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

Open AccessPublished:May 12, 2007DOI:https://doi.org/10.1074/mcp.M700072-MCP200
      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 β1, 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 (
      • Chen R.
      • Pan S.
      • Brentnall T.A.
      • Aebersold R.
      Proteomic profiling of pancreatic cancer for biomarker discovery.
      ,
      • Chen R.
      • Pan S.
      • Crispin D.A.
      • Brentnall T.A.
      Gene expression and proteomic analysis of pancreatic cancer: a recent update.
      ). However, there are few large scale investigations at the protein level. A recent study by Shen et al. (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      ) identified 40 differentially expressed proteins in pancreatic cancer using two-dimensional gel electrophoresis and mass spectrometry. In another study, Crnogorac-Jurcevic et al. (
      • Crnogorac-Jurcevic T.
      • Gangeswaran R.
      • Bhakta V.
      • Capurso G.
      • Lattimore S.
      • Akada M.
      • Sunamura M.
      • Prime W.
      • Campbell F.
      • Brentnall T.A.
      • Costello E.
      • Neoptolemos J.
      • Lemoine N.R.
      Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma.
      ) 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 (
      • Gygi S.P.
      • Rist B.
      • Gerber S.A.
      • Turecek F.
      • Gelb M.H.
      • Aebersold R.
      Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.
      ,
      • Han D.K.
      • Eng J.
      • Zhou H.
      • Aebersold R.
      Quantitative profiling of differentiation-induced microsomal proteins using isotope-coded affinity tags and mass spectrometry.
      )-based quantitative proteomics to study protein expression profiles of pancreatic cancer tissues and normal pancreas (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      ) 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)
      The abbreviations used are: IHC, immunohistochemistry; IGFBP-2, insulin-like growth factor-binding protein 2.
      1The abbreviations used are: IHC, immunohistochemistry; IGFBP-2, insulin-like growth factor-binding protein 2.
      were also used to validate the relevancy of the proteomics results to the development of biomarker candidates for pancreatic cancer.

      RESULTS AND DISCUSSION

      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).
      Table IProteins with at least 2-fold change in abundance in chronic pancreatitis compared with normal pancreas
      Database IDGene symbolProtein descriptionRatio CP/NLS.D.Unique peptidesCA ICAT studyOther CA studyOther CP study
      More abundant by at lease 2-fold
       IPI00178744
      Protein group, only one is listed.
      ACADVLAcyl-CoA dehydrogenase (splice isoform 2)14.296.122Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      )
       IPI00419237LAP3LAP3 protein9.090.832
       IPI00029039REG1ARegenerating islet-derived protein 3α (pancreatitis-associated protein 1)8.332.782Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Patard L.
      • Lallemand J.Y.
      • Stoven V.
      An insight into the role of human pancreatic lithostathine.
      )
       IPI00010274
      Protein group, only one is listed.
      TPSAB1Tryptase α-1 precursor (splice isoform 1)7.141.532Yes (
      • Ferrero S.
      Serum levels of mast cell tryptase, vascular endothelial growth factor, and basic fibroblast growth factor in patients with acute pancreatitis.
      )
       IPI00219713
      Protein group, only one is listed.
      FGGγ-A of fibrinogen γ chain precursor (splice isoform)6.672.224Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Lu Z.
      • Hu L.
      • Evers S.
      • Chen J.
      • Shen Y.
      Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue.
      ,
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00032179
      Protein group, only one is listed.
      SERPINC1Antithrombin III variant, SERPINC1 protein6.253.133Yes (
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00641737HPHaptoglobin precursor6.251.172Yes (
      • Quilliot D.
      • Walters E.
      • Guerci B.
      • Fruchart J.C.
      • Duriez P.
      • Drouin P.
      • Ziegler O.
      Effect of the inflammation, chronic hyperglycemia, or malabsorption on the apolipoprotein A-IV concentration in type 1 diabetes mellitus and in diabetes secondary to chronic pancreatitis.
      )
       IPI00102821PACAPPituitary adenylate cyclase-activating polypeptide protein6.252.735
       IPI00019004TLOC1Translocation protein-16.251.172
       IPI00384428BPHLValacyclovir hydrolase precursor5.881.042
       IPI00021854APOA2Apolipoprotein A-II precursor5.562.477
       IPI00639937
      Protein group, only one is listed.
      CFBComplement B-factor5.561.232Yes (
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      ,
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
      • Radlwimmer B.
      • Dohner H.
      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      )
       IPI00375676FTLFerritin light chain5.261.113Yes (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      )
       IPI00328113FBN1Fibrillin-1 precursor4.761.5915Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Buchholz M.
      • Kestler H.A.
      • Bauer A.
      • Bock W.
      • Rau B.
      • Leder G.
      • Kratzer W.
      • Bommer M.
      • Scarpa A.
      • Schilling M.K.
      • Adler G.
      • Hoheisel J.D.
      • Gress T.M.
      Specialized DNA arrays for the differentiation of pancreatic tumors.
      ,
      • Grutzmann R.
      • Boriss H.
      • Ammerpohl O.
      • Luttges J.
      • Kalthoff H.
      • Schackert H.K.
      • Kloppel G.
      • Saeger H.D.
      • Pilarsky C.
      Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes.
      )
       IPI00298994TLN1Talin-14.554.134Yes (
      • Aguirre A.J.
      • Brennan C.
      • Bailey G.
      • Sinha R.
      • Feng B.
      • Leo C.
      • Zhang Y.
      • Zhang J.
      • Gans J.D.
      • Bardeesy N.
      • Cauwels C.
      • Cordon-Cardo C.
      • Redston M.S.
      • DePinho R.A.
      • Chin L.
      High-resolution characterization of the pancreatic adenocarcinoma genome.
      )
       IPI00021263YWHAZ14-3-3 protein ζ/δ4.353.785Yes (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      )
       IPI00022463TFSerotransferrin precursor4.351.8950Yes (
      • Gronborg M.
      • Kristiansen T.Z.
      • Iwahori A.
      • Chang R.
      • Reddy R.
      • Sato N.
      • Molina H.
      • Jensen O.N.
      • Hruban R.H.
      • Goggins M.G.
      • Maitra A.
      • Pandey A.
      Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach.
      )
       IPI00017601CPCeruloplasmin precursor4.171.222Yes (
      • Crnogorac-Jurcevic T.
      • Gangeswaran R.
      • Bhakta V.
      • Capurso G.
      • Lattimore S.
      • Akada M.
      • Sunamura M.
      • Prime W.
      • Campbell F.
      • Brentnall T.A.
      • Costello E.
      • Neoptolemos J.
      • Lemoine N.R.
      Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma.
      ,
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00003348GNB3Guanine nucleotide-binding protein Gi/Gs/Gt β subunit 24.000.962Yes (
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      ,
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
      • Radlwimmer B.
      • Dohner H.
      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      )
       IPI00022488HPXHemopexin precursor4.001.4411Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      ,
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00293303
      Protein group, only one is listed.
      LGMNLegumain precursor4.000.804Yes (
      • Missiaglia E.
      • Blaveri E.
      • Terris B.
      • Wang Y.H.
      • Costello E.
      • Neoptolemos J.P.
      • Crnogorac-Jurcevic T.
      • Lemoine N.R.
      Analysis of gene expression in cancer cell lines identifies candidate markers for pancreatic tumorigenesis and metastasis.
      )
       IPI00645078UBE1LUbiquitin-activating enzyme E14.000.804Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      )
       IPI00022371HRGHistidine-rich glycoprotein precursor3.851.184Yes (
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      ,
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
      • Radlwimmer B.
      • Dohner H.
      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      )
       IPI00418336
      Protein group, only one is listed.
      INTS3Hypothetical protein DKFZp686E19503.850.892
       IPI00465313
      Protein group, only one is listed.
      A2M163-kDa protein, α2-macroglobulin3.701.5110Yes (
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00298828APOHβ2-Glycoprotein I precursor3.701.3712Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
       IPI00477090
      Protein group, only one is listed.
      IGHG1Full-length cDNA clone CS0DD006YL02 of neuroblastoma of Homo sapiens3.700.554
       IPI00643041
      Protein group, only one is listed.
      RANGTP-binding nuclear protein RAN3.700.822Yes (
      • Lu Z.
      • Hu L.
      • Evers S.
      • Chen J.
      • Shen Y.
      Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue.
      )
       IPI00022434ALBSerum albumin3.701.78225Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Sitek B.
      • Luttges J.
      • Marcus K.
      • Kloppel G.
      • Schmiegel W.
      • Meyer H.E.
      • Hahn S.A.
      • Stuhler K.
      Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.
      )
       IPI00304840COL6A22C2 of collagen α2(VI) chain precursor (splice isoform)3.571.024Yes (
      • Crnogorac-Jurcevic T.
      • Efthimiou E.
      • Nielsen T.
      • Loader J.
      • Terris B.
      • Stamp G.
      • Baron A.
      • Scarpa A.
      • Lemoine N.R.
      Expression profiling of microdissected pancreatic adenocarcinomas.
      )
       IPI00215611CRIP1Cysteine-rich protein 13.571.532
       IPI00301987FLJ20323Hypothetical protein FLJ203233.571.282
       IPI00298497FGBFibrinogen β chain precursor3.451.664Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00010157MAT2AS-Adenosylmethionine synthetase γ form3.450.713Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      ,
      • Grutzmann R.
      • Boriss H.
      • Ammerpohl O.
      • Luttges J.
      • Kalthoff H.
      • Schackert H.K.
      • Kloppel G.
      • Saeger H.D.
      • Pilarsky C.
      Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes.
      ,
      • Iacobuzio-Donahue C.A.
      • Maitra A.
      • Shen-Ong G.L.
      • van Heek T.
      • Ashfaq R.
      • Meyer R.
      • Walter K.
      • Berg K.
      • Hollingsworth M.A.
      • Cameron J.L.
      • Yeo C.J.
      • Kern S.E.
      • Goggins M.
      • Hruban R.H.
      Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.
      )
      Yes (
      • Lu S.C.
      • Gukovsky I.
      • Lugea A.
      • Reyes C.N.
      • Huang Z.Z.
      • Chen L.
      • Mato J.M.
      • Bottiglieri T.
      • Pandol S.J.
      Role of S-adenosylmethionine in two experimental models of pancreatitis.
      )
       IPI00473011
      Protein group, only one is listed.
      HBDHemoglobin δ subunit3.230.9410
       IPI00376798RPL11Ribosomal protein L113.233.644
       IPI00513698
      Protein group, only one is listed.
      TPM2Tropomyosin 23.230.733Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      ,
      • Aguirre A.J.
      • Brennan C.
      • Bailey G.
      • Sinha R.
      • Feng B.
      • Leo C.
      • Zhang Y.
      • Zhang J.
      • Gans J.D.
      • Bardeesy N.
      • Cauwels C.
      • Cordon-Cardo C.
      • Redston M.S.
      • DePinho R.A.
      • Chin L.
      High-resolution characterization of the pancreatic adenocarcinoma genome.
      ,
      • Logsdon C.D.
      • Simeone D.M.
      • Binkley C.
      • Arumugam T.
      • Greenson J.K.
      • Giordano T.J.
      • Misek D.E.
      • Hanash S.
      Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer.
      )
      Yes (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      )
       IPI00382950HBBβ-Globin gene from a thalassemia patient3.130.8845Yes (
      • Aguirre A.J.
      • Brennan C.
      • Bailey G.
      • Sinha R.
      • Feng B.
      • Leo C.
      • Zhang Y.
      • Zhang J.
      • Gans J.D.
      • Bardeesy N.
      • Cauwels C.
      • Cordon-Cardo C.
      • Redston M.S.
      • DePinho R.A.
      • Chin L.
      High-resolution characterization of the pancreatic adenocarcinoma genome.
      )
       IPI00465343
      Protein group, only one is listed.
      ADH1CAlcohol dehydrogenase3.030.287Yes (
      • Homann N.
      • Stickel F.
      • Konig I.R.
      • Jacobs A.
      • Junghanns K.
      • Benesova M.
      • Schuppan D.
      • Himsel S.
      • Zuber-Jerger I.
      • Hellerbrand C.
      • Ludwig D.
      • Caselmann W.H.
      • Seitz H.K.
      Alcohol dehydrogenase 1C*1 allele is a genetic marker for alcohol-associated cancer in heavy drinkers.
      )
       IPI00216650HNMTHistamine N-methyltransferase3.030.642
       IPI00215631
      Protein group, only one is listed.
      CSPG2Versican core protein precursor (splice isoform Vint)3.030.642Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      ,
      • Iacobuzio-Donahue C.A.
      • Maitra A.
      • Shen-Ong G.L.
      • van Heek T.
      • Ashfaq R.
      • Meyer R.
      • Walter K.
      • Berg K.
      • Hollingsworth M.A.
      • Cameron J.L.
      • Yeo C.J.
      • Kern S.E.
      • Goggins M.
      • Hruban R.H.
      Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.
      )
       IPI00479925AGRINAgrin2.860.492
       IPI00176193
      Protein group, only one is listed.
      COL14A1Collagen α1 chain (splice isoform)2.860.412Yes (
      • Lohr M.
      • Hummel F.
      • Martus P.
      • Cidlinsky K.
      • Kroger J.C.
      • Hahn E.G.
      • Oesterling C.
      • Emmrich J.
      • Schuppan D.
      • Liebe S.
      Serum levels of extracellular matrix in acute pancreatitis.
      )
       IPI00556148CFHComplement factor H2.780.3915Yes (
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00215997CD9CD9 antigen2.700.662Yes (
      • Crnogorac-Jurcevic T.
      • Efthimiou E.
      • Capelli P.
      • Blaveri E.
      • Baron A.
      • Terris B.
      • Jones M.
      • Tyson K.
      • Bassi C.
      • Scarpa A.
      • Lemoine N.R.
      Gene expression profiles of pancreatic cancer and stromal desmoplasia.
      )
       IPI00289800
      Protein group, only one is listed.
      G6PDGlucose-6-phosphate dehydrogenase2.700.292Yes (
      • Van Driel B.E.
      • Van Gulik T.M.
      • Strum P.D.
      • Johan G.
      • Offerhaus A.
      • Gouma D.J.
      • van Noorden C.J.
      Differential diagnosis of chronic pancreatitis and pancreatic cancer in brush cytology specimens.
      )
       IPI00012011CFL1Cofilin, non-muscle isoform2.630.6917Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      ,
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
      • Radlwimmer B.
      • Dohner H.
      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      )
      Yes (
      • Crnogorac-Jurcevic T.
      • Gangeswaran R.
      • Bhakta V.
      • Capurso G.
      • Lattimore S.
      • Akada M.
      • Sunamura M.
      • Prime W.
      • Campbell F.
      • Brentnall T.A.
      • Costello E.
      • Neoptolemos J.
      • Lemoine N.R.
      Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma.
      )
       IPI00549682ALDOAFructose-bisphosphate aldolase A2.631.1111Yes (
      • Logsdon C.D.
      • Simeone D.M.
      • Binkley C.
      • Arumugam T.
      • Greenson J.K.
      • Giordano T.J.
      • Misek D.E.
      • Hanash S.
      Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer.
      )
       IPI00216298TXNThioredoxin2.630.352Yes (
      • Cecconi D.
      • Scarpa A.
      • Donadelli M.
      • Palmieri M.
      • Hamdan M.
      • Astner H.
      • Righetti P.G.
      Proteomic profiling of pancreatic ductal carcinoma cell lines treated with trichostatin-A.
      ,
      • Cecconi D.
      • Astner H.
      • Donadelli M.
      • Palmieri M.
      • Missiaglia E.
      • Hamdan M.
      • Scarpa A.
      • Righetti P.G.
      Proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2′-deoxycytidine.
      )
       IPI00005160ARPC1BActin-related protein 2/3 complex subunit 1B2.560.722
       IPI00005171HLA-DRAHuman leukocyte antigen class II histocompatibility antigen2.561.256Yes (
      • Grutzmann R.
      • Boriss H.
      • Ammerpohl O.
      • Luttges J.
      • Kalthoff H.
      • Schackert H.K.
      • Kloppel G.
      • Saeger H.D.
      • Pilarsky C.
      Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes.
      ,
      • Aguirre A.J.
      • Brennan C.
      • Bailey G.
      • Sinha R.
      • Feng B.
      • Leo C.
      • Zhang Y.
      • Zhang J.
      • Gans J.D.
      • Bardeesy N.
      • Cauwels C.
      • Cordon-Cardo C.
      • Redston M.S.
      • DePinho R.A.
      • Chin L.
      High-resolution characterization of the pancreatic adenocarcinoma genome.
      ,
      • Han H.
      • Bearss D.J.
      • Browne L.W.
      • Calaluce R.
      • Nagle R.B.
      • Von Hoff D.D.
      Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray.
      )
       IPI00028091ACTR3Actin-like protein 32.500.254
       IPI00004656B2Mβ2-Microglobulin precursor2.500.564Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
       IPI00025721COPS3COP9 signalosome complex subunit 32.500.313
       IPI00027827SOD3Extracellular Cu,Zn-superoxide dismutase precursor2.500.562
       IPI00410714HBA1Hypothetical protein2.501.0010
       IPI00000760DDAH2NG-Dimethylarginine dimethylaminohydrolase 22.500.882Yes (
      • Aguirre A.J.
      • Brennan C.
      • Bailey G.
      • Sinha R.
      • Feng B.
      • Leo C.
      • Zhang Y.
      • Zhang J.
      • Gans J.D.
      • Bardeesy N.
      • Cauwels C.
      • Cordon-Cardo C.
      • Redston M.S.
      • DePinho R.A.
      • Chin L.
      High-resolution characterization of the pancreatic adenocarcinoma genome.
      )
       IPI00216318YWHAB14-3-3 protein β/α2.440.365Yes (
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
      • Radlwimmer B.
      • Dohner H.
      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      )
       IPI00556258
      Protein group, only one is listed.
      AKR1A1Aldo-keto reductase family 1, member B1 variant2.441.073
       IPI00465121
      Protein group, only one is listed.
      GNAI2i2 protein, GNAI2 protein2.440.483Yes (
      • Iacobuzio-Donahue C.A.
      • Maitra A.
      • Shen-Ong G.L.
      • van Heek T.
      • Ashfaq R.
      • Meyer R.
      • Walter K.
      • Berg K.
      • Hollingsworth M.A.
      • Cameron J.L.
      • Yeo C.J.
      • Kern S.E.
      • Goggins M.
      • Hruban R.H.
      Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.
      )
       IPI00032575C17orf25Hypothetical protein2.440.832
       IPI00294739SAMHD1Sterile α motif domain and histidine aspartate domain-containing protein 12.440.892
       IPI00465436CATCatalase2.380.573
       IPI00382696
      Protein group, only one is listed.
      FLNBFilamin-B (splice isoform)2.380.797Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Buchholz M.
      • Kestler H.A.
      • Bauer A.
      • Bock W.
      • Rau B.
      • Leder G.
      • Kratzer W.
      • Bommer M.
      • Scarpa A.
      • Schilling M.K.
      • Adler G.
      • Hoheisel J.D.
      • Gress T.M.
      Specialized DNA arrays for the differentiation of pancreatic tumors.
      )
       IPI00328415
      Protein group, only one is listed.
      CYB5R3NADH-cytochrome b5 reductase2.380.344
       IPI00383500
      Protein group, only one is listed.
      PLEKHC1Pleckstrin homology domain-containing family C member 12.380.172
       IPI00479186
      Protein group, only one is listed.
      PKM2Pyruvate kinase 32.331.306Yes (
      • Logsdon C.D.
      • Simeone D.M.
      • Binkley C.
      • Arumugam T.
      • Greenson J.K.
      • Giordano T.J.
      • Misek D.E.
      • Hanash S.
      Molecular profiling of pancreatic adenocarcinoma and chronic pancreatitis identifies multiple genes differentially regulated in pancreatic cancer.
      )
       IPI00555616SOD2Superoxide dismutase, Mn-superoxide dismutase, mitochondrial precursor2.330.592Yes (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      ,
      • Buchholz M.
      • Kestler H.A.
      • Bauer A.
      • Bock W.
      • Rau B.
      • Leder G.
      • Kratzer W.
      • Bommer M.
      • Scarpa A.
      • Schilling M.K.
      • Adler G.
      • Hoheisel J.D.
      • Gress T.M.
      Specialized DNA arrays for the differentiation of pancreatic tumors.
      ,
      • Grutzmann R.
      • Boriss H.
      • Ammerpohl O.
      • Luttges J.
      • Kalthoff H.
      • Schackert H.K.
      • Kloppel G.
      • Saeger H.D.
      • Pilarsky C.
      Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes.
      )
      Yes (
      • Crnogorac-Jurcevic T.
      • Gangeswaran R.
      • Bhakta V.
      • Capurso G.
      • Lattimore S.
      • Akada M.
      • Sunamura M.
      • Prime W.
      • Campbell F.
      • Brentnall T.A.
      • Costello E.
      • Neoptolemos J.
      • Lemoine N.R.
      Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma.
      )
       IPI00431795KBTBD9Kelch repeat and BTB (POZ) domain-containing 92.270.413
       IPI00007321LYPLA1Splice isoform 1 of acyl-protein thioesterase 12.270.672
       IPI00514020
      Protein group, only one is listed.
      GRHPRGlyoxylate reductase/hydroxypyruvate reductase2.220.203
       IPI00293276MIFMacrophage migration-inhibitory factor2.220.893Yes (
      • Sakai Y.
      • Masamune A.
      • Satoh A.
      • Nishihira J.
      • Yamagiwa T.
      • Shimosegawa T.
      Macrophage migration inhibitory factor is a critical mediator of severe acute pancreatitis.
      )
       IPI00019580PLGPlasminogen precursor2.170.522Yes (
      • Lugea A.
      • Nan L.
      • French S.W.
      • Bezerra J.A.
      • Gukovskaya A.S.
      • Pandol S.J.
      Pancreas recovery following cerulein-induced pancreatitis is impaired in plasminogen-deficient mice.
      )
       IPI00289862SCRN1Secernin-12.170.332
       IPI00003865HSPA8Heat shock cognate 71-kDa protein (splice isoform 1)2.131.008Yes (
      • Person M.D.
      • Shen J.
      • Traner A.
      • Hensley S.C.
      • Lo H.H.
      • Abbruzzese J.L.
      • Li D.
      Protein fragment domains identified using 2D gel electrophoresis/MALDI-TOF.
      )
       IPI00022078
      Protein group, only one is listed.
      NDRG1NDRG1 protein, hypothetical protein FLJ383302.130.363Yes
       IPI00017672NPPurine-nucleoside phosphorylase2.130.272
       IPI00218733SOD1Superoxide dismutase2.130.639Yes (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      ,
      • Crnogorac-Jurcevic T.
      • Efthimiou E.
      • Capelli P.
      • Blaveri E.
      • Baron A.
      • Terris B.
      • Jones M.
      • Tyson K.
      • Bassi C.
      • Scarpa A.
      • Lemoine N.R.
      Gene expression profiles of pancreatic cancer and stromal desmoplasia.
      ,
      • Cecconi D.
      • Scarpa A.
      • Donadelli M.
      • Palmieri M.
      • Hamdan M.
      • Astner H.
      • Righetti P.G.
      Proteomic profiling of pancreatic ductal carcinoma cell lines treated with trichostatin-A.
      ,
      • Cecconi D.
      • Astner H.
      • Donadelli M.
      • Palmieri M.
      • Missiaglia E.
      • Hamdan M.
      • Scarpa A.
      • Righetti P.G.
      Proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2′-deoxycytidine.
      ,
      • Prasad N.B.
      • Biankin A.V.
      • Fukushima N.
      • Maitra A.
      • Dhara S.
      • Elkahloun A.G.
      • Hruban R.H.
      • Goggins M.
      • Leach S.D.
      Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells.
      )
       IPI00005159
      Protein group, only one is listed.
      ACTR2Actin-like protein 22.080.227
       IPI00002243
      Protein group, only one is listed.
      GGTLA1γ-Glutamyltransferase 52.080.302
       IPI00383046CMBLHypothetical protein FLJ236172.080.562
       IPI00031545ITPR2Inositol 1,4,5-trisphosphate receptor type 2 (splice isoform long)2.080.302Yes (
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
      • Radlwimmer B.
      • Dohner H.
      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      )
       IPI00028416
      Protein group, only one is listed.
      PBX2Pre-B-cell leukemia transcription factor 22.080.522Yes (
      • Aguirre A.J.
      • Brennan C.
      • Bailey G.
      • Sinha R.
      • Feng B.
      • Leo C.
      • Zhang Y.
      • Zhang J.
      • Gans J.D.
      • Bardeesy N.
      • Cauwels C.
      • Cordon-Cardo C.
      • Redston M.S.
      • DePinho R.A.
      • Chin L.
      High-resolution characterization of the pancreatic adenocarcinoma genome.
      )
       IPI00017376
      Protein group, only one is listed.
      SEC23BProtein transport protein Sec23B, Sec23 (Saccharomyces cerevisiae) homolog B2.080.222
       IPI00164623C3Complement C3 precursor2.040.924Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      ,
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
      Yes (
      • Shalimov S.A.
      • Fedishin P.S.
      • Biletskii V.I.
      • Podpriatov S.E.
      Circulating immune complexes in the blood serum in acute pancreatitis.
      )
       IPI00021408
      Protein group, only one is listed.
      NUDT4Diphosphoinositol-polyphosphate phosphohydrolase2.040.672
       IPI00025427RNASE3Eosinophil cationic protein precursor2.040.292
       IPI00013219ILKIntegrin-linked protein kinase 12.040.293
       IPI00010471LCP1L-plastin2.040.875Yes (
      • Lu Z.
      • Hu L.
      • Evers S.
      • Chen J.
      • Shen Y.
      Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue.
      ,
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      )
       IPI00018195MAP2K3Mitogen-activated protein kinase 32.040.333
       IPI00246058PDCD6IPProgrammed cell death 6-interacting protein2.040.923Yes (
      • Lu Z.
      • Hu L.
      • Evers S.
      • Chen J.
      • Shen Y.
      Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue.
      ,
      • Bloomston M.
      • Zhou J.X.
      • Rosemurgy A.S.
      • Frankel W.
      • Muro-Cacho C.A.
      • Yeatman T.J.
      Fibrinogen gamma overexpression in pancreatic cancer identified by large-scale proteomic analysis of serum samples.
      )
       IPI00009634SQRDLSulfide:quinone oxidoreductase, mitochondrial precursor2.040.252
       IPI00293305
      Protein group, only one is listed.
      ITGB1Integrin β1 (splice isoform)2.000.364Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Crnogorac-Jurcevic T.
      • Gangeswaran R.
      • Bhakta V.
      • Capurso G.
      • Lattimore S.
      • Akada M.
      • Sunamura M.
      • Prime W.
      • Campbell F.
      • Brentnall T.A.
      • Costello E.
      • Neoptolemos J.
      • Lemoine N.R.
      Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma.
      )
       IPI00098902OGDHOxoglutarate2.000.322
       IPI00003817ARHGDIBRho GDP-dissociation inhibitor 22.000.242Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      )
      Less abundant by at lease 2-fold
       IPI00640411
      Protein group, only one is listed.
      HGDH2GD protein, homogentisate 1,2-dioxygenase0.220.032Yes (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      )
       IPI00216085
      Protein group, only one is listed.
      COX6B1Cytochrome c oxidase subunit VIb0.230.042
       IPI00397808
      Protein group, only one is listed.
      LOC388720Similar to bA92K2.2 (similar to ubiquitin)0.240.223
       IPI00382733
      Protein group, only one is listed.
      LRRFIP1Transcription repressor0.260.032
       IPI00478208
      Protein group, only one is listed.
      LOC64529660 S ribosomal protein L17, 21-kDa protein0.270.023
       IPI00178440EEF1B2Elongation factor 1-β0.290.037
       IPI00550689C22orf28HSPC117 protein0.370.042
       IPI00012772RPL860 S ribosomal protein L80.390.025
       IPI00022232CLPSColipase precursor0.400.0626Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      ,
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      ,
      • Grutzmann R.
      • Boriss H.
      • Ammerpohl O.
      • Luttges J.
      • Kalthoff H.
      • Schackert H.K.
      • Kloppel G.
      • Saeger H.D.
      • Pilarsky C.
      Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes.
      )
       IPI00477874
      Protein group, only one is listed.
      TATDN1Hepatocarcinoma high expression protein, TatD DNase domain-containing 10.410.183Yes (
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
      • Radlwimmer B.
      • Dohner H.
      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      )
       IPI00218568PCBD1Pterin-4-α-carbinolamine dehydratase0.410.083
       IPI00007676HSD17B12Steroid dehydrogenase homolog0.410.103
       IPI00219156RPL3060 S ribosomal protein L300.440.102
       IPI00023048
      Protein group, only one is listed.
      EEF1DElongation factor 1-δ0.440.082
       IPI00412579
      Protein group, only one is listed.
      RPL10A60 S ribosomal protein L10a0.450.054
       IPI00027626
      Protein group, only one is listed.
      CCT6AT-complex protein 10.480.073
       IPI00140420SND1Staphylococcal nuclease domain-containing protein 10.490.235
       IPI00010790
      Protein group, only one is listed.
      BGNBiglycan precursor0.500.052Yes (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      )
      Yes (
      • Iacobuzio-Donahue C.A.
      • Maitra A.
      • Shen-Ong G.L.
      • van Heek T.
      • Ashfaq R.
      • Meyer R.
      • Walter K.
      • Berg K.
      • Hollingsworth M.A.
      • Cameron J.L.
      • Yeo C.J.
      • Kern S.E.
      • Goggins M.
      • Hruban R.H.
      Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.
      ,
      • Qian J.
      • Niu J.
      • Li M.
      • Chiao P.J.
      • Tsao M.S.
      In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.
      )
       IPI00013452EPRSHypothetical protein DKFZp3313B0470.500.153
      a Protein group, only one is listed.

      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 (α2-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 3α (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 × 10−12), 2) response to wounding (p value = 1.24 × 10−11), 3) response to external stimulus (p value = 1.50 × 10−9), and 4) inflammatory response (p value = 1.93 × 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 (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      ) (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 β1, 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-κB1, and p53), suggesting their common roles in pancreatic cancer and chronic pancreatitis (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Biological 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, α2-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 β1—

      We previously reported overexpression of integrin β1 in pancreatic cancer (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      ). In the current study, integrin β1 was also overexpressed by 2.0-fold in chronic pancreatitis tissue. To further validate the overexpression of integrin β1 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) (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      ). In summary, the IHC analysis showed that primary and secondary chronic pancreatitis displayed some increased integrin β1 expression over normal pancreas, validating the result by ICAT analysis. Together these results suggest that integrin β1 expression is increased in chronic pancreatitis but to a lower degree compared with pancreatic cancer. Integrin β1 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 β1 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 β1 would be even further exacerbated in cancer, which has enhanced cell movement, tissue damage, and associated matrix response.
      Table IIIHC analysis of integrin β1, cathepsin D, and annexin A2
      Staining score
      Staining score: 0, negative; 1+, mild; 2+, moderate; 3+, strong.
      Normal pancreasPancreatic cancerChronic pancreatitis
      PrimarySecondary
      SESASESE
      Integrin β1
       01228637302312
       +101018490104
       +20010220103
       +300250000
      Cathepsin D
       01202632001
       +10239211117
       +20516112176
       +305381004553
      Annexin A2
       01201500000
       +10124753223
       +2002840096
       +30035118111410
      a Staining score: 0, negative; 1+, mild; 2+, moderate; 3+, strong.

      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 (
      • Shen J.
      • Person M.D.
      • Zhu J.
      • Abbruzzese J.L.
      • Li D.
      Protein expression profiles in pancreatic adenocarcinoma compared with normal pancreatic tissue and tissue affected by pancreatitis as detected by two-dimensional gel electrophoresis and mass spectrometry.
      ,
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      ,
      • Gronborg M.
      • Kristiansen T.Z.
      • Iwahori A.
      • Chang R.
      • Reddy R.
      • Sato N.
      • Molina H.
      • Jensen O.N.
      • Hruban R.H.
      • Goggins M.G.
      • Maitra A.
      • Pandey A.
      Biomarker discovery from pancreatic cancer secretome using a differential proteomic approach.
      ,
      • Lu Z.
      • Hu L.
      • Evers S.
      • Chen J.
      • Shen Y.
      Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue.
      ).
      Figure thumbnail gr2
      Fig. 2Identification 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 thumbnail gr3
      Fig. 3Cathepsin 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 (
      • Nomura T.
      • Katunuma N.
      Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells.
      ). 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 (
      • Berchem G.
      • Glondu M.
      • Gleizes M.
      • Brouillet J.P.
      • Vignon F.
      • Garcia M.
      • Liaudet-Coopman E.
      Cathepsin-D affects multiple tumor progression steps in vivo: proliferation, angiogenesis and apoptosis.
      ,
      • Liaudet-Coopman E.
      • Beaujouin M.
      • Derocq D.
      • Garcia M.
      • Glondu-Lassis M.
      • Laurent-Matha V.
      • Prebois C.
      • Rochefort H.
      • Vignon F.
      Cathepsin D: newly discovered functions of a long-standing aspartic protease in cancer and apoptosis.
      ). 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 (
      • Liaudet-Coopman E.
      • Beaujouin M.
      • Derocq D.
      • Garcia M.
      • Glondu-Lassis M.
      • Laurent-Matha V.
      • Prebois C.
      • Rochefort H.
      • Vignon F.
      Cathepsin D: newly discovered functions of a long-standing aspartic protease in cancer and apoptosis.
      ). Up-regulated cathepsin D has also been detected in the inflammatory setting (
      • Hausmann M.
      • Obermeier F.
      • Schreiter K.
      • Spottl T.
      • Falk W.
      • Scholmerich J.
      • Herfarth H.
      • Saftig P.
      • Rogler G.
      Cathepsin D is up-regulated in inflammatory bowel disease macrophages.
      ). 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 (
      • Chen R.
      • Pan S.
      • Cooke K.
      • Moyes K.W.
      • Bronner M.P.
      • Goodlett D.R.
      • Aebersold R.
      • Brentnall T.A.
      Comparison of pancreas juice proteins from cancer versus pancreatitis using quantitative proteomic analysis.
      ). 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 (
      • Syrovets T.
      • Simmet T.
      Novel aspects and new roles for the serine protease plasmin.
      ). 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 (
      • Andreasen P.A.
      • Egelund R.
      • Petersen H.H.
      The plasminogen activation system in tumor growth, invasion, and metastasis.
      ,
      • Schmitt M.
      • Harbeck N.
      • Thomssen C.
      • Wilhelm O.
      • Magdolen V.
      • Reuning U.
      • Ulm K.
      • Hofler H.
      • Janicke F.
      • Graeff H.
      Clinical impact of the plasminogen activation system in tumor invasion and metastasis: prognostic relevance and target for therapy.
      ).
      Figure thumbnail gr4
      Fig. 4Expression 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 β1, 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 γ, 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 (
      • Crnogorac-Jurcevic T.
      • Gangeswaran R.
      • Bhakta V.
      • Capurso G.
      • Lattimore S.
      • Akada M.
      • Sunamura M.
      • Prime W.
      • Campbell F.
      • Brentnall T.A.
      • Costello E.
      • Neoptolemos J.
      • Lemoine N.R.
      Proteomic analysis of chronic pancreatitis and pancreatic adenocarcinoma.
      ,
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      ,
      • Lu Z.
      • Hu L.
      • Evers S.
      • Chen J.
      • Shen Y.
      Differential expression profiling of human pancreatic adenocarcinoma and healthy pancreatic tissue.
      ,
      • Buchholz M.
      • Kestler H.A.
      • Bauer A.
      • Bock W.
      • Rau B.
      • Leder G.
      • Kratzer W.
      • Bommer M.
      • Scarpa A.
      • Schilling M.K.
      • Adler G.
      • Hoheisel J.D.
      • Gress T.M.
      Specialized DNA arrays for the differentiation of pancreatic tumors.
      ,
      • Sitek B.
      • Luttges J.
      • Marcus K.
      • Kloppel G.
      • Schmiegel W.
      • Meyer H.E.
      • Hahn S.A.
      • Stuhler K.
      Application of fluorescence difference gel electrophoresis saturation labelling for the analysis of microdissected precursor lesions of pancreatic ductal adenocarcinoma.
      ). 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 (
      • Chen R.
      • Yi E.C.
      • Donohoe D.
      • Pan S.
      • Eng J.
      • Crispin D.A.
      • Lane Z.
      • Goodlett D.A.
      • Bronner M.P.
      • Aebersold R.
      • Brentnall T.A.
      Pancreatic cancer proteome: the proteins that underlie invasion, metastasis, and immunologic escape.
      ). 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) (
      • Chen R.
      • Pan S.
      • Yi E.C.
      • Donohoe S.
      • Bronner M.P.
      • Potter J.D.
      • Goodlett D.R.
      • Aebersold R.
      • Brentnall T.A.
      Quantitative proteomic profiling of pancreatic cancer juice.
      ). 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 (
      • Flyvbjerg A.
      • Mogensen O.
      • Mogensen B.
      • Nielsen O.S.
      Elevated serum insulin-like growth factor-binding protein 2 (IGFBP-2) and decreased IGFBP-3 in epithelial ovarian cancer: correlation with cancer antigen 125 and tumor-associated trypsin inhibitor.
      ,
      • Muller H.L.
      • Oh Y.
      • Lehrnbecher T.
      • Blum W.F.
      • Rosenfeld R.G.
      Insulin-like growth factor-binding protein-2 concentrations in cerebrospinal fluid and serum of children with malignant solid tumors or acute leukemia.
      ,
      • Ranke M.B.
      • Maier K.P.
      • Schweizer R.
      • Stadler B.
      • Schleicher S.
      • Elmlinger M.W.
      • Flehmig B.
      Pilot study of elevated levels of insulin-like growth factor-binding protein-2 as indicators of hepatocellular carcinoma.
      ). In pancreatic cancer, the overexpression of IGFBP-2 has been reported in a cDNA microarray study (
      • Nakamura T.
      • Furukawa Y.
      • Nakagawa H.
      • Tsunoda T.
      • Ohigashi H.
      • Murata K.
      • Ishikawa O.
      • Ohgaki K.
      • Kashimura N.
      • Miyamoto M.
      • Hirano S.
      • Kondo S.
      • Katoh H.
      • Nakamura Y.
      • Katagiri T.
      Genome-wide cDNA microarray analysis of gene expression profiles in pancreatic cancers using populations of tumor cells and normal ductal epithelial cells selected for purity by laser microdissection.
      ). 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 β1, 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.

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