inactivated fetal calf serum (Autogen Bioclear).

Establishment of stable cell linesThe pCEP4 AGR2 vector was constructed by excising AGR2

frompCMV-SPORT6-AGR2 (MRCGeneservice) usingKpnI andNotI. AGR2 cDNA was cloned into the KpnI/NotI site of pCEP4(Invitrogen). MiaPaCa2 cells (2 � 106 cells per 10-cm plate)were transfected with FuGENE6 (Roche Diagnostics) in a 3:1ratio with pCEP4 AGR2 or pCEP4. Stable cell lines wereestablished following selection with 300 mg/mL hygromycinB (Merck) and single-cell clones isolated.

Gene silencingCells were seeded at 2 � 105 cells per well in a 6-well plate

and transfected with 1, 5, 10, or 50 nmol/L of siGENOME ON-TARGETplus SMARTpool siRNA specific for human AGR2,CTSB, or CTSD gene or siGENOME Non-Targeting siRNA pool#2 (Dharmacon) using INTERFERin (PeqLab) according tomanufacturer's instructions.

RNA extraction and semiquantitative real-time PCRTotal RNA was extracted using RNAqueous RNA extraction

kit (Ambion). First-strand cDNA was prepared from 1 mg oftotal RNA with Quantitect Reverse Transcription Kit (Qiagen).

Real-time PCR was carried out on a 7500 Real-Time PCRsystem (Applied Biosystems) using SYBR Green dye (ThermoFisher Scientific). The primers used were S16, forward 50

GTCACGTGGCCCAGATTTAT 30 and reverse 50 TCTCCTTCT-TGGAAGCCTCA 30; CTSB, forward 50 CACTGACTGGGGTGA-CAATG 30 and reverse 50 GCCACCACTTCTGATTCGAT 30; andCTSD, forward 50 GCGAGTACATGATCCCCTGT 30 and reverse50 CTCTGGGGACAGCTTGTAGC 30. All samples were tested in3 independent experiments. Relative changes of expressionwere expressed after normalization to the human ribosomalS16 gene.

Western blottingCell lysis was done using NP40 buffer (1% NP40, 50 mmol/L

Tris, pH 7.4, 150mmol/L NaCl) with protease inhibitors (RocheDiagnostics). For secretome analyses, cells were serum starvedfor 16 hours and culture supernatants centrifuged at 5,000 rpmfor 15 minutes at 4�C. Secretome samples were concentratedusing Amicon Ultra Centrifugal filters Ultracel 3 kDa (Milli-pore). Twenty-five micrograms of protein lysate or 5 mg ofsecretome proteins were analyzed by SDS-PAGE as previouslydescribed (14). Primary antibodies were rabbit anti-AGR2(1:250; Abcam), goat anti-actin (1:2,000; Santa Cruz Biotech-nology), mouse anti-CTSD (1:5,000) and rabbit anti-CTSB(1:1,000; Abcam).

ImmunofluorescenceCells were seeded on coverslips (5 � 104 per well in 24-well

plate) and cultured for 48 hours. After fixing in 4% parafor-maldehyde, permeabilization with 0.1% Triton X, and blockingin 2% bovine serum albumin (BSA), cells were incubated withmouse anti-AGR2 (1:500; Santa Cruz Biotechnology), rabbitanti-giantin (1:1,000), rabbit anti-calreticulin (1:200), and rab-bit anti-LAMP1 (1:100; Abcam). Secondary antibodies wereAlexa Fluor 568/488-conjugated anti-mouse or anti-rabbit IgG(1:2,000; Invitrogen). DNA was stained with 50 mg/mL 40,6-diamidino-2-phenylindole (DAPI; Invitrogen), and imagingdone with LSM 710 confocal microscope (Zeiss).

ImmunohistochemistryStaining was done on 4-mm thick paraffin sections using

rabbit anti-AGR2 antibody (Abcam) diluted 1:30 with DABMapkit, following protocols for the Ventana Discovery System.Counterstaining was done with hematoxylin. The intensity ofimmunoreactivity was graded on a scale from 0 to 3 and theextent according to the percentage of stained cells (0 points forno staining, 1 point, <20%; 2 points, 20%–50%; and 3 points,>50%). The total scorewas the product of intensity and extent ofstaining. Negative or weakly positive cases scored 0 to 3, mod-erately positive as 4 to 6, and strongly positive as more than 6.

Flow cytometrySubconfluent cells were harvested by trypsin/EDTA (0.25%

w:v, 5mmol/L) and resuspended in DMEM, 0.1% BSA, and 0.1%

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Cancer Res; 71(22) November 15, 2011 Cancer Research7092




sodium azide. AGR2 was detected with rabbit antibody (SantaCruz Biotechnology; 1:10) on ice for 45 minutes. Bound anti-bodies were detected with Alexa Fluor 488–conjugated sec-ondary antibody (Invitrogen). Labeled cells were scanned on aBD FACSAria II Cell sorter (BD Biosciences) and analyzedusing CellQuest Pro software.

Functional assaysMTT, invasion, and wound-healing assays were conducted

as described previously (14). For migration assays, Biocoat CellCulture Inserts with 8-mm pores (BD Biosciences) were used.Seven hundred and fifty microliters of DMEM media supple-mented with 10% fetal calf serum was added to the lowerchamber and 2.5 � 104 cells in 500 mL serum-free medium tothe upper chamber. The assays were set up 48 hours aftertransfection and cells were incubated for 24 hours. Cells thatmoved through the pores were fixed in 100% methanol andstained with 1% Giemsa Blue (Sigma-Aldrich) before counting.All functional assays were carried out in triplicate in at least 3individual experiments.

Protein expression profiling by 2D-DIGE/MSMiaPaCa2-pCEP4 and pCEP4 AGR2 were lysed and labeled

in triplicate with either NHS-Cy3 or NHS-Cy5 dyes and run infirst and second dimension as described previously (14). Intotal, 6 gels were run generating 18 images that were exportedinto DeCyder software v5.0 (GE Healthcare). Spots displaying agreater than 1.4 average-fold increase or decrease in abun-

dance,matching across all images, and having values ofP< 0.05(Student t test) were selected. Spot picking, tryptic digestion,and protein identification using liquid chromatography/tan-dem mass spectrometry (LC/MS-MS) were done as describedpreviously (15). Nano-HPLC-electrospray ionization-collision-induced dissociation MS/MS was done on an Ultimate HPLCwith a PepMap C18 75 mm inner diameter column (bothDionex) at a flow rate of 300 nL/min, coupled to a Q-TOF1mass spectrometer (Micromass). Spectrawere processed usingMassLynx (Micromass) software and submitted to Mascotdatabase search routines against the Human IPI database.Positive identifications were made when at least 3 peptidesequences matched an entry and MOWSE scores were abovethe significance threshold value (P ¼ 0.05).

Zebrafish embryo xenograft modelZebrafish (Danio rerio) were handled in compliance with

local animal care regulations and standard protocols. Fishwere kept at 28�C in aquaria with day/night light cycles(10-hour dark/14-hour light periods). The developing embryoswere kept in an incubator at constant temperature.

Cancer cells in suspension were stained with 10 mmol/LCMTMR or CMFDA (Invitrogen) and resuspended in serum-free medium before injecting into 48-hour-old zebrafishembryos. Injections were done using a manual injector (Picos-pritzer III, Parker Hannifin Instruments). Embryos weredechorionated and anesthetized with tricaine (Sigma-Aldrich)prior to injection. After injections, embryos were incubated at

Figure 1. AGR2protein expressionin pancreatic tissues.Immunohistochemical analysis ofAGR2 expression in normalpancreas (A), familial PanIN1,PanIN2, PanIN3, and PDAC (B–E,respectively); perineural invasion,circulating tumor cells, lymph node,and liver metastasis from sporadicpancreatic cancer samples (F–I,respectively). Scale bars, 50 mm.

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35�C. Three separate experiments were carried out for eachprotein of interest. In total, 103 embryos were injected forAGR2 experiments, 109 for CTSB, and 107 for CTSD.

Counting of disseminated cells was done 24 hours afterinjections; embryos were assessed using a Zeiss Axioplanepifluorescence microscope and disseminated cells countedunder high magnification.

Statistical analysisThe statistical analysis was done with the Student t test

using Prism software; a value of P < 0.05 was statisticallysignificant.

Results

AGR2 expression is induced from the earliest lesions ofpancreatic neoplasia and is retained in all DTCs

Immunohistochemistry confirmed that AGR2 is notexpressed in normal pancreas (Fig. 1A) but is induced in allsporadic PanIN lesions. Here, we additionally show that AGR2is similarly expressed in familial cases; representative images offamilial PanIN1–3 lesions are shown on Fig. 1B–D. High levelsof AGR2 expression were seen in almost all PDAC specimens(73 of 84, 87%; Fig. 1E) except the rare cases with squamoiddifferentiation, which were negative (Supplementary Fig. S1,AI), and undifferentiated PDACs,which showed low expression(data not shown). AGR2 expression was retained in perineuralinvasion (Fig. 1F), in circulating tumor cells (Fig. 1G) and all

metastatic samples, namely, lymph node, liver (Fig. 1H and I),and lungmetastases (Supplementary Fig. S1, AII). The data aresummarized in Table 1. The immunochemistry thereforerevealed that AGR2 is expressed in almost all in situ ordisseminated cancer cells, suggesting that itmay be implicatedin all steps of PDAC development and spreading. As expected,due to the almost invariable expression of AGR2 across thecases tested, there was no correlation with any of the clinico-pathologic data. Furthermore, using a multiorgan tissue array,we have seen that AGR2 is expressed in a limited number ofnormal human tissues (Supplementary Fig. S1 B and Supple-mentary Table S1). Of note, most adenocarcinomas of variousorgans show AGR2 expression, in contrast to squamous cellcarcinomas.

AGR2 is an endoplasmic reticulum and a cell surfaceantigen

Both cytoplasmic and membranous immunoreactivity forAGR2 were noted in PDAC cells (Fig. 2A). The precise subcel-lular localization of AGR2 was further explored in vitro usingconfocal microscopy. Immunofluorescent staining in permea-bilized PaTu 8988s pancreatic cancer cells, which express highlevels of AGR2 (Fig. 2B), showed that AGR2 is predominantlylocalized in a fine reticular network around the nucleus (Fig.2C). The extensive colocalization with calreticulin confirmedthat AGR2 is localized in the ER in PDAC cells. However, AGR2was not seen in the Golgi apparatus and lysosomes (Supple-mentary Fig. S2A). Immunostaining of 3 nonpermeabilized

Table 1. Summary of immunohistochemical findings in human pancreatic tissues and pancreatic cancermetastases

Specimens AGR2-positivecores/total analyzed

Expression level Positivecases, %

0 1–3 4–6 7–9

Normal 0/42 42 0 0 0 0PanIN1All cases 17/17 0 4 8 5 100Sporadic 7/7 0 2 4 1Familial 10/10 0 2 4 4

PanIN2All cases 23/23 0 0 13 10 100Sporadic 14/14 0 0 7 7Familial 9/9 0 0 6 3

PanIN3All cases 8/8 0 0 1 7 100Sporadic 4/4 0 0 1 3Familial 4/4 0 0 0 4

PDACAll cases 73/84 11 15 26 32 87Sporadic 70/81 11 15 23 32Familial 3/3 0 0 3 0

Perineural invasion 30/30 0 0 0 30 100Lymph node metastasis 8/8 0 0 0 8 100Liver metastasis 9/9 0 0 0 9 100Lung metastasis 1/1 0 0 0 1 100

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Figure 2. Localization of AGR2 in pancreatic cancer cells. A, immunohistochemical analysis of AGR2 in a representative human PDAC sample showing bothcytoplasmic and membranous (arrows) immunoreactivity. B, Western blot analysis showing AGR2 expression in a panel of pancreatic cancer and normalHPDE cells. C, coimmunostaining of AGR2 (green) and actin or calreticulin (red) in permeabilized PaTu 8988s cells showing that AGR2 is situated in theendoplasmic reticulum. D, cell surface immunostaining for AGR2 (green) on nonpermeabilized PaTu 8988s cells. Scale bars, 10 mm. E, i, flow cytometricanalysis of AGR2 cell surface expression on intact pancreatic cells in the sameorder (as in D) and (ii) sorting of AGR2-positive–gated cells.MiaPaCa2 cells thatdo not endogenously express AGR2 were used as a negative control.






AGR2-expressing cell lines, PaTu 8988s, FA6, and CFPAC1 (Fig.2D), indicated that AGR2 also localized at the external surfaceof the plasma membrane. This finding was further confirmedon nonpermeabilized cells by flow cytometry [Fig. 2E (i)],which also permitted the physical isolation of cell surfaceAGR2-expressing cells [Fig. 2E (ii)]. This was not observed inthe MiaPaCa2 cell line that does not express AGR2 endoge-nously and thus served as a negative control. Of note, similarsurface expression was also seen on breast cancer cells (Sup-plementary Fig. S2B), indicating that AGR2 also might be a cellsurface antigen in other tumor types. Furthermore, Westernblotting (Supplementary Fig. S2C) showed the presence ofAGR2 in culture supernatants of PaTu 8988s and CFPAC1 butnot in FA6 or BxPC3 that also expresses AGR2 (Fig. 2B, D, andE), suggesting that AGR2 might be shed from the cell surfacerather than being actively secreted.

Taken together, our data indicate that in pancreatic tumorcells, the PDI protein AGR2 not only has a conserved ERlocalization but also is a cell surface marker.

AGR2 regulates the invasiveness of PDAC cancer cellsTo assess the functional roles of AGR2 in pancreatic cells,

long-term stable expression of AGR2 was achieved in Mia-PaCa2 cells, an undifferentiated pancreatic cancer cell line.AGR2 expression was confirmed byWestern blot inMiaPaCa2-pCEP4 AGR2 cells compared with cells transfected with emptycontrol vector [pCEP4; Fig. 3A (i)]. The recombinant AGR2protein had the same subcellular localization as endogenousAGR2, as it was detected in the ER, at the cell surface, and in theculture supernatant of pCEP4 AGR2–transfected cells (Sup-plementary Fig. S3A–S3C, respectively). In parallel, a complete

loss of AGR2 protein expression was seen in FA6 cells (whichexpress high endogenous levels of AGR2) 48 hours after trans-fection with 50 nmol/L of AGR2-specific siRNA [Fig. 3A (ii)]. Nomorphologic change was observed after alteration of AGR2expression in these cells (Supplementary Fig. S4A). Moreover,no differencewas observed in cell proliferation, wound healing,or migration (Supplementary Fig. S4B–S4D, respectively), fol-lowing overexpression or knockdown of AGR2. However, ininvasion assays, a significant increase (P < 0.05) in the numberof invading cells was observed in AGR2-expressing MiaPaCa2cells, compared with vector-only controls [Fig. 3B (i)], and asignificant decrease (P < 0.05) in the number of invading cellswas seen following siRNA-mediated knockdown of AGR2 inFA6 cells [Fig. 3B (ii)]. The invasive capabilities of pancreaticcancer cells are therefore proportional to levels of AGR2expression.

AGR2 induces changes in cancer cell proteomeTo assess the molecular mechanisms underlying the proin-

vasive activity of AGR2, proteomic analysis of AGR2-expressingMiaPaCa2 versus vector control–transfected cells was doneusing 2-dimensional difference in gel electrophoresis (2D-DIGE). A representative gel is shown in Fig. 4. We identified15 upregulated and 24 downregulated proteins (represented by15 and 36 spots, respectively) with a cutoff of 1.4-fold at P < 0.05(Table 2).

Two ER chaperones, CALU and RCN1, were identified as thehighest upregulated proteins (both 2-fold), in addition to AGR2(8.5-fold upregulated). The archetypal cellular ER PDI, PDI/P4HB, was also upregulated, in addition to deregulation ofseveral proteins of the ubiquitin-proteasome degradationpathway (HIP2, PSMB2, PSMA3, PSMC3, and PSMB4) thatplay a role in ER-associated degradation of improperly pro-cessed proteins. The expression levels of a number of structuralproteins (LMNA, VIM, KRT1, KRT8, KRT18, and KRT19) werealso altered. The functional interactions among themajority ofthese differentially expressed proteins mapped in a networkare shown on Supplementary Fig. S5.

AGR2 is a posttranscriptional regulator of CTSB andCTSD

Interestingly, the proteases CTSB and CTSD were bothupregulated (1.79- and 1.52-fold, respectively) in AGR2-expres-sing cells. As CTSB and CTSD are known to be frequentlyoverexpressed and hypersecreted in several cancers, includingpancreatic (16, 17) cancer, they were selected for furtheranalyses.

CTSB/D overexpression was confirmed by Western blotting[Fig. 5A (i); Fig. 5A (ii) shows densitometry quantification],whereas no change was detected at the mRNA level using real-time PCR [Fig. 5A (iii)]. A large increase in the expression of theprecursor isoforms, pro-CTSB and CTSD, in AGR2-expressingcells was also observed. This was further confirmed in addi-tional pancreatic cells, in AGR2-expressing FA6 cells whencompared with normal HPDE cells [Fig. 5B (i) and (iii)] and inPaTu 8988s compared with PaTu 8988t cells, which originatefrom livermetastases of the same patient (18). Metastatic PaTu8988s cells express AGR2 and higher levels of CTSB/CTSD

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Figure 3. Functional roles of AGR2. A, Western blot analysis of AGR2expression in (i) MiaPaCa2 cells stably transfected with empty vectorcontrol (pCEP) or pCEP-AGR2 vector and (ii) in FA6 cells 48 hours aftertransfection with specific AGR2 siRNA or nontargeting (NT) controlsiRNA. Actin was used as a loading control. B, invasion assays using (i)stably transfected MiaPaCa2 cells and (ii) siRNA-transfected FA6 cells.Mean values of triplicate experiments are shown. �, P < 0.05.

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isoforms than nonmetastatic PaTu 8988t cells that do notexpress AGR2 [Fig. 5B (ii) and (iv)]. These results indicate thatAGR2 may be an important posttranscriptional regulator ofCTSB and CTSD in PDAC cells.As overexpression of CTSB and CTSD is often associated

with an increase in their secretion, the culture supernatants ofPDAC cells were analyzed: higher levels of pro-CTSD weredetected in the supernatants of AGR2-expressing MiaPaCa2than in vector-only transfected cells, and inversely, silencing ofAGR2 in FA6 cells strongly inhibited pro-CTSD secretion (Fig.5C). AGR2 is thus an important regulator of pro-CTSD secre-tion.Wewere not able to detect proCTSBorCTSB/CTSD in thesupernatants, which might be due to their binding to theplasma membrane rather than being secreted (19).

CTSB and CTSD are involved in AGR2-mediateddissemination of pancreatic cancer cellsTo examine whether cathepsins are involved in the AGR2-

induced increase in cell invasion, CTSB and CTSD weresilenced inAGR2-expressingMiaPaCa2 cells [Fig. 5D (i)]. About50% (P < 0.005) and 80% (P < 0.001) reduction in the number ofinvading cells was observed after silencing of CTSB and CTSD,respectively [Fig. 5D (ii)], thus reducing the invasiveness ofMiaPaCa2-pCEP4 AGR2 cells close to the level observed incontrol MiaPaCa2-pCEP4. The same dramatic effect wasobserved in FA6 cells after silencing of CTSB (P < 0.05) andCTSD [P < 0.001; Fig. 5D (iii) and (iv)]. AGR2 thus regulates theexpression, and potentially secretion, of CTSB and CTSD,which in turn mediate the AGR2-induced invasiveness ofpancreatic cancer cells.To analyze whether AGR2, CTSB, and CTSD are important

regulators of the dissemination of pancreatic cancer cellsin vivo, a zebrafish embryo xenotransplant model was used.PaTu 8988s cells were stained in vitro with red or greenfluorescent dyes and were coinjected into the yolk sack of

48-hour-old zebrafish embryos (Fig. 6A). After 24 hours, bothred and green tumor cells were equally observed as dissem-inated into the tail of the embryo [Fig. 6B (i) and (iii)] orlocalized in the vicinity of the injection site in the yolk sack[Fig. 6B (ii) and (iv)]. Silencing of AGR2 in PaTu 8988s cellssignificantly lowered the number of DTC than siRNA-control–transfected cells (P < 0.001; Fig. 6C andD). A similar decrease ofDTC was observed after silencing of CTSB (P < 0.001) or CTSD(P < 0.001; Fig. 6E and F, respectively). These results show thatall 3 proteins are major regulators of the in vivo ability of PDACcells to disseminate.

Discussion

AGR2 expression has previously been shown to be inducedfrom the earliest precursor lesions of pancreatic cancer, PanINs,aswell as in PDACs (9, 10, 20). Here, weprovide a comprehensiveanalysis of AGR2 expression in both pancreas and extrapan-creatic tissues, showing that it is widely expressed in bothsporadic and familial PanINs, PDACs, and metastatic lesions.In contrast, AGR2 is not widely expressed in normal organs, inwhich it is mostly confined to mucin-secreting cells (21).

We show that in PDAC cells, AGR2 localizes to the ER, asobserved in normal intestinal epithelial cells (12), suggestingthat AGR2 could exert a PDI activity on presecretory proteinsalso in transformed cells. Furthermore, we provide the firstevidence that AGR2 also localizes to the external surface ofAGR2-expressing pancreatic cancer cells; accumulation at thecell surface has previously been shown for several ER proteins(22, 23). Therefore, AGR2 may be used for the detection ofcirculating tumor cells in the peripheral blood of patients withpancreatic cancer and could also be a novel tumor cell surfaceantigen for the development of antibody-targeting strategies(24). An increasing number of recent studies report that PDIproteins have important roles on the cell surface, asmajority of

Figure 4. Proteomic profiling ofMiaPaCa2 AGR2-expressing cells.A, Representative gel imagedisplaying protein spots withAGR2-dependent changes inexpression is shown. Magnifiedregions showing severaldifferentially expressed proteins.CALU, calumenin; RCN1,reticulocalbin 1.

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753

969

109511461105

124511821262

13021318

1455

1009

360

590

878

569 581592

210kDa

753

P4HB

2394

2459

2473

2428

10kDa

24592099

2119

pCEP4pCEP4 AGR2

1410

1455

CALU and RCN1

AGR2CTSB and CTSD






surface proteins contain disulfide bonds (reviewed inrefs. 23, 25) in which they can modulate the activity of mem-brane receptors (and thus activate and regulate signalingpathways; ref. 26), adhesion molecules integrins (27), or evenproteases such as ADAM17 (28). We and others show thatAGR2 can also be found in cell culture supernatants (10) andpancreatic juice (29). It remains to be established whetherAGR2 functions also at the cell surface or when secreted andwhether it is immunogenic as shown for PDI proteins in renalcell carcinoma (30).

Protein profiling of AGR2-expressing MiaPaCa2 cells iden-tified upregulation of several ER chaperones (PDI, CALU, and

RCN1), suggesting activation of the ER stress response. Thiscorrelates with a previous report on Agr2�/�mice that showedthat Agr2 is involved in the ER stress response as well as beingitself induced by ER stress (31). Stressful conditions such ashypoxia, nutrient deprivation, and pH changes encountered bytumor cells are known to induce ER stress (32), characterizedby the upregulation of ER chaperones. This enables cells toadapt to an unfavorable microenvironment and avoid ERstress–induced apoptosis. The main cellular PDI, PDI/P4HB,in which upregulation was seen here, is a well-establishedexecutor of the ER stress response and has been shown toprotect melanoma cancer cells against ER stress–induced

Table 2. Deregulated proteins identified by 2D-DIGE analysis comparing MiaPaCa2-pCEP AGR2 withMiaPaCa2-pCEP cells

Spot ID Name Symbol Fold change

2459 IPI00007427 Anterior gradient 2 AGR2 8.551410 IPI00789155 Calumenin CALU 2.071455 IPI00015842 Reticulocalbin 1 RCN1 2.032144 IPI00176455 Synovial sarcoma, X breakpoint 9 SSX9 1.832119 IPI00011229 Cathepsin D CTSD 1.791285 IPI00009634 Sulfide quinone reductase-like SQRDL 1.711135 IPI00418471 Vimentin VIM 1.692428 IPI00375676 Ferritin, light polypeptide FTL 1.681562 IPI00220685 Heterogeneous nuclear ribonucleoprotein D HNRPD 1.661293 IPI00784347 Keratin 18 KRT18 1.552310 IPI00021370 Huntingtin interacting protein 2 HIP2 1.552099 IPI00295741 Cathepsin B CTSB 1.52753 IPI00010796 Proline 4-hydroxylase, b polypeptide PDI/P4HB 1.502473 IPI00175923 Synovial sarcoma, X breakpoint 6 SSX6 1.50878 IPI00219049 Trans-golgi network protein 2 TGOLN2 �1.471302 IPI00550488 Transaldolase 1 TALDO1 �1.471033 IPI00298423 Pyruvate dehydrogenase complex, component X PDHX �1.491108 IPI00019376 Septin 11 SEPT11 �1.492176 IPI00216318 Tyrosine 3-/tryptophan5-monooxygenase activation protein b YWHAB �1.492394 IPI00028006 Proteasome subunit b type 2 PSMB2 �1.50985 IPI00337370 Pre-B-cell colony enhancing factor 1 PBEF1 �1.511631 IPI00014873 Cyclin-dependent kinase 10 CDK10 �1.532160 IPI00171199 Proteasome subunit a type 3 PSMA3 �1.532186 IPI00003815 Rho GDP dissociation inhibitor (GDI) a ARHGDIA �1.54875 IPI00795040 Heat shock 70kDa protein 8 HSPA8 �1.551216 IPI00465248 Enolase 1 ENO1 �1.551966 IPI00021700 Proliferating cell nuclear antigen PCNA �1.561969 IPI00329801 Annexin A5 ANXA5 �1.561123 IPI00018398 Proteasome 26S subunit, ATPase, 3 PSMC3 �1.571413 IPI00479145 Keratin 19 KRT19 �1.581654 IPI00220327 Keratin 1 KRT1 �1.612241 IPI00220301 Peroxiredoxin 6 PRDX6 �1.611753 IPI00455315 Annexin A2 ANXA2 �1.661095 IPI00554648 Keratin 8 KRT8 �1.671281 IPI00337494 Solute carrier family 25, member 24 SLC25A24 �1.70590 IPI00514204 Lamin A/C LMNA �1.712045 IPI00296913 Nucleoside diphosphate linked moiety X-type motif 5 NUDT5 �1.752295 IPI00555956 Proteasome subunit b type 4 PSMB4 �1.791182 IPI00418471 Vimentin VIM �1.93

Dumartin et al.





cell death (33). CALU and RCN1 are EF-hand members of theCREC protein family (34) which is associated with variousCa2þ-dependent processes in the secretory pathway; in the ER,these proteins interact with the protein translocase (35) thatguides the transport of nascent presecretory proteins into theER lumen. The regulation of these 2 ER chaperones by AGR2could thus directly affect one of the very first steps in theprotein secretion process (35).Interestingly, Denoyelle and colleagues (36) showed that the

induction of ER stress chaperones is an early event in theinitiation of melanoma. Similarly, the universal AGR2 expres-sion in all precursor lesions in both sporadic and familial

settings suggests that activation of ER stress response couldbe one of the earliest events in PDAC development and couldprovide a survival advantage to premalignant cells. Although inthe present study, we report on the consequences of AGR2induction in the pancreas, the nature of the initial trigger thatcauses early AGR2 induction is not known and currently underinvestigation as it could potentially lead to a better under-standing of pancreatic cancer initiation.

The expression of PDI proteins and ER chaperones has alsobeen correlated with cancer invasion andmetastasis in severaltumor types (33, 37), and we here provide similar evidence forthe role of AGR2 in PDAC.

Ai

kDa

52 Pre-pro CTSD

pC

EP

4

pC

EP

4 A

GR

2

HP

DE

PaT

u 8

988t

PaT

u 8

988s

FA6

pCEP4pCEP4 AGR2

pro CTSDpro-CTSD

Rel

ativ

e ex

pres

sion

(den

sito

met

ry)

Rel

ativ

e ex

pres

sion

(den

sito

met

ry)

Rel

ativ

e ex

pres

sion

(den

sito

met

ry)

Inva

ding

cel

ls (

% c

trl)

Inva

ding

cel

ls (

% c

trl)

Tran

scrip

t rat

io(p

CE

P4

AG

R2/

pCE

P4)

10

6

2

3

2

1

0

21.4

1

0CTSD

CTSB CTSD

pro CTSB CTSBpro CTSD CTSD pro CTSB CTSB

pro CTSD

siNT siCTSB siCTSD

siNT siCTSB siCTSD

CTSD pro CTSB CTSB

P = 0.0007

P = 0.0027

P = 0.0018

P = 0.015

pro CTSB

CTSD

CTSB

AGR2

Actin

MiaPaCa2 MiaPaCa2-pCEP4 AGR2

pro-CTSDSupernatant

pro

-CT

SD

leve

l(d

ensi

tom

etry

)

pro

-CT

SD

leve

l(d

ensi

tom

etry

)

pCEP4 siNT siAGR2

10,000

8,000

6,000

4,000

2,000

0

8,000

6,000

4,000

2,000

0

150

100

50

0

150

100

50

0pCEP4 AGR2

pC

EP

4

pC

EP

4 A

GR

2

siN

T

siA

GR

2

siN

T

siC

TS

B

siN

T

siC

TS

D

siN

T

siC

TS

B

siN

T

siC

TS

D

FA6

FA6

Pre-pro CTSDpro CTSD

pro CTSB

CTSD

HPDE

22

12

22

1.41

0

10

6

22

1.41

0

FA6

PaTu 8988tPaTu 8988s

CTSB

AGR2

Actin

34

42

26

24

52

26

52

CTSB

Actin

CTSB

Actin

CTSD

Actin

CTSD

Actin

34

52

26

52

34

52

kDa

52

34

42

26

24

52

ii

ii

ii

ii

iii

*****

***

iv

i ii iii

iviii

B

C D

Figure 5. CTSB and CTSD are functional downstream targets of AGR2. A, Western blot analysis (i) and densitometry quantification (ii) showing increasedexpression of precursor and mature CTSB and CTSD in MiaPaCa2-pCEP4 and pCEP4 AGR2 lysates. Actin was used as a loading control. III,semiquantitative real-time PCR analysis of CTSB and CTSD gene expression in MiaPaCa2-pCEP4 and pCEP4 AGR2 showing no change in theirtranscript levels. S16 was used as a reference gene. B, Western blot (i and ii) and densitometry (iii and iv) showing increased protein levels of precursorand mature CTSB and CTSD in FA6 pancreatic cancer cells in comparison with HPDE cells and in metastatic PaTu 8988s in comparison withnonmetastatic PaTu 8988t. C, levels of pro-CTSD in the culture supernatant of MiaPaCa2-pCEP4 and pCEP4 AGR2 and in FA6 cells 48 hours aftertransfection with AGR2 or nontargeting (NT) siRNA. D, after siRNA silencing of CTSB and CTSD for 48 hours (i and iii), decreased invasion of MiaPaCa2-pCEP4 AGR2 cells (ii) and FA6 cells (iv) was seen. Mean values of triplicate experiments are shown. �, P < 0.05; ��, P < 0.005; ��, P < 0.0001.






AGR2 expression induced an increase in the levels of CTSBand CTSD, 2 disulfide-containing thiol proteases that havepreviously been reported to be upregulated in pancreaticcancer (16, 38, 39) and are known to play a role in the dis-semination of cancer cells (40–42). Our in vitro data indicatedthat AGR2-induced invasion was mediated through the actionof these proteases rather than by increased cell motility, as nodifference in migration and wound-healing assays wasobserved. The AGR2-induced increase in CTSB and CTSDlevels could be the direct result of AGR2 PDI activity in theER during the processing of pro-cathepsins, as previouslyreported for the production of MUC2 in enterocytes (12),especially because we also observed an increase in the levelsof cathepsin precursor forms. This is also in accordance with anumber of other reports of PDI activity in protein folding beinga limiting factor for protein synthesis and secretion (43, 44).The concept of a posttranslational regulation of cathepsins asobserved previously (45), and here reported to be mediated byAGR2, is additionally supported by the absence of change in

cathepsins mRNA levels upon AGR2 upregulation. Identifyingthe formation of mixed disulfide bonds between AGR2 andCTSB and CTSD would confer a formal evidence of thismechanism in pancreatic cancer cells; however, the speed andthe transient nature of this type of interaction potentiated bythe presence of only 6 and 4 disulfide bonds in CTSB and CTSD,respectively (46), rendered such experimental confirmationchallenging. Our in vivo studies using transparent zebrafishembryos, which provide an elegant short-term invasion assayand allows quantification of the DTCs with a single-cell res-olution (47), faithfully recapitulated in vitro data and furthersubstantiated that the role of AGR2 in increased invasion ofpancreatic cancer cells is largely mediated by the 2 cathepsins.

Finally, we observed that AGR2 expression in PDAC cells isalso involved in regulation of pro-CTSD secretion; overexpres-sion of CTSD in cancer cells was reported previously to lead tothe hypersecretion of its proteolytically inactive proform (48).Furthermore, increased levels of pro-CTSD have been found inthe plasma of patients with metastatic breast carcinoma (49)

Ai

Posterior Anterior

ii

i

i

kDa

AGR2

Actin

siNT

siAGR2

PaTu 8988s - CMTMRPaTu 8988s - CMFDA

siNT

100

80

60

40

20

0

100

80

60

40

20

0

100

80

60

40

20

0

50

40

30

20

10

0

40

30

20

10

0

100

80

60

40

20

0siAGR2

No.

of D

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No.

of D

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No.

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)/co

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bryo

(%

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siNT siAGR2

siNT siCTSB

siNT siCTSD siNT siCTSD

siNT siCTSB

P < 0.0001

P < 0.0001P = 0.0026

P = 0.0074P = 0.0008

P < 0.0001***

***

***

**

**

***

24

52

i ii

iii

ii

siN

T

siA

GR

2

i ii iii

kDa

CTSB

Actin

26

52

siN

T

siC

TS

Bi ii iii

kDa

CTSD

Actin

34

52

siN

T

siC

TS

D

iii

ii

iii iv

Day

0D

ay 1

C

B

D

E

F

Figure 6. AGR2 and CTSB/CTSD are major regulators of pancreatic cancer cell dissemination in vivo. A, fifty to 100 PaTu 8988s cells stained with CMTMRor CMFDA dyes were injected into the yolk sack of 48-hour-old embryos and assessed by epifluorescence microscopy (i) of the whole embryo andconfocal microscopy (ii) of the injection area. B, twenty-four hours after injection, disseminated cancer cells were observed in the tail of the embryo (i) or inthe yolk sack (ii). Elongated cells imaged by confocal microscopy in the tail (iii) and the yolk sack (iv) of the embryo. Scale bars, 50 mm. C, labeledPaTu 8988s cells transfectedwith AGR2or nontargeting (NT) siRNA (i) were assessed by epifluorescencemicroscopy 24 hours after injection. The numberof disseminated cells was statistically significantly decreased after AGR2 silencing (ii and iii). D, representative image of decreased tumor celldissemination in zebrafish tail after AGR2 silencing (i). The enlarged respective images of the 2 marked areas in tail are shown on ii and iii. E and F, aftersilencing of CTSB/CTSD (i), a significant decrease in the number of disseminated cells was also seen (ii and iii), respectively. ��, P < 0.005; ��, P < 0.0001.

Dumartin et al.





and antibodies recognizing pro-CTSD in the serum of patientswith ovarian cancer (50). If a similar situation is established inPDAC, pro-CTSD might constitute a potential target for newdetection strategies.In summary, we provide new insights into the mechanisms

of action of AGR2 and its role in dissemination of pancreaticcancer cells through regulation of CTSB and CTSD both in vitroand in vivo. In addition, we show that AGR2 can also beimmunodetected at the surface of cancer cells, which couldopen new avenues for both the early detection and the devel-opment of novel immunotherapeutic strategies in pancreaticadenocarcinoma.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Dr. Guglielmo Rosignoli for technical assistance with flowcytometric analyses.

Grant Support

The work was supported by funding from CR-UK (L. Dumartin, H.J.Whiteman, and N.R. Lemoine), EU-FW6 (M.E. Weeks and D. Hariharan),and HEFCE (T. Crnogorac-Jurcevic). Part of the proteomic work (2D-DIGE)was undertaken at UCLH/UCL and supported by funding to J.F. Timms fromthe Department of Health's NIHR Biomedical Research Centres fundingscheme.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received May 2, 2011; revised August 31, 2011; accepted September 15, 2011;published OnlineFirst September 26, 2011.

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Dumartin et al.





2011;71:7091-7102. Published OnlineFirst September 26, 2011.Cancer Res Laurent Dumartin, Hannah J. Whiteman, Mark E. Weeks, et al. Regulation of Cathepsins B and DDissemination of Pancreatic Cancer Cells through AGR2 Is a Novel Surface Antigen That Promotes the

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http://cancerres.aacrjournals.org/content/suppl/2011/09/26/0008-5472.CAN-11-1367.DC1

http://cancerres.aacrjournals.org/content/71/22/7091.full#ref-list-1

http://cancerres.aacrjournals.org/content/71/22/7091.full#related-urls

http://cancerres.aacrjournals.org/cgi/alerts

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