interactionbetweencancercellsandstromalfibroblasts … ·...

InteractionbetweenCancerCells andStromalFibroblastsIsRequiredforActivationof theuPAR-uPA-MMP-2CascadeinPancreaticCancerMetastasisYu He, Xiang-de Liu, Zhi-yu Chen, Jin Zhu,Yan Xiong, Kun Li, Jia-hong Dong, and Xiaowu Li

Abstract Purpose: Interaction between tumor cells and surrounding stromal fibroblast (SF) plays a criticalrole in tumor growth and invasion.The aim of the study is to determine the role of SF in regulatingthe invasive behaviors of pancreatic cancer by evaluating the mode of SF activating theurokinase plasminogen activator (uPA)-plasmin-matrix metalloproteinase (MMP)-2cascade.Experimental Design: The expression patterns of uPA, MMP-2, and uPA receptor (uPAR) inhuman metastatic pancreatic cancer were analyzed by immunohistochemistry and the roles ofSF in activation of the uPA-plasmin-MMP-2 cascade were evaluated by coculturing pancreaticcancer cell lines with SF.Results: uPA expression and fibroblastic uPAR expression were correlated with liver metastasisof human pancreatic cancer. MMP-2 rather than MMP-9 was activated in the metastatic pancre-atic cancer. In the in vitro culture system, the coculture of peritumor fibroblasts with metastaticpancreatic cancer BxPc3 cells resulted in activation of MMP-2 and up-regulation of uPARexpression. In this coculture system, the uPA-plasminogen cascade was involved in MMP-2activation. This activation required a direct interaction between SF and cancer cells. In thecoculture system, intergrin a6h1 expression was increased in BxPc3 cells, and blocking thefunction of integrin a6h1 decreased the activation of uPA and MMP-2. This suggests thatinteraction between integrins of cancer cells and the uPARs of the SF might be involved in theactivation of the uPAR-uPA-MMP-2 cascade.Conclusion: Our results suggest that SF plays a role in promoting pancreatic cancer metastasisvia activation of the uPA-plasminogen-MMP-2 cascade.

Cancer metastasis is a complex process, which results from theinteraction of cancer cells with host cells and extracellularmatrices (1). In this process, cancer cells and stromal cellsexchange enzymes and cytokines and then constantly modifylocal extracellular matrix. This modified extracellular matrixinteracts with cell-surface receptors and thereby promotes cellmigration and invasion (2, 3).Invasive tumor cells have a marked ability to degrade

extracellular matrix via activation of matrix metalloproteinase

(MMP)-2. MMP-2 is secreted as an inactive zymogen andrequires distinct activation processes to be converted into anactive MMP-2 (4). Two principal mechanisms are involved inMMP-2 activation. One proposed mechanism is through theMT1-MMP pathway, in which tissue inhibitor of metallopro-teinase 2 (TIMP-2), a bifunctional molecule (5, 6), is capable ofinteracting with pro-MMP-2 via its NH2-terminal domain anddocking MT1-MMP via its COOH-terminal domain, therebyformatting a ternary complex. This complex is a prerequisite foreffective activation of pro-MMP-2 by an adjacent TIMP-freeactive MT1-MMP (7, 8). Another mechanism of MMP-2activation is through plasminogen activator/plasmin system,in which pro-urokinase plasminogen activator (pro-uPA) bindsto its receptor, uPA receptor (uPAR), through a specific NH2-terminal sequence of its noncatalytic chain (9–11). Thisbinding results in uPA activation, accelerates the conversionof plasminogen to plasmin on the cell surface, and localizesthese enzymes to focal contact sites (12–14). Although plasminhas been shown to principally activate MMP-1, MMP-3, and, toa certain extent, MMP-9 (15), increasing evidence proves thatuPA and plasmin can activate pro-MMP-2 and therebypromoting tumor invasion and metastasis (16–19).There is growing evidence indicating that interaction between

tumor cells and surrounding stromal fibroblast (SF) plays acritical role in growth, invasion, metastasis, and angiogenesis of

Human Cancer Biology

Authors’Affiliation: Hepatobiliary Surgery Institute, Southwest Hospital, ThirdMilitary Medical University, Chongqing, P.R. ChinaReceived 8/20/06; revised 2/26/07; accepted 3/8/07.Grant support: National Natural Science Foundation of China grant 30371587andMilitary Medical Science Foundation of China grant 06H032.The costs of publication of this article were defrayed in part by the payment of pagecharges.This article must therefore be hereby marked advertisement in accordancewith18 U.S.C. Section1734 solely to indicate this fact.Note:Y. He and X-d. Liu contributed equally to this work.Requests for reprints: Xiaowu Li, Hepatobiliary Surgery Hospital and Institute,Southwest Hospital, Third Military Medical University, Chongqing 400038,P.R. China. Phone: 86-23-6875-4156; Fax : 86-23-6531-7637; E-mail:[email protected].

F2007 American Association for Cancer Research.doi:10.1158/1078-0432.CCR-06-2088

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tumor (20–22). It has been shown that the invasive potentialof pancreatic cancer cells can be greatly enhanced by cocultur-ing with SF (23). Although an intimate relationship clearlyexists between the growing tumor and surrounding stromalenvironment, the molecular mechanisms of tumor-stromalinteraction in promoting cancer progression have not been wellcharacterized. Several molecules have been identified partici-pating in tumor-stromal interactions, including hepatocytegrowth factor (21, 24), transforming growth factor h (24), andseveral MMPs (25, 26). In pancreatic cancer, uPA activation anduPAR overexpression are involved in the systemic dissemina-tion of pancreatic cancer (27, 28). Recently, our preliminarystudy found that fibroblastic uPAR expression was significantlyincreased in metastatic pancreatic cancers, implying that the SFmight participate in the metastatic process of pancreatic cancervia the uPA pathway. To evaluate the role of SF in promotingpancreatic cancer metastasis, we analyzed the expressionpatterns of uPA, MMP-2, and uPAR in human metastaticpancreatic cancer. Via an in vitro coculture system, bycoculturing two pancreatic cancer cell lines, highly metastaticBxPc3 cells and low metastatic PaCa2 cells, with peritumorfibroblasts, we observed the effects of the cocultures on theexpression of uPAR and evaluated the roles of cocultures inactivation of the uPA-plasmin-MMP-2 cascade.

Materials andMethods

Tissue samples. A total of 20 pancreatic adenocarcinoma patientsfrom Hepatobiliary Surgery Institute, Southwest Hospital, ThirdMilitary Medical University, were randomized and case-pair controlledselection (including age, tumor size, location, and classification) in thisstudy. The tumor specimens included 6 metastatic pancreatic cancer(liver metastasis) specimens and 10 nonmetastatic pancreatic cancerspecimens obtained from surgery, as well as 4 liver metastasisspecimens obtained from formalin-fixed, paraffin-embedded tissues(Table 1). Each pancreatic tumor specimen was reviewed by patholo-

gists. The protocol was approved by the Institutional Review Board andthe patients gave written consent.

Antibodies and reagents. Antibodies and reagents included anti–MT1-MMP, TIMP-2, and MMP-2 (Oncogene Science); anti–plasmino-gen activator inhibitor (PAI-1 380), uPA-specific antibody 3471, anduPAR-specific antibody (399RAmericanDiagnostica); gelatin, aprotinin,plasminogen, plasmin, glycine, andH-D-Val-Leu-Lys-pNA (S 2051 SigmaChemical Co.); amiloride, active uPA, andMMP-2 (Calbiochem);mousemonoclonal antibodies anti-a6h1 (GoH3), anti-h3 (LM609), and anti-h5(P1F5; PharMingen); and anti-h1 (P4C10), anti-h4 (3E1), anti-a2(P1E6), anti-a3 (P1B5), and anti-a5 (P1D6; Life Technologies, Inc.).

Immunohistochemistry. The details of the procedure have previously

been described (19). Briefly, the deparaffinized sections were trypsi-

nized (0.05% trypsin with 0.05% Triton X-100 in TBS) for 20 min and

blocked with 10% goat serum in Superblock, where each section was

incubated separately with monoclonal antibodies uPA at 20 Ag/mL,

uPAR at 10 Ag/mL, MMP-9 at 8 Ag/mL, and MMP-2 at 10 Ag/mL at 4jCfor 18 to 24 h. After washing four to five times (15 min each) with

Triton-TBS, the slides were processed in the Ventana-automated stainer

according to the manufacturer’s instructions. The immunoperoxidase-

3,3-diaminobenzidine– stained slides were subsequently counter-

stained with hematoxylin and mounted with a coverslip. Normal

pancreases were used as controls.Cell cultures. The metastatic human pancreatic carcinoma cell line

BxPc3 and nonmetastatic human pancreatic carcinoma cell line PaCa2,described by Sawai H et al. (29), were purchased from the AmericanType Culture Collection. SF was isolated from the pancreatic carcinomatissues (from surgery in our Institute) and epithelial cell contaminationexcluded by light microscopy. These cells were maintained in DMEMwith 10% FCS.

Flow cytometry. Cultured cells were harvested by trypsinization andwashed with PBS containing 1% normal goat serum. Cells were in-cubated with primary antibodies at 4jC for 1 h, followed by secondaryantibodies conjugated to FITC for 30 min. The stained cells were resus-pended in 100 AL of PBS and analyzed by Becton Dickinson FACSort.

Acid treatment of cells. The cells were treated with glycine buffer todeplete the membrane-bound uPA. Briefly, the cells were harvested with0.25% trypsin and 2 mmol/L EDTA, treated with glycine buffer (pH4.0) at 4jC for 3 min, and neutralized by incubation in Tris buffer atpH 7.0 for 10 min for further use.

Table 1. Immunohistochemical analyses of pancreatic tumors

Case no. Age (y) Tumor size (cm) Location Classification uPA uPAR MMP-9 MMP-2 MT1-MMP

1 45 2 Head Adenocarcinoma 2+ 2+ 0 2+ 1+2 48 2.5 Head and liver Adenocarcinoma 3+ 3+ 1+ 2+ 03 52 3 Head and liver Adenocarcinoma 3+ 2+ 0 2+ 1+4 54 3.5 Head Adenocarcinoma 1+ 1+ 0 2+ 05 48 3.5 Body and liver Adenocarcinoma 3+ 2+ 1+ 3+ 06 46 3.2 Body Adenocarcinoma 0 0 2+ 3+ 07 57 5.5 Body and liver Adenocarcinoma 3+ 2+ 0 2+ 08 59 5.6 Body Adenocarcinoma 1+ 2+ 1+ 2+ 09 63 3 Body and liver Adenocarcinoma 2+ 2+ 1+ 2+ 0

10 65 2.8 Body Adenocarcinoma 1+ 1+ 0 1+ 011 66 3.9 Tail Adenocarcinoma 1+ 1+ 0 2+ 012 69 4.0 Tail and liver Adenocarcinoma 0 2+ 0 2+ 1+13 60 5.2 Head and liver Adenocarcinoma 2+ 3+ 1+ 3+ 1+14 62 5.4 Head Adenocarcinoma 1+ 1+ 2+ 1+ 1+15 56 4.4 Tail and liver Adenocarcinoma 3+ 2+ 1+ 2+ 016 54 4.7 Tail Adenocarcinoma 1+ 2+ 1+ 1+ 1+17 44 4.2 Head and liver Adenocarcinoma 3+ 3+ 2+ 2+ 2+18 47 4.5 Head Adenocarcinoma 1+ 1+ 2+ 1+ 2+19 53 5.3 Head and liver Adenocarcinoma 2+ 2+ 1+ 2+ 1+20 56 5.1 Head Adenocarcinoma 0 0 1+ 1+ 1+


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Cell lysis and Western blotting. The cells were lysed in NP40 lysisbuffer (1.5% NP40, 150 mmol/L NaCl, 0.2% SDS, 1 mmol/L EDTA,20mmol/L Tris-HCl, 1 mmol/L phenylmethylsulfonyl fluoride, 10 Ag/mLleupeptin, 10 Ag/mL aprotinin, 1 mmol/L Na3VO4, 50 mmol/L NaF).Protein concentrations were determined with a BCA protein assay kit(Pierce). For Western blotting, conditioned medium and cell lysatescontaining equal amounts of protein were separated by SDS-PAGE andtransferred to nylon membranes. Membranes were probed with primaryantibodies followed by peroxidase-labeled secondary antibodies andvisualized by enhanced chemiluminescence detection system (Amersham)according to the manufacturer’s instructions.

Gelatin zymography. MMP-2 and MMP-9 were analyzed by using10% SDS-gelatin substrate gel. The conditioned medium was collectedunder serum-free conditions and subjected to a gelatin-SDS-PAGEelectrophoresis. The gels were treated with 2.5% Triton X-100 at 37jCfor 30 min to remove SDS and then incubated at 37jC for 16 h insubstrate buffer (50 mmol/L Tris-HCl and 5 mmol/L CaCl2 at pH 8.0).The gels were stained with 0.15% Coomassie blue R250 (Bio-Rad) in50% methanol, 10% glacial acetic acid at room temperature for 20 min,and then destained in the same solution without Coomassie blue. Theactivities of enzymes were identified as clear gelatin-degrading bandsagainst the blue background.

Colorimetric plasminogen activation assay. The cells were grown inDMEM with 10% plasminogen-depleted FCS in 100-mm dishes for24 h. The activities of uPA in conditioned media were determined byconverting plasminogen to plasmin by using a coupled colorimetricplasminogen activation assay. Briefly, the conditioned media wererecovered and concentrated by Centricon concentrators (Amicon).Conditioned media of 10 AL were incubated with 2.8 Ag ofplasminogen in 65 AL of uPA buffer [100 mmol/L Tris-HCl (pH8.8), 0.5% Triton X-100] in 96-well plates at room temperature for4 h; then, the plasmin activity was determined by adding 25-ALsubstrates of plasmin containing 50 Ag of H-D-Val-Leu-Lys-pNA (S-2251; Sigma), with absorbance of 405 nm using a Titertek multiscanplate reader. The conditioned media without plasminogen were usedas control. Results were presented as plough units determined by aplasmin standard.

Reverse transcription-PCR analysis. The RNA was extracted frompancreatic cancer cells and SF by using RNA mini-Kit (Qiagen) and thenreverse transcribed with Moloney murine leukemia virus reversetranscriptase in the presence of random primers. The reverse-transcribed

cDNA was amplified further by 30 cycles of PCR in the presence of10 pmol of sense and antisense primers. The primers used were asfollows: for uPAR, 5¶-ACAGGAGCTGCCCTCGCGAC-3¶ and 5¶-GAGG-GGGATTTCAGGTTTAGG-3¶; for GAPDH, 5¶-ACGGATTTGGTCGTATT-GGG-3¶ and 5¶-TGATTTTGGAGGGATCTCGC-3¶. Each set of primerscorresponded to sequences located on different exons to allow thedetection of genomic DNA contamination. Each PCR cycle included adenaturation step at 94jC for 1 min, an annealing step at 61jC for1 min, and an extension step at 72jC for 1 min. The PCR products wereanalyzed by electrophoresis on 2% agarose gel containing ethidiumbromide and visualized under UV light. Adobe Photoshop software 7.0was used to quantitate the densitometry of visualized bands andcalculate the relative value units of densitometry.

Statistical analysis. Statistical analysis was done by using SPSSsoftware for Windows version 10.0. Correlation analysis was conductedby Spearman’s rho test. P < 0.05 was considered as statisticallysignificant.

Results

uPA expression and MMP-2 activation were correlated withliver metastasis of human pancreatic cancer. To determine therelationship between liver metastasis of pancreatic cancer andthe expressions of MMPs and uPA, we applied immunohisto-chemistry to analyze the expressions of uPA, MT1-MMP, uPAR,and MMPs in 10 metastatic pancreatic cancers and 10nonmetastatic pancreatic cancers. As shown in Table 1, incomparison with normal pancreatic tissues, there was increasedexpression of MMP-2 in all human pancreatic cancers,moderate expression of MMP-9 in four pancreatic cancers,and increased expression of MT1-MMP in two. The expressionof MMPs was not correlated with pancreatic cancer metastasis(Table 3). In contrast, expression of uPA was found in 17 of 20(85%) examined tumors, with failed expression in only threetumors (Table 1). The stronger staining for uPA was detected atcancer nests of the metastatic pancreatic cancers compared withnonmetastatic pancreatic cancers (Fig. 1A, left versus right). Of10 cases of metastatic pancreatic cancer, 7 expressed high levelsof uPA (Table 1). The expression of uPA was correlated withliver metastases of pancreatic cancers (P < 0.001, r = 0.714).These results suggest that up-regulation of uPA expression maybe involved in pancreatic cancer metastasis.We next detected the active forms of MMP-2 and MMP-9 in

six metastatic and six nonmetastatic pancreatic cancer tissues byusing gelatin zymograph. As shown in Fig. 2, the active forms ofMMP-2 but not MMP-9 were found in liver metastases as wellas in situ metastatic pancreatic cancers (Fig. 2A and B). Incontrast, all nonmetastatic pancreatic cancers expressed equallevels of MMP-2 (Fig. 2C). These results suggest that MMP-2 isactivated in the metastatic pancreatic cancers.

Fibroblastic uPAR expression was increased in metastaticpancreatic cancer. The expression patterns of fibroblasticuPA, MMP-2, and uPAR in pancreatic cancer tissues wereanalyzed by immunohistochemistry; the expressions of MMP-2and uPA were also seen in fibroblasts (Table 2; Fig. 1A and D)but the expressions of MMP-2 and uPA in fibroblasts wereunrelated to liver metastasis of pancreatic cancer (P = 0.177 and0.065, respectively). In contrast, the fibroblastic uPAR expres-sion was significantly increased in the metastatic pancreaticcancers compared with nonmetastatic pancreatic cancers(Fig. 1C, left versus right). Statistical analysis showed that thefibroblastic uPAR expression was significantly associated with

Table 2. Fibroblastic expression of uPA, MMP-2,and uPAR in pancreatic cancer

Case no. fuPA fuPAR fMMP-2

1 1+ 1+ 1+2 1+ 3+ 2+3 2+ 2+ 2+4 1+ 1+ 2+5 2+ 2+ 2+6 0 0 2+7 1+ 2+ 2+8 1+ 1+ 2+9 1+ 2+ 2+

10 1+ 1+ 1+11 1+ 1+ 1+12 0 2+ 2+13 1+ 3+ 2+14 1+ 1+ 1+15 1+ 2+ 1+16 0 1+ 1+17 2+ 2+ 1+18 1+ 1+ 1+19 1+ 1+ 1+20 0 0 1+

uPA-MMP-2 Cascade and Pancreatic CancerMetastasis




the liver metastases of pancreatic cancers (P < 0.001, r = 0.857;Table 3), suggesting that fibroblasts may play a role inactivating uPA cascades and promoting pancreatic cancermetastasis.

Activation of MMP-2 required direct interaction between tumorcells and fibroblasts in an in vitro culture system. A coculturewas used to determine whether activation of MMP-2 required adirect cell-cell interaction. Specifically, two pancreatic cancercell lines, the highly metastatic cell line BxPc3 and the lowmetastatic cell line PaCa2, were cocultured with SF and normalfibroblast (NF) for evaluating the activation of MMP-2 in theconditioned medium by using gelatin substrate zymography.As shown in Fig. 3, pancreatic cancer cells and fibroblastsproduced equivalent levels of pro-MMP-2 and pro-MMP-9.However, when pancreatic cancer cells were cocultured with SF,an active form of MMP-2 was detected in BxPc3/SF coculturesbut not in PaCa/SF (Fig. 3A, top, lane 4 versus lane 3) or BxPc3/NF (bottom, lane 3 versus lane 2) cocultures, although MMP-2expression was eventually unchanged. These results indicatethat MMP-2 is activated by the coculture of pancreatic cancerBxPc3 cells with SF.In the coculture, pancreatic cancer cells or SF can produce

some soluble cell factors to regulate gene expression of theapposing cells. To determine whether this regulatory mecha-nism resulted in MMP-2 activation in BxPC3/SF cocultures,BxPc3 cells were indirectly cocultured with SF by using aTranswell filter. We found that MMP-2 was not activated in theindirect coculture of BxPc3/SF (Fig. 3A, bottom, lane 5). Theseresults suggest that MMP-2 activation requires the interaction ofcancer cells with SF.

The uPA-plasminogen pathway was responsible for MMP-2activation in the coculture system. To identify which pathwaywas responsible for MMP-2 activation in BxPc3/SF coculture,we examined expressions of MT1-MMP, TIMP-2, and uPA incell lysates by Western blotting and evaluated uPA activities inthe conditioned media using a coupled colorimetric plasmin-ogen activation assay. Two pancreatic cancer cells expressedequal levels of MT1-MMP, TIMP-2, and uPA. The expressionsof MT1-MMP and TIMP-2 were not changed in BxPc3/SFcoculture and PaCa2/SF coculture (Fig. 3B, rows 1 and 2, lane4 versus lane 5). However, uPA activities determined byplasmin as well as the protein levels of uPA were significantlyincreased in BxPc3/SF coculture but not in PaCa2/SF coculture(Fig. 3B, bottom, lane 4 versus lane 5 and Fig. 3C). uPA wasnot activated in the indirect BxPc3/SF coculture or in BxPc3/NF coculture. These results indicate that the uPA-plasminogenpathway, but not MT1-MMP, is activated in BxPc3/SFcoculture, which requires a direct interaction of SF withcancer cells.We next used two approaches to confirm whether MMP-2

activation is uPA-plasminogen dependent. First, we addedamiloride (an uPA inhibitor), aprotinin (a specific inhibitor of

plasmin), and anti–MT1-MMP antibodies to BxPc3/SF cocul-ture and found that addition of 25 Amol/L amiloride to theBxPc3/SF coculture prevented uPA activation, decreased theconversion of plasminogen to plasmin (Fig. 3C), and sup-pressed active MMP-2 (Fig. 3D, top, lane 3), but anti–MT1-MMPdid not suppress activation of MMP-2 (Fig. 3D, top, lane 2).Furthermore, addition of 40 Ag/mL aprotinin to BxPc3/SFcoculture also suppressed MMP-2 activation (Fig. 3D, top, lane4), suggesting that the uPA-plasmin cascade is essential forMMP-2 activation in this coculture. Second, because uPA andMMP-2 were not activated in PaCa2/SF coculture, we exoge-nously added active uPA to PaCa2/SF coculture to observe theconversion of plasminogen to plasmin and the activation ofMMP-2. Our preliminary study showed that 50 nmol/L uPA wasthe optimal condition for stimulating MMP-2 activation. In thisstudy, we found that exogenously adding active uPA intoPaCa2/SF coculture promoted the conversion of plasminogen toplasmin (Fig. 3C) as well as the activation of MMP-2 (Fig. 3D,

Fig. 1. Immunoperoxidase staining of pancreatic cancer. A, immunoperoxidase staining of adenocarcinomas of the pancreas (Table 1, case no. 2 versus no. 1) withanti-uPA. Metastatic pancreatic cancer revealed strong immunoperoxidase staining of uPA in nests of adenocarcinoma cells (arrow) as well as SF. The nonmetastaticpancreatic cancer only revealed weak staining in adenocarcinoma cells [arrow ; row1, left versus right (Table 1, case no. 2 versus no. 1, respectively)]. Magnification, �100.B, immunoperoxidase staining of adenocarcinomas of the pancreas (Table 1, case no. 3 versus no. 4) with anti ^ MT1-MMP. Both metastatic pancreatic cancer andnonmetastatic pancreatic cancer showed weak staining with MT1-MMP antibody at cancer cells (arrow) and stromal cells. Magnification, �100. C, immunoperoxidasestaining of adenocarcinomas of the pancreas (Table 1, case no. 5 versus no. 6) with anti-uPAR. Metastatic pancreatic cancers showed intensive staining in SF (left, arrow)but the nonmetastatic pancreatic cancer revealed moderate staining in both cancer cells (arrow) and SF (right). Magnification, �100. D, immunoperoxidase staining ofadenocarcinomas of the pancreas (Table 1, case no. 12 versus no. 11) with anti-MMP-2. Both metastatic and nonmetastatic pancreatic cancer revealed intensive stainingof MMP-2 in nests of cancer. A weaker staining of SF was seen. Magnification, �100.

Fig. 2. MMP-2 is activated in metastatic pancreatic cancers. Liver metastases(A), primary cancers of metastatic pancreatic cancers (B ; Table 1, case nos. 2, 3, 5,7, 9, and12), and nonmetastatic pancreatic cancers (C ; Table1, case nos.1, 4, 6, 8,10, and11) were hemolyzed and analyzed by gelatin zymography as describedin Materials and Methods. MMP-9 and MMP-2 were identified as clear gelatin-degrading bands against the blue background. Con-2, active MMP-2; Con-1,pro-MMP-2.





bottom, lane 3). These results suggest that MMP-2 activation isdependent on the uPA-plasminogen pathway.

Up-regulation of uPAR in the SF of BxPc3/SF coculture wasresponsible for activation of uPA. Interaction of pro-uPA with

uPA receptor on the cell surfaces leads to uPA activation (30).To determine whether uPAR is involved in uPA activation, weobserved the protein levels of uPAR in SF, pancreatic cancercells, and the coculture byWestern blotting. As shown in Fig. 4A,

Table 3. Statistics analyses for liver metastases and expression of uPA, uPAR, and MMPs

Liver metastasis uPA uPAR MMP-9 MMP-2 MT1-MMP fuPA fuPAR fMMP-2

Correlation coefficient, r 0.714 0.740 0.112 0.677 0.00 0.420 0.857 0.314P 0.000 0.000 0.639 0.001 1.000 0.065 0.000 0.177

Fig. 3. MMP-2 is activated through the uPA/plasminogenpathway but not theMT1-MMP/TIMP-2 pathway inpancreatic cancer BxPc3/SF coculture.A, BxPc3/SF cocultureresults in activationofMMP-2 but notMMP-9. Conditionedmedia fromnonmetastatic pancreatic cancer PaCa2 cells (top, lane1), metastatic pancreatic cancer BxPc3 cells(top, lane 2), PaCa2/SF1coculture (top, lane 3), BxPc3/SF coculture (top, lane 4), SF (top, lane 5), BxPc3/NF (bottom, lane 2), BxPc3/SF-2 (bottom, lane 3), and BxPc3/SF/SE indirect coculture (bottom, lane 5) were collected and analyzedby gelatin zymography as described inMaterials andMethods. MMP-9 andMMP-2were identified asclear gelatin-degrading bands against the blue background. SE, indirect coculture.B, BxPc3/SF cocultures result in activationof uPA but not inup-regulationofMT1-MMP.SFcells (lane1) or pancreatic cancer cells (lanes 2 and3) and the cocultures (lanes 4 and5)were grownovernight. Whole-cell lysateswere collectedas described inMaterialsandMethods.Twentymicrograms of whole-cell lysateswere analyzedbyMT1-MMPblotting (row1),TIMP-2 blotting (row2), anduPA blotting (row3). Focal adhesion kinase(FAK) blotting (row 4) was used as loading control.C, the uPA/plasminogenpathway is activated in BxPc3/SF coculture but not in PaCa2/SF coculture.The cells acceptedacid treatment with glycine buffer to deplete themembrane-bounduPA andwere grown in DMEMwith10% plasminogen-depleted FCS overnight and treatedwith amiloride(Ami ; 25 Amol/L), aprotinin (Apro ; 50 Ag/mL), andactive uPA (50 nmol/L).The activities ofuPA in conditionedmediawere determinedby a coupled colorimetric plasminogenactivation assay as described inMaterials andMethods.The conditionedmedia of BxPc3/SF coculturewithout plasminogenwere used as control. Resultswere presentedas ploughunits determinedby a plasmin standard. Columns, mean from three individual experiments.D,MMP-2 is activated throughuPA but notMT1-MMP. BxPc3 cells/SFcoculturewas treatedwith immunoglobulin G (IgG), anti ^ MT1-MMP, amiloride (25 Amol/L), and aprotinin (50 Ag/mL; row1), or PaCa2/SF cocultures were treatedwithinactive uPA (control) or active uPA 50 nmol/L (rows 3 and 4). Whole-cell lysates were collected as described inMaterials andMethods.Twenty micrograms of whole-celllysates were analyzedbyMMP-2 blotting (rows1and 3) anduPA blotting (row 4). Focal adhesion kinase blottings (rows 2 and 5) were used as loading control.





SF, PaCa2, and BxPc3 expressed equivalent levels of uPAR (Fig.4A, lanes 1-3 , respectively). However, expression of uPAR wasincreased in the coculture of BxPc3 cells with SF but not in thecoculture of PaCa2 with SF (Fig. 4A, lane 4 versus lane 6),suggesting that the coculture of BxPc3/SF up-regulates uPARexpression.In the coculture, pancreatic cancer cells or SF can produce

some soluble cell factors to regulate the gene expression of theapposing cells. To determine whether this regulatory mecha-nism resulted in the up-regulation of uPAR in BxPC3/SFcoculture, BxPc3 cells were indirectly cocultured with SF by aTranswell filter or directly cocultured with SF to evaluate uPARexpression by reverse transcription-PCR. We found that, in theindirect BxPc3/SF coculture, the up-regulated mRNA expressionof uPAR was found in SF but not in BxPc3 cells (Fig. 4B, lane 5versus lane 6). Similarly, the up-regulated mRNA expression ofuPAR was also found in the direct BxPc3/SF coculture (Fig. 4B,lane 4). In contrast, mRNA expression of uPAR was notincreased in the direct and indirect BxPc3/NF cocultures(Fig. 4B, lanes 2 and 3). These results have confirmed ourprevious results of Western blotting (Fig. 4A) and also suggestthat the metastatic pancreatic cancer BxPc3 cells may producesome soluble cell factors to up-regulate uPAR expression in SFbut not in NF.In the coculture, the activation of uPA and MMP-2 requires a

direct cell-cell contact between SF and cancer cells, whereasuPAR expression is on the contrary. To further explain thisphenomenon, we observed the expression of PAI-1 in thesecocultures by Western blotting. As shown in Fig. 4C, PAI-1showed no change in direct or indirect cocultures, suggestingthat the mechanism for tumor cells activating uPA may be morecomplicated.

Integrin a6b1 and uPAR coordinately activated uPA andresulted in MMP-2 activation. It has been shown that integrinsare involved in MMP-2 activation (31). We then analyzedintegrin profiles in two pancreatic cancer cell lines by flowcytometric analysis. As shown in Fig. 5A, the expressions of a6and h1 were significantly increased in BxPc3 cells rather than inPaCa2 cells. The expression levels of a3, h4, av, a5, h3, and h5integrins were equivalent in both cell lines. These resultsindicate that integrin a6h1 is up-regulated in the pancreaticcancer BxPc3 cells.To determine whether integrin a6h1 played a role in

activation of MMP-2 in this complicated coculture, BxPc3 cellswere incubated with the specific a6h1-blocking antibody GoH3and then cocultured with SF in the presence of GoH3. MMP-2activation and uPA activities were determined in BxPc3/SFcoculture via Western blotting and plasminogen activationassay. Our previous study showed that 10 Ag/mL GoH3 was theoptimal condition for functional inhibition of a6h1. In thisstudy, we found that preincubation of GoH3 with BxPc3 cellsdecreased plasmin production by 50% (Fig. 5B) and subse-quently inhibited MMP-2 activation in BxPc3/SF coculture(Fig. 5C), suggesting that a6h1 functions in the activation ofuPA and MMP-2 in BxPc3/SF coculture.

Discussion

In this study, we investigated the role of SF in activating theuPA-plasmin-MMP-2 cascade and regulating the invasivebehaviors of pancreatic cancer cells. We found that expressions

of uPA and fibroblastic uPAR were increased in metastaticpancreatic cancer. The metastatic pancreatic cancer expressedthe active forms of MMP-2, but the nonmetastatic pancreaticcancer did not. In the in vitro coculture, the coculture of SF with

Fig. 4. Up-regulation of uPAR results in activation of uPA in the coculture ofBxPc3/SF.A, protein levels of uPARare increased in SF cells coculturedwithmetastatic pancreatic cancer BxPc3 cells. Pancreatic cancer cells were culturedalone, directly coculturedwith SF cells, or indirectly coculturedwith SF cells by a0.1-AmTranswell filter. Whole-cell lysates were collected as described inMaterialsandMethods.Twenty micrograms of whole-cell lysates were analyzed by uPARblotting (row1). Focal adhesion kinaseblotting (row2)wasusedas loading control.B, the coculture of BxPc3 cells with SF cells up-regulatedmRNA expression ofuPAR.The cells were treated as mentioned above.The RNAwas extracted frompancreatic cancer cells, SF, or cocultures using RNAmini-Kit (Qiagen) and thensubjected to reverse transcription-PCRanalysis as described inMaterials andMethods.The PCR products were analyzed by electrophoresis on 2% agarose gelcontaining ethidium bromide and visualized and photographedunder UV light.The densitometry of visualized bandswas quantitated byAdobe Photoshop 7.0software.The relative value units of densitometry were calculated as uPAR/GAPDHratio.The size of the PCR products (arrows) is1,046 bp for uPARand 230 bp forGAPDH.C, PAI-1expression is not changed in the cocultures. Pancreatic cancer cellswere directly coculturedwith SF cells or indirectly coculturedwith SF cells by a0.1-AmTranswell filter.The conditionedmediawere collected.Twenty micrograms ofproteins of conditionedmediawere analyzed by PAI-1blotting.





metastatic pancreatic cancer BxPc3 cells resulted in activation ofMMP-2 and up-regulated expression of uPAR. In this coculture,the uPA-plasminogen cascade, but not the MT1-MMP-TIMP-2pathway, was involved in MMP-2 activation that required directinteraction between SF and cancer cells. We also found that, inthe coculture, the interaction between integrins of cancer cellsand the uPARs of SF may be involved in the activation of the

uPAR-uPA-MMP-2 cascade. Our results suggest that SF regulatesthe activation of the uPA-plasminogen-MMP-2 cascade, therebypromoting pancreatic cancer metastasis.Overexpressions of uPA and MMPs have been a consistent

finding in adenocarcinomas of the colon, lung, breast, prostate,and ovary and melanoma (9, 21–27), but relatively littleattention has been paid to the pancreatic cancer. We found that

Fig. 5. The integrin a6h1 and uPARcoordinately activate uPA, thus leading toMMP-2 Activation. A, expression ofintegrins in the pancreatic cancer cells.The metastatic pancreatic cancer BxPc3cells and the nonmetastatic pancreaticcancer PaCa2 cells were subjected tofluorescence-activated cell sorting analysisafter incubation with antibodies toa6 (GoH3), h1 (P4C10), a3 (P1B5),h4 (clone 3E1), av (L230), a5 (P1D6),h3 (LM609), and h5 (P1F5). Arrows,representative histograms for BxPc3and PaCa2. The unstaining BxPc3 cellswere used as control (Con). B, a6h1

function-blocking antibodies suppressuPA activation. BxPc3 cells werepretreated with glycine buffer andcocultured with SF in the presenceof rat immunoglobulin G and a6h1

function-blocking antibody GoH3(10 Ag/mL) for 24 h. The activities of uPAin conditioned media were determined bya coupled colorimetric plasminogenactivation assay as described in Materialsand Methods. Results were presented asplough units determined by a plasminstandard. Columns, mean from threeindividual experiments; bars, SD.C, a6h1 function-blocking antibodiessuppress MMP-2 activation. BxPc3 cellspretreated with glycine buffer andcocultured with SF in the presence ofrat immunoglobulin G and a6h1

function-blocking antibody GoH3(10 Ag/mL) for 24 h. The whole-celllysates were collected and analyzed byWestern blotting as described in Materialsand Methods. Twenty micrograms ofwhole-cell lysate proteins were analyzedby MMP-2 blotting (row 1). Focal adhesionkinase blotting (row 2) was used asloading control.





uPA and MMP-2 were expressed in most pancreatic tumors.However, uPA /uPAR expression and MMP-2 activation weresignificantly increased in metastatic pancreatic cancers com-pared with nonmetastatic pancreatic cancers. In the metastaticpancreatic cancers, the fibroblastic uPAR expression wassignificantly increased (Fig. 1C, left ; Table 3) and associatedwith the liver metastases of pancreatic cancers (P = 0.000,r = 0.857; Table 3), suggesting that fibroblasts may play a rolein promoting pancreatic cancer metastasis.In the in vitro coculture, the activation of MMP-2 in the

highly metastatic pancreatic cancer BxPc3 cells requires theparticipation of peritumor fibroblasts and activation of uPA-plasmin cascade. In fact, MMP-2 was not activated in eithermetastatic pancreatic cancer BxPc3 cells or nonmetastaticpancreatic cancer PaCa2 cells. When these pancreatic cancercells were cocultured with SF, the activation of MMP-2 was onlydetected in BxPc3/SF coculture but not in PaCa/SF coculture,although the coculture did not enhance the levels of MMP-2expression. In BxPc3/SF coculture, MMP-2 activation was linkedto the activation of the uPA-plasmin cascade because chemicalinhibitors of either uPA or plasmin prevented MMP-2activation. In addition, addition of active uPA to PaCa2/SFcoculture with no active uPA detected resulted in MMP-2activation. These findings are in agreement with these studiesshowing that uPA-plasmin cascade is involved in pro-MMP-2processing (16–18, 32, 33) and uPA helps release active MMP-2from the matrix-bound pro-MMP-2 (34). Besides the uPA-plasmin cascade, the MT1-MMP/TIMP-2 pathway is thought tobe another important mechanism to activate pro-MMP-2(35, 36). However, in our cocultures, neither MT1-MMP norTIMP-2 was increased in BxPc3/SF coculture. Addition of TIMP-2 or anti–MT1-MMP to the coculture failed to activate MMP-2.Therefore, in the BxPc3/SF coculture, MT1-MMP and TIMP-2may format ternary complexes without leaving TIMP-free activeMT1-MMP to activate MMP-2, suggesting that MMP-2 activa-tion via uPA-plasmin cascade may be a mechanism involved inthe role of SF in regulating pancreatic cancer metastasis.Our observations also suggest that a direct interaction

between pancreatic cancer cell and peritumor fibroblast isrequired for activating the uPA/plasmin cascade. In thecocultures, uPA was supplied by pancreatic cancer cells as wellas SF, but uPAR and pro-MMP-2 were expressed principally inSF. Yet, uPAR expression was up-regulated in SF when

cocultured with pancreatic cancer BxPc3 cells by using directand indirect cocultures. These results are consistent with thefindings by Seghezzi et al. (37). However, uPA activation wasonly found in the direct coculture but not in the indirectcoculture of BxPc3 cells with SF. In this indirect coculture, thepro-uPA was also secreted in the condition media and the PAI-1expression was not changed. Therefore, in the cocultures, thereexists a more complicated mechanism for tumor cells solicitingtheir surrounding stromal cells to activate uPA.It has been known that MMP-2 is secreted and stored in

extracellular depots as precursor zymogens and activated byspecific proteases at cell surface. The clustering of integrin h1 byfibronectin and binding of MMP-2 to integrin avh3 facilitateMMP-2 activation (31). Previous reports have indicated thatuPAR can physically interact with multiple integrins, includingh1, h2 (38, 39), and a5h1 (40), and to regulate cell migration tofibronectin (41). In the cocultures, we found that BxPc3 cellsexpressed an increased level of integrin a6h1. In the cocultures,uPA activation was significantly increased in a6h1-positiveBxPc3/SF coculture but not in a6h1-negative PaCa2/SF cocul-ture. In addition, addition of the a6h1 function-blockingantibodies suppressed uPA activation in BxPc3/SF coculture.Therefore, in the cocultures, the interaction between integrinsof cancer cells and the uPARs of the SF may be a mechanism topromote activation of the uPAR-uPA-MMP-2 cascade.In conclusion, during the process of migration of pancreatic

cancers, the SF not only supports cancer cell growth of theprimary tumor but also participates in activation of the uPA-plasminogen-MMP-2 cascade, thereby promoting pancreaticcancer invasion and metastasis. Thus, we can envision an in vivoscenario where the a6h1-positive pancreatic cancer cells interactwith stromal cells, which results in up-regulation of uPAR, andintegrin a6h1 interacts with uPAR, which results in activation ofthe uPA-plasminogen-MMP-2 cascade, facilitating the activateduPA-plasminogen-MMP-2 cascade focused on the focal adhe-sions of integrin a6h1 to matrix. The activated uPA and MMP-2may help the dissemination of these cells from the primarytumor.

Acknowledgments

We thank David Kramer for helpful discussions and critical reading of the manu-script and Dr. Guo-dong Liu for editing the manuscript.



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2007;13:3115-3124. Clin Cancer Res Yu He, Xiang-de Liu, Zhi-yu Chen, et al. Pancreatic Cancer MetastasisRequired for Activation of the uPAR-uPA-MMP-2 Cascade in Interaction between Cancer Cells and Stromal Fibroblasts Is

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