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Originally published In Press as doi:10.1074/mcp.M700072-MCP200 on May 12, 2007.
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Molecular & Cellular Proteomics 6:1331-1342, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

Quantitative Proteomics Analysis Reveals That Proteins Differentially Expressed in Chronic Pancreatitis Are Also Frequently Involved in Pancreatic Cancer*,S

Ru Chen{ddagger},§, Teresa A. Brentnall{ddagger}, Sheng Pan, Kelly Cooke||, Kara White Moyes{ddagger}, Zhaoli Lane**, David A. Crispin{ddagger}, David R. Goodlett{ddagger}{ddagger}, Ruedi Aebersold§§ and Mary P. Bronner**

From the {ddagger} Division of Gastroenterology, Department of Medicine, and Departments of Pathology and {ddagger}{ddagger} Medicinal Chemistry, University of Washington, Seattle, Washington 98195, || Institute for Systems Biology, Seattle, Washington 98103, ** Department of Anatomic Pathology, Cleveland Clinic, Cleveland, Ohio 44195, and §§ Institute of Molecular Systems Biology, Swiss Federal Institute of Technology Zurich and Faculty of Science, University of Zurich, Zurich CH-8093, Switzerland


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The effective treatment of pancreatic cancer relies on the diagnosis of the disease at an early stage, a difficult challenge. One major obstacle in the development of diagnostic biomarkers of early pancreatic cancer has been the dual expression of potential biomarkers in both chronic pancreatitis and cancer. To better understand the limitations of potential protein biomarkers, we used ICAT technology and tandem mass spectrometry-based proteomics to systematically study protein expression in chronic pancreatitis. Among the 116 differentially expressed proteins identified in chronic pancreatitis, most biological processes were responses to wounding and inflammation, a finding consistent with the underlining inflammation and tissue repair associated with chronic pancreatitis. Furthermore 40% of the differentially expressed proteins identified in chronic pancreatitis have been implicated previously in pancreatic cancer, suggesting some commonality in protein expression between these two diseases. Biological network analysis further identified c-MYC as a common prominent regulatory protein in pancreatic cancer and chronic pancreatitis. Lastly five proteins were selected for validation by Western blot and immunohistochemistry. Annexin A2 and insulin-like growth factor-binding protein 2 were overexpressed in cancer but not in chronic pancreatitis, making them promising biomarker candidates for pancreatic cancer. In addition, our study validated that cathepsin D, integrin ß1, and plasminogen were overexpressed in both pancreatic cancer and chronic pancreatitis. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. Altogether our study provides some insights into the molecular events in chronic pancreatitis that may lead to diverse strategies for diagnosis and treatment of these diseases.

Pancreatitis is an inflammatory condition of the pancreas that shares many molecular features with pancreatic cancer. Many of the abnormally expressed proteins present in the setting of pancreatic cancer are also abnormally expressed in chronic pancreatitis, providing an unacceptably low level of specificity for use as protein biomarkers and cancer screening. Thus, a major obstacle for the development of biomarkers for early diagnosis of pancreatic cancer has been the dual expression of potential biomarkers in the neoplastic and non-neoplastic setting. It is therefore important to understand the proteins expressed in pancreatitis because they could be a source of false positive biomarkers for pancreatic cancer. Moreover chronic pancreatitis is a risk factor for eventual neoplastic progression. Patients with chronic pancreatitis have a 2-fold increased risk of pancreatic cancer. Understanding the molecular events involved in both diseases may lead to a better understanding of the mechanisms that link them.

The DNA and gene expression profile of pancreatic cancer has been detailed by multiple technologies, including RNA expression arrays, DNA microarray, differential display, and serial analysis of gene expression (1, 2). However, there are few large scale investigations at the protein level. A recent study by Shen et al. (3) identified 40 differentially expressed proteins in pancreatic cancer using two-dimensional gel electrophoresis and mass spectrometry. In another study, Crnogorac-Jurcevic et al. (4) used PowerBlot Western array screening to investigate protein expression in pancreatic cancer and pancreatitis. The study identified dysregulated proteins in disease states compared with normal: 30 proteins in chronic pancreatitis and 102 proteins in pancreatic cancer.

We previously used ICAT (5, 6)-based quantitative proteomics to study protein expression profiles of pancreatic cancer tissues and normal pancreas (7) and identified a number of new biomarker candidates associated with pancreatic cancer. In this report, we extend our investigation to study protein profiles in chronic pancreatitis. We identified differentially expressed proteins in chronic pancreatitis and compared them with the differentially expressed genes and proteins identified in pancreatic cancer reported in the literature. The differentially expressed proteins identified in chronic pancreatitis were further investigated to reveal the biological pathways of these proteins in association with the pathogenesis of chronic pancreatitis and pancreatic cancer. Western blotting and immunohistochemistry (IHC)1 were also used to validate the relevancy of the proteomics results to the development of biomarker candidates for pancreatic cancer.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Specimens—
Tissue specimens were obtained from patients with histologically proven pancreatic cancer or chronic pancreatitis and were collected in accordance with approved human subject guidelines at the University of Washington, Virginia Mason Hospital, and the Cleveland Clinic. For proteomics analysis, the chronic pancreatitis specimens were from patients who had no clinical or histological findings of pancreatic cancer at the time of diagnosis. To obtain a relatively homogeneous sample, we selected the chronic pancreatitis specimens with moderate fibrosis and occasional lymphocytes. It is difficult to obtain completely normal patients because they do not undergo surgery; thus we selected the 10 control specimens from patients who had as normal a pancreas as possible. The 10 normal controls were pooled from specimens derived from patients who had benign lesions of the pancreas such as pseudocyst, serous cystadenoma, serous microcystic adenoma, and non-pancreatic cancers such as cholangiocarcinoma and periampullary adenocarcinoma. All the control specimens were histologically verified normal pancreas specimens with one exception that had some periductal fibrosis but without significant inflammation. For the validation studies, we included specimens that were from patients who had ductal adenocarcinoma, primary pancreatitis, secondary pancreatitis associated with pancreatic cancer, and normal tissues. All of the specimens were obtained from surgical resections intraoperatively, immediately processed to minimize enzymatic destruction of the proteins, and frozen at –70 °C in minimal essential medium with 10% DMSO until use. DMSO was used to maintain cell architecture by reducing ice crystals within the cell during freezing.

ICAT Labeling and Mass Spectrometry—
The ICAT labeling and mass spectrometry analysis were described previously (7). Briefly frozen tissue was first rinsed with cold PBS, and 0.5–1.0 ml of T-Per buffer (Pierce) with 1x protease inhibitors was added to the tissue followed by tissue homogenization. The lysate was then centrifuged for 10 min at 10,000 rpm, and the debris were discarded. A Bradford assay (Sigma-Aldrich) was used to determine protein concentration. Ten normal pancreas samples were pooled together using an equal amount of protein from each individual sample as a pooled normal pancreas sample. Similarly 10 chronic pancreatitis samples were pooled together as a pooled chronic pancreatitis sample. For each pooled sample, 0.5 mg of protein was labeled with the acid-cleavable ICAT reagents, either the isotopically light (pooled normal pancreas) or heavy (pooled chronic pancreatitis) forms (Applied Biosystems, Foster City, CA) (7). The labeled normal sample and the matching labeled pancreatitis sample were combined and digested into peptides by trypsin (Promega, Madison, WI). ICAT-labeled peptides were subsequently fractionated by cation-exchange chromatography and purified by avidin affinity chromatography. The resulting 40 cation-exchange fractions were then combined into 17 fractions based on the peak intensity, e.g. multiple original cation-exchange fractions could be further combined into one fraction if the peak intensities were low. The final 17 fractions were then analyzed by microcapillary HPLC-ESI-MS/MS using an ion trap mass spectrometer (LTQ, ThermoFinnigan, San Jose, CA) as described previously (7).

Data Analysis—
The raw data were converted to mzXML using ReAdW. MS/MS scans were then exported as .dta files without further processing using the program msxlm2other. MS/MS spectra were searched against the International Protein Index (IPI) human sequence database (IPI.HUMAN.v3.13.fasta, 57,032 entries) using SEQUEST (version 27) (7). The SEQUEST database search criteria included a static modification of cysteine residues of 227 Da (light cleavable ICAT reagent) and a variable modification of 9 Da for cysteines (for the heavy cleavable ICAT reagent). The identified peptides were processed and analyzed through the mass spectrometry Trans-Proteomic Pipeline (TPP). In Trans-Proteomic Pipeline, the database search results were validated using the PeptideProphet program (8), which uses various SEQUEST scores and a number of other parameters to calculate a probability score for each identified peptide. The peptides were then assigned for protein identification using the ProteinProphet software (9). ProteinProphet allows filtering of large scale data sets with assessment of predictable sensitivity and false positive identification error rates. In this study, we used a ProteinProphet probability score ≥0.9 to ensure an overall false positive rate below 0.9%. Furthermore proteins with single peptide identification were also excluded in this study. Quantification of the ratio of each protein, isotopically heavy (pooled chronic pancreatitis) versus light (pooled normal), was achieved using the ASAPRatio program (10). Information about PeptideProphet, ProteinProphet, and ASAPRatio programs and other programs in Trans-Proteomic Pipeline can be obtained from the Seattle Proteome Center.

Western Blot—
Fifteen micrograms of protein from each specimen were used for SDS-PAGE. The gels were then transferred to nitrocellulose membrane according to the manufacturer's protocol (Amersham Biosciences). Antibodies were used at the following dilutions: anti-annexin A2 (Santa Cruz Biotechnology, Santa Cruz, CA), 1:1000 dilution; anti-plasminogen (US Biological, Swampscott, MA), 1:1000 dilution; and glyceraldehyde-3-phosphate dehydrogenase (R&D Systems, Minneapolis, MN), 1:2000 dilution. Proteins were detected using an ECL Plus kit (Amersham Biosciences).

IHC Analysis—
The tissue microarray was constructed from representative regions of paraffin-embedded tissue samples fixed in formalin and Hollande's fixative from 71 patients' surgical resections from the Cleveland Clinic between the years 1993 and 2004. Single 1-mm-diameter-sized cores of each diagnosis were re-embedded as a tissue microarray using a standard microarray instrument (Beecher Instruments, Silver Spring, MD). The microarray included 128 core samples from 47 sporadic pancreatic ductal adenocarcinoma patients, six core samples from four primary benign chronic pancreatitis patients without cancer and 12 core samples from two normal pancreatic control patients. From the above indicated 47 patients with adenocarcinoma, additional so-called "secondary" chronic pancreatitis was separately sampled in 24 core samples from a subset of 16 of the cancer patients. The term secondary chronic pancreatitis is used here to distinguish the chronic pancreatitis observed in patients with synchronous pancreatic ductal adenocarcinoma as opposed to primary benign chronic pancreatitis without evidence of malignancy.

IHC staining on tissue sections was performed as described previously (7). Briefly the slides were immunohistochemically stained using primary antibodies specific for annexin A2, cathepsin D, and integrin ß1 from BD Biosciences. Slides were processed for antigen retrieval using microwave heating in citrate buffer (0.1 M, pH 6.0) for 20 min followed by cooling to room temperature and then primary antibody incubation for 32 min. Secondary antibody and streptavidin-peroxidase were applied (ES autostains, Ventana Medical Systems, Tucson, AZ). Diaminobenzidine substrate was applied using the autostains and Iview detection chemistry. Results were scored as diffuse or focal and were graded (from 0, negative to 3+, intensely positive) for both neoplasm, admixed benign epithelial elements (ducts, acini, and islets), and surrounding stroma by an experienced pancreatic pathologist (M. P. B.).

Network Analysis Using MetaCore—
MetaCore (GeneGo, St. Joseph, MI) was used to map the differentially expressed proteins into biological networks. MetaCore is an integrated software suite for functional analysis of experimental data. It is based on a proprietary manually curated database of human protein-protein, protein-DNA, and protein-compound interactions; metabolic and signaling pathways; and the effects of bioactive molecules. Differentially expressed proteins were converted into gene symbols and uploaded into MetaCore for analysis. The biological process enrichment was analyzed based on Gene Ontology processes. For network analysis, two algorithms were used: 1) the direct interaction algorithm to map direct protein-protein interaction and 2) the shortest path algorithm to map the shortest path for interaction.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Proteomics Profiling of Pancreatitis
Using ICAT labeling and MS/MS, 657 proteins were identified and quantified in the comparison of pooled chronic pancreatitis tissues with pooled normal pancreas tissues. These identified proteins had a ProteinProphet score ≥0.9 with error rate ≤0.9% for protein identification. For the purpose of this study, single peptide-based protein identifications were further excluded, resulting in 498 proteins with a ProteinProphet score ≥0.9 and at least two-peptide identification (see the supplemental table for the complete list). In addition to protein identification, quantification of protein abundance ratios between pancreatitis and normal pancreas could also be achieved using ASAPRatio software. A total of 116 proteins showed an abundance change of at least 2-fold in chronic pancreatitis tissues compared with normal pancreas: 96 were overexpressed and 20 were underexpressed in chronic pancreatitis compared with normal pancreas (Table I).


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  		TABLE I Proteins with at least 2-fold change in abundance in chronic pancreatitis compared with normal pancreas

CP, chronic pancreatitis; CA, pancreatic cancer; NL, normal pancreas.

 
Differentially Expressed Proteins in Chronic Pancreatitis
Among the proteins identified in chronic pancreatitis, a wide array of inflammatory proteins were up-regulated. Thirteen inflammatory proteins were identified through the ICAT MS/MS profiling of pancreatitis tissues, including three proteins ({alpha}2-macroglobulin, macrophage migration-inhibitory factor, and apolipoprotein A-II) that were up-regulated by at least 2-fold, another three proteins that were up-regulated by at least 1.5-fold (annexin A1, peroxiredoxin 5, and toll-interacting protein), and another five proteins with up-regulation less than 1.5-fold in the chronic pancreatitis tissue. The three proteins with a greater than 2-fold expression change between pancreatitis and normal pancreas are included in Table I. The identification of these inflammatory proteins overexpressed in pancreatitis tissue is consistent with the inflammation processes of chronic pancreatitis. We also detected proteins, such as regenerating islet-derived protein 3{alpha} (pancreatitis-associated protein-1), which was overexpressed by 8.3-fold, that have been previously associated with pancreatitis (Table I).

To further interpret the differentially expressed proteins in chronic pancreatitis, we used the MetaCore pathway mapping tool to analyze and build the biological networks involved in these differentially expressed proteins. The top four biological processes associated with the differentially expressed proteins in chronic pancreatitis were all centered around inflammatory processes, which included 1) physiological response to wounding (p value = 3.63 x 10–12), 2) response to wounding (p value = 1.24 x 10–11), 3) response to external stimulus (p value = 1.50 x 10–9), and 4) inflammatory response (p value = 1.93 x 10–9). Many other associated processes were also related to inflammation occurring in chronic pancreatitis. A complete list of biological processes associated with these differentially expressed proteins is presented in Supplemental Fig. 1. In addition, using the direct interaction algorithm of MetaCore, a total of 15 direct interactions were identified between these differentially expressed proteins, demonstrating the functional relevance between them (data not shown).

Comparison of the Proteins Identified in the Current Study with the Differentially Expressed Proteins in Pancreatic Cancer and Pancreatitis Reported in the Literature
Of the 116 differentially expressed proteins in chronic pancreatitis, 15 proteins were also differentially regulated in pancreatic cancer tissue in our previous proteomics study of pancreatic cancer (7) (Table I). Moreover almost half of these differentially expressed proteins in chronic pancreatitis have been reported previously in pancreatic cancer tissue or pancreatitis tissue: n = 47 (40%) in pancreatic cancer and n = 13 (11%) in pancreatitis. Altogether 60 proteins from this study have not been reported in prior studies of pancreatitis or pancreatic cancer and thus provide novel targets for further study of biomarkers and the pathogenesis of chronic pancreatitis.

It has been well noted that pancreatic cancer is often associated with chronic pancreatitis. Moreover some patients with chronic pancreatitis have an increased risk for pancreatic cancer development. To analyze the common pathways of pancreatic cancer and chronic pancreatitis, we next used the MetaCore tool to map the biological networks of the 47 commonly and differentially expressed proteins in pancreatic cancer and chronic pancreatitis. In contrast to the enrichment of inflammation-related processes in chronic pancreatitis, the 47 commonly, differentially expressed proteins were mostly involved in the processes of cell organization, biosynthesis, and metabolism, which were essentially related to cell growth (Supplemental Fig. 1). Using the shortest path algorithm to map the shortest paths of interaction among these differentially expressed proteins, 21 of these proteins were brought together in the networks with interactions (Fig. 1). One of the most prominent regulatory proteins in the networks was c-MYC, which interacts with five of the differentially expressed proteins in chronic pancreatitis and pancreatic cancer (including HBB protein, integrin ß1, NDRG1 protein, thioredoxin, and tropomyosin 2). c-MYC is an oncoprotein that is involved in over 20% of all human cancers. Identification of c-MYC in the networks suggests that it may participate in the transcriptional activation of key pathways common in chronic pancreatitis and pancreatic cancer. In addition, four other transcriptional factors were also identified in the networks (c-FOS, c-JUN, NF-{kappa}B1, and p53), suggesting their common roles in pancreatic cancer and chronic pancreatitis (Fig. 1).


Figure 1
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  		FIG. 1. Biological network analysis of differentially expressed proteins in both pancreatic cancer and chronic pancreatitis using MetaCore mapping tool. The network was generated using the shortest path algorithm to map interaction between the proteins. Nodes represent proteins; lines between the nodes indicate direct protein-protein interaction. A small red circle denotes an overexpressed protein, whereas a small blue circle denotes an underexpressed protein. PKC, protein kinase C; PKB, protein kinase B; MHC, major histocompatibility complex; PI3K, phosphoinositide 3-kinase; cat, catalytic; reg, regulatory; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; Erk, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MEKK, MEK kinase; IL-1, interleukin-1; IL-1RI, type I IL-1 receptor; A2M, {alpha}2-macroglobulin; IRAK, IL-1R-associated kinase; HPRG, histidine-proline-rich-glycoprotein; TABP, truncated actin-binding protein; SITPEC (ECSIT), signaling intermediate in Toll pathway-evolutionarily conserved (evolutionarily conserved signaling intermediate in toll pathways); IP3R2, inositol 1,4,5-trisphosphate receptor type 2; JNK, c-Jun NH2-terminal kinase; SOS, son of sevenless; HPRG, histidine-rich glycoprotein; FCGRT, IgG receptor FcRn large subunit p51.

 
Proteins that are held in common between pancreatitis and pancreatic cancer do not necessarily reflect pathways that cause one disease (pancreatitis) to eventually lead to the other (cancer). It is noteworthy that the majority of patients with chronic pancreatitis do not develop pancreatic cancer even though there is an increased incidence of pancreatic cancer among this population. The corollary is also true: the majority of pancreatic cancer patients do not have overt clinical chronic pancreatitis as an underlying etiology. In addition, the picture is further complicated by the fact that there may be some histological changes of chronic pancreatitis in the setting of pancreatic cancer that may also contribute to identification of chronic pancreatitis-associated proteins in cancer specimens. Thus, the shared proteins discovered in this analysis of pancreatitis and pancreatic cancer may only reflect certain common biological processes, such as cell growth, in these two diseases. Nevertheless it is this commonality in protein expression between the two diseases that may be contributing to the false positive biomarkers for pancreatic cancer; such information can be important for researchers interested in highly specific methods of early cancer detection.

Validation of Proteins That Are Overexpressed in Both Pancreatic Cancer and Chronic Pancreatitis
Of the 116 differentially regulated proteins identified in chronic pancreatitis, 40% of them have been reported previously to be also involved in pancreatic cancer (Table I). This highlights the concept that there are many shared molecular events between chronic inflammatory diseases and neoplastic transformation. From the standpoint of biomarker development for pancreatic cancer, these shared proteins should be excluded to avoid false positive results.

Integrin ß1—
We previously reported overexpression of integrin ß1 in pancreatic cancer (7). In the current study, integrin ß1 was also overexpressed by 2.0-fold in chronic pancreatitis tissue. To further validate the overexpression of integrin ß1 in chronic pancreatitis, we performed IHC analysis on pancreatic tissues, including normal, pancreatic cancer, primary chronic pancreatitis, and pancreatitis associated with cancer (secondary chronic pancreatitis). In normal pancreas, all stromal components were negative (12 of 12 were at score 0), whereas most of the epithelial cells had minimal staining (12 of 12 were at score ≤+1) (Table II). In primary chronic pancreatitis, staining was negative for the stroma (three of three were at score 0) and was increased in epithelial cells (one of two was at score +1, and one of two was at score +2). For the secondary chronic pancreatitis (complicated by pancreatic adenocarcinoma), there was increased staining limited to the epithelial cells as well (seven of 12 at score ≥+2). In contrast, cancer samples displayed increased staining in the stroma and in the cancer cells (12 of 116 and 27 of 113 at score ≥+2, respectively) (7). In summary, the IHC analysis showed that primary and secondary chronic pancreatitis displayed some increased integrin ß1 expression over normal pancreas, validating the result by ICAT analysis. Together these results suggest that integrin ß1 expression is increased in chronic pancreatitis but to a lower degree compared with pancreatic cancer. Integrin ß1 acts as a fibronectin receptor that is involved in cell adhesion and recognition in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response, and movement of tumor cells. Increased expression of integrin ß1 in chronic pancreatitis is consistent with the nature of continuous tissue repair and immune response processes from the chronic inflammation. One can envision that the abnormal levels of integrin ß1 would be even further exacerbated in cancer, which has enhanced cell movement, tissue damage, and associated matrix response.


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  		TABLE II IHC analysis of integrin ß1, cathepsin D, and annexin A2

S, stroma; E, epithelia (or ductal cells); A, adenocarcinoma.

 
Cathepsin D—
Identification of two peptides, LLDIACWIHH and AIGAVPLIQGEYMIPCEK (Fig. 2), in the proteomics analysis led to an explicit identification of cathepsin D in the pooled chronic pancreatitis samples compared with pooled normal pancreas (1.8-fold overexpression in chronic pancreatitis). Cathepsin D has been shown previously to be overexpressed in pancreatic cancer tissues, in our previous ICAT analysis of pancreatic cancer study, and in other reports as well (3, 7, 11, 12).


Figure 2
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  		FIG. 2. Identification and quantification of cathepsin D in chronic pancreatitis. A, identification of two peptides, AIGAVPLIQGEYMIPCEK (A) and LLDIACWIHH (MS/MS spectrum not shown), led to identification of cathepsin D in the pooled chronic pancreatitis sample. Quantification of cathepsin D was determined by the ratios of these two peptides using ASAPRatio software. B, the reconstructed ion chromatogram of the precursor ion from peptide AIGAVPLIQGEYMIPCEK in its light and heavy versions using ASAPRatio. The ratio of heavy (pooled pancreatitis) versus light (pooled normal) was determined to be 1.8 (note the difference in the y axis between the two chromatograms).

 
To validate and further investigate the expression of cathepsin D in pancreatic tissue, we performed IHC analysis on pancreatic tissues. In normal pancreas, most of the ductal cells were positive at various degrees, whereas all of the stromal cells were negative for cathepsin D (Table II and Fig. 3A). In pancreatic cancer, there was increased expression in the stromal cells and marked overexpression in the ductal cancer cells. In primary chronic pancreatitis, there was increased expression in the stroma and ducts compared with normal pancreas with a similar degree of expression compared with pancreatic cancer. A similar pattern was also observed in the chronic pancreatitis associated with pancreatic cancer. Using a cutoff of strong staining (3+) versus non-strong staining (≤+2), none (zero of 12) of the stroma and 42% (five of 12) of the ductal epithelia of normal pancreas had strong expression; 44% (four of nine) of stroma and 71% (five of seven) of epithelia of primary chronic pancreatitis had strong expression; 22% (five of 23) of stroma and 18% (three of 17) of ductal epithelia of secondary chronic pancreatitis had strong expression; and 32% (38 of 119) of stroma and 86% (100 of 116) of epithelia of pancreatic carcinoma cells had strong staining. In summary, the tissue array data suggest that pancreatic cancer exhibits strong expression of cathepsin D in the stroma and cancer cells, whereas chronic pancreatitis also shows a similar degree of increased expression in the same cells compared with normal pancreas.


Figure 3
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  		FIG. 3. Cathepsin D IHC analysis in pancreatic cancer and chronic pancreatitis. In normal pancreas (A), only the ductal cells were positive (2+ and 3+), whereas all the stromal cells were negative. In chronic pancreatitis (B), there is increased staining in the stroma (1+ to 3+) and ductal cells (2+ and 3+). In pancreatic cancer (C), there is marked staining in the adenocarcinoma cells (majority at 3+). The scale of the IHC score was as follows: 0, negative; 1+, mild; 2+, moderate; 3+ strong.

 
Up-regulation of cathepsin D has been found in many malignancies, including colon cancer, pancreatic cancer, prostate cancer, uterine cancer, and ovarian cancer (13). Cathepsin D is secreted by malignant cells and is believed to be involved in the breakdown of the extracellular matrix. It has been implicated in cancer progression and metastasis, playing an essential role in stimulating cancer cell proliferation, fibroblast outgrowth, and angiogenesis as well as in inhibiting tumor apoptosis (14, 15). In breast cancer, cathepsin D is overexpressed from 2- to 50-fold compared with its concentration in other cell types such as fibroblasts or normal mammary glands (15). Up-regulated cathepsin D has also been detected in the inflammatory setting (16). The role of cathepsin D may make it a possible biomarker for diagnosis and prognosis of some cancers; however, the data in this study suggest that it is also up-regulated in chronic pancreatitis, limiting its use as biomarker for pancreatic cancer.

Plasminogen—
We previously identified overexpression of plasminogen in the pancreatic juice from patients with chronic pancreatitis (17). In the current study, plasminogen was also overexpressed in chronic pancreatitis tissues by 2.17-fold, validating our previous finding. Furthermore we compared expression of plasminogen in normal pancreas, chronic pancreatitis, and pancreatic cancer (Fig. 4). Plasminogen was not expressed in all nine normal pancreas tissues. In contrast, it was overexpressed in most of the chronic pancreatitis (eight of nine) and pancreatic cancer (seven of eight). The pathophysiological importance of the plasminogen/plasmin system in classical inflammation diseases has been well established by previous studies (18). Our finding that it is also up-regulated in pancreatic cancer is consistent with the fact that pancreatic cancer is often accompanied by inflammation in the pancreas and supports its potential role in tumor cell invasiveness and metastasis (19, 20).


Figure 4
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  		FIG. 4. Expression of plasminogen and annexin A2 in pancreatic tissues by Western blot analysis. A, plasminogen. No expression of plasminogen was detected in all nine normal pancreas tissues, whereas expression in chronic pancreatitis and pancreatic cancer was apparent in most of the samples. B, annexin A2. Expression of annexin A2 is very low in all nine normal pancreas. In chronic pancreatitis, two of nine have increased annexin A2 expression, whereas the other seven show a low expression level similar to that in normal pancreas. In pancreatic cancer, eight of nine cancer tissues have marked overexpression compared with normal pancreas and chronic pancreatitis. C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was used as loading control. NL, normal pancreas; CP, chronic pancreatitis; CA, pancreatic cancer.

 
In addition to integrin ß1, cathepsin D, and plasminogen, this study also identified many other proteins that were both overexpressed in pancreatic cancer and chronic pancreatitis. These included fibrillin-1, tropomyosin 2, biglycan, cofilin, versican, and fibrinogen {gamma}, which were identified to be overexpressed in pancreatic cancer and chronic pancreatitis by our previous ICAT proteomics study and were also validated by other studies (Table I).

Validation of Proteins That Are Overexpressed in Pancreatic Cancer but Not in Chronic Pancreatitis
To reduce the potential false positive rate of biomarkers for pancreatic cancer, it is logical to develop biomarkers on the proteins that show overexpression in pancreatic cancer but not in chronic pancreatitis. Below we present validation of two such proteins.

Annexin A2—
Overexpression of annexin A2 in pancreatic cancer has been reported previously (4, 7, 12, 21, 22). Our data in this study revealed that annexin A2 is not overexpressed in the primary chronic pancreatitis: the expression ratio of annexin A2 in chronic pancreatitis compared with normal pancreas is 1.1-fold. We validated the expression of annexin A2 in pancreatic tissues using Western blot and by IHC. As shown in Fig. 4, annexin A2 expression in normal pancreas was low. In pancreatic cancer, it was overexpressed in essentially all of the nine pancreatic cancer specimens: one cancer tissue showed moderated overexpression, whereas the other eight cancer tissues showed marked overexpression. In primary chronic pancreatitis, two of the nine tissues showed moderate overexpression, whereas the rest of the seven tissues showed low annexin A2 expression similar to that in normal pancreas. In IHC staining (Table II), all ductal cells from normal pancreas were mildly positive (12 of 12), whereas in primary chronic pancreatitis, two of three tissues showed similar mild staining, and the other exhibited strong annexin A2 expression in the ductal cells. For secondary chronic pancreatitis associated with cancer, the annexin A2 expression was strong in 14 of 25 stroma (56%) and 10 of 19 ductal cells (53%). Lastly in the cancer samples, the staining for cancer cells was greatly increased (118 of 127, or 93% showing strong staining). These data suggest that annexin A2 is overexpressed in pancreatic cancer and secondary chronic pancreatitis, although its expression in primary chronic pancreatitis is similar to that in normal pancreas. We previously reported overexpression of annexin A2 in pancreatic cancer by ICAT proteomics study (increased 2.6-fold in pancreatic cancer) and IHC analysis (7). The current study extended the investigation to chronic pancreatitis and revealed that annexin A2 is not overexpressed in primary chronic pancreatitis in most of the cases analyzed by proteomics, Western blot, and IHC. Together these results suggest that annexin A2 may be a promising candidate for further biomarker development for pancreatic cancer.

Insulin-like Growth Factor-binding Protein 2 (IGFBP-2)—
We recently reported elevated levels of IGFBP-2 in the pancreatic juice from a pancreatic cancer patient by ICAT proteomics analysis (overexpressed by 4.6-fold) (23). Moreover the same study showed that IGFBP-2 was only marginally expressed in two of eight normal pancreatic tissues and in four of eight pancreatitis samples. In contrast to the low levels of expression in normal and pancreatitis, marked overexpression of IGFBP-2 was detected in most of the cancer tissues (seven of eight). In the current study, we confirmed some of these findings and showed that IGFBP-2 is not overexpressed in chronic pancreatitis (ratio = 0.9 compared with normal pancreas).

IGFBP-2 is overexpressed in many malignant tissues and has been found to be elevated in serum and the cerebrospinal fluid of patients with various malignancies (24–26). In pancreatic cancer, the overexpression of IGFBP-2 has been reported in a cDNA microarray study (27). Using proteomics analysis and Western blotting, our study demonstrated that at the protein level IGFBP-2 is overexpressed in pancreatic cancer relative to chronic pancreatitis and normal pancreas. Moreover the expression level of IGFBP-2 in chronic pancreatitis is similar to that in normal pancreas. These findings suggest that IGFBP-2 may be another potential biomarker candidate for pancreatic cancer.

Summary
In this study, we used ICAT-based quantitative proteomics to analyze protein expression in chronic pancreatitis in comparison with normal pancreas. 116 proteins were shown to be differentially expressed by at least 2-fold in chronic pancreatitis of which 15 proteins were also shown to be differentially expressed in our previous ICAT proteomics analysis of pancreatic cancer, and 47 and 13 proteins have been previously shown in the literature to be involved in pancreatic cancer and chronic pancreatitis, respectively. Our findings reinforce the concept that chronic pancreatitis shares many protein signatures with pancreatic cancer. Sixty of the proteins from this study have not been reported in prior studies of pancreatic cancer and/or pancreatitis and thus provide novel targets for further study of the pathogenesis of pancreatitis. Biological network analysis identified c-MYC as a prominent regulator in the networks of differentially expressed proteins common in pancreatic cancer and chronic pancreatitis. Five proteins that were identified in the proteomics studies were selected for further validation by Western blot and IHC analysis. Integrin ß1, cathepsin D, and plasminogen were shown to be overexpressed in chronic pancreatitis and pancreatic cancer by ICAT analysis and IHC staining of pancreatic tissue sections or Western blot. The positive involvement of these proteins in chronic pancreatitis and pancreatic cancer will potentially lower the specificity of these proteins as biomarker candidates for pancreatic cancer. On the other hand, annexin A2 and IGFBP-2 were demonstrated to be mainly up-regulated in pancreatic cancer but not in chronic pancreatitis by ICAT analysis, and the observation was further validated by Western blotting and/or IHC staining of pancreatic tissue sections. Together the results presented in this study reveal that proteins differentially expressed in chronic pancreatitis are also frequently involved in pancreatic cancer, suggesting that this commonality in protein expression between the two diseases may be contributing to the false positive biomarkers for pancreatic cancer.


   ACKNOWLEDGMENTS
 
We acknowledge GeneGo for providing access to the MetaCore software suite and John Metz for technical assistance using the software.


  FOOTNOTES
 
Received, February 16, 2007, and in revised form, April 30, 2007.

Published, MCP Papers in Press, May 12, 2007, DOI 10.1074/mcp.M700072-MCP200

1 The abbreviations used are: IHC, immunohistochemistry; IGFBP-2, insulin-like growth factor-binding protein 2. Back

* This work was supported by National Institutes of Health Grant 1R01CA107209; funding from the Canary Foundation, the Concern Foundation, the Gene and Mary Ann Walters Pancreatic Cancer Foundation; the American Association for Cancer Research PanCAN Career Development Award for Pancreatic Cancer Research; and federal funds from the NHLBI, National Institutes of Health, under Contract NOI-HV-28179. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material. Back

§ To whom correspondence should be addressed: Dept. of Medicine, Division of Gastroenterology, University of Washington, Seattle, WA 98195. Tel.: 206-221-4109; Fax: 206-685-9478; E-mail: ruc{at}medicine.washington.edu


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