Exploration of Global Gene Expression Patterns in Pancreatic Adenocarcinoma Using cDNA Microarrays
2003; Elsevier BV; Volume: 162; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)63911-9
ISSN1525-2191
AutoresChristine A. Iacobuzio‐Donahue, Anirban Maitra, Mari Olsen, Anson W. Lowe, N. Tjarda van Heek, Christophe Rosty, Walter Kim, Norihiro Sato, Antony R. Parker, Raheela Ashfaq, Elizabeth M. Jaffee, Byungwoo Ryu, Jessa Jones, James R. Eshleman, Charles J. Yeo, John L. Cameron, Scott E. Kern, Ralph H. Hruban, Patrick O. Brown, Michael Goggins,
Tópico(s)14-3-3 protein interactions
ResumoPancreatic cancer is the fifth leading cause of cancer death in the United States. We used cDNA microarrays to analyze global gene expression patterns in 14 pancreatic cancer cell lines, 17 resected infiltrating pancreatic cancer tissues, and 5 samples of normal pancreas to identify genes that are differentially expressed in pancreatic cancer. We found more than 400 cDNAs corresponding to genes that were differentially expressed in the pancreatic cancer tissues and cell lines as compared to normal pancreas. These genes that tended to be expressed at higher levels in pancreatic cancers were associated with a variety of processes, including cell-cell and cell-matrix interactions, cytoskeletal remodeling, proteolytic activity, and Ca++ homeostasis. Two prominent clusters of genes were related to the high rates of cellular proliferation in pancreatic cancer cell lines and the host desmoplastic response in the resected pancreatic cancer tissues. Of 149 genes identified as more highly expressed in the pancreatic cancers compared with normal pancreas, 103 genes have not been previously reported in association with pancreatic cancer. The expression patterns of 14 of these highly expressed genes were validated by either immunohistochemistry or reverse transcriptase-polymerase chain reaction as being expressed in pancreatic cancer. The overexpression of one gene in particular, 14-3-3σ, was found to be associated with aberrant hypomethylation in the majority of pancreatic cancers analyzed. The genes and expressed sequence tags presented in this study provide clues to the pathobiology of pancreatic cancer and implicate a large number of potentially new molecular markers for the detection and treatment of pancreatic cancer. Pancreatic cancer is the fifth leading cause of cancer death in the United States. We used cDNA microarrays to analyze global gene expression patterns in 14 pancreatic cancer cell lines, 17 resected infiltrating pancreatic cancer tissues, and 5 samples of normal pancreas to identify genes that are differentially expressed in pancreatic cancer. We found more than 400 cDNAs corresponding to genes that were differentially expressed in the pancreatic cancer tissues and cell lines as compared to normal pancreas. These genes that tended to be expressed at higher levels in pancreatic cancers were associated with a variety of processes, including cell-cell and cell-matrix interactions, cytoskeletal remodeling, proteolytic activity, and Ca++ homeostasis. Two prominent clusters of genes were related to the high rates of cellular proliferation in pancreatic cancer cell lines and the host desmoplastic response in the resected pancreatic cancer tissues. Of 149 genes identified as more highly expressed in the pancreatic cancers compared with normal pancreas, 103 genes have not been previously reported in association with pancreatic cancer. The expression patterns of 14 of these highly expressed genes were validated by either immunohistochemistry or reverse transcriptase-polymerase chain reaction as being expressed in pancreatic cancer. The overexpression of one gene in particular, 14-3-3σ, was found to be associated with aberrant hypomethylation in the majority of pancreatic cancers analyzed. The genes and expressed sequence tags presented in this study provide clues to the pathobiology of pancreatic cancer and implicate a large number of potentially new molecular markers for the detection and treatment of pancreatic cancer. Most pancreatic adenocarcinomas share similar genetic alterations and the clinical course of the disease is relatively homogenous.1Hruban RH Iacobuzio-Donahue C Wilentz RE Goggins M Kern SE Molecular pathology of pancreatic cancer.Cancer J. 2001; 7: 251-258PubMed Google Scholar The majority of pancreatic adenocarcinomas contain activating point mutations in the K-ras gene (>90% in most studies), and a significant number of these neoplasms also exhibit genetic inactivation of the p16 gene. A sizable group of genes has been found that are mutated less frequently in pancreatic cancers.2Goggins M Schutte M Lu J Moskaluk CA Weinstein CL Petersen GM Yeo CJ Jackson CE Lynch HT Hruban RH Kern SE Germline BRCA2 gene mutations in patients with apparently sporadic pancreatic carcinomas.Cancer Res. 1996; 56: 5360-5364PubMed Google Scholar, 3Su GH Hilgers W Shekher M Tang D Yeo CJ Hruban RH Kern SE Alterations in pancreatic, biliary, and breast carcinomas support MKK4 as a genetically targeted tumor-suppressor gene.Cancer Res. 1998; 58: 2339-2342PubMed Google Scholar Other molecular mechanisms that may contribute to carcinogenesis of the pancreas include overexpression of growth factors or their receptors, or changes in activity of signal transduction pathways.4Ebert M Yokoyama M Friess H Kobrin MS Buchler MW Korc M Induction of platelet-derived growth factor A and B chains and over-expression of their receptors in human pancreatic cancer.Int J Cancer. 1995; 62: 529-535Crossref PubMed Scopus (119) Google Scholar, 5Friess H Yamanaka Y Buchler M Berger HG Kobrin MS Baldwin RL Korc M Enhanced expression of the type II transforming growth factor beta receptor in human pancreatic cancer cells without alteration of type III receptor expression.Cancer Res. 1993; 53: 2704-2707PubMed Google Scholar Despite significant progress, much remains to be learned regarding the fundamental changes that occur in the development and progression of adenocarcinomas of the pancreas. Gene expression methodologies have shown promising utility in identifying novel markers or genes of interest in solid tumors, particularly in the study of pancreatic ductal adenocarcinoma. Compared to only 5 years ago, we are now aware of hundreds of genes with potential importance in the biology of pancreatic cancer.6Gress TM Wallrapp C Frohme M Muller-Pillasch F Lacher U Friess H Buchler M Adler G Hoheisel JD Identification of genes with specific expression in pancreatic cancer by cDNA representational difference analysis.Genes Chromosom Cancer. 1997; 19: 97-103Crossref PubMed Scopus (66) Google Scholar, 7Wallrapp C Muller-Pillasch F Micha A Wenger C Geng M Solinas-Toldo S Lichter P Frohme M Hoheisel JD Adler G Gress TM Novel technology for detection of genomic and transcriptional alterations in pancreatic cancer.Ann Oncol. 1999; 10: 64-68Crossref PubMed Scopus (6) Google Scholar, 8Han H Bearss DJ Browne LW Calaluce R Nagle RB Von Hoff DD Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray.Cancer Res. 2002; 62: 2890-2896PubMed Google Scholar, 9Gardner-Thorpe J Ito H Ashley SW Whang EE Differential display of expressed genes in pancreatic cancer cells.Biochem Biophys Res Commun. 2002; 293: 391-395Crossref PubMed Scopus (34) Google Scholar, 10Crnogorac-Jurcevic T Efthimiou E Nielsen T Loader J Terris B Stamp G Baron A Scarpa A Lemoine NR Expression profiling of microdissected pancreatic adenocarcinomas.Oncogene. 2002; 21: 4587-4594Crossref PubMed Scopus (192) Google Scholar, 11Iacobuzio-Donahue CA Maitra A Shen-Ong GL van Heek T Ashfaq R Meyer R Walter K Berg K Hollingsworth MA Cameron JL Yeo CJ Kern SE Goggins M Hruban RH Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.Am J Pathol. 2002; 160: 1239-1249Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar Strategies for identifying differentially expressed genes in pancreatic cancer have progressed from initial studies using gridded cDNA libraries,12Gress TM Muller-Pillasch F Geng M Zimmerhackl F Zehetner G Friess H Buchler M Adler G Lehrach H A pancreatic cancer-specific expression profile.Oncogene. 1996; 13: 1819-1830PubMed Google Scholar and later representational difference analysis of cDNAs,7Wallrapp C Muller-Pillasch F Micha A Wenger C Geng M Solinas-Toldo S Lichter P Frohme M Hoheisel JD Adler G Gress TM Novel technology for detection of genomic and transcriptional alterations in pancreatic cancer.Ann Oncol. 1999; 10: 64-68Crossref PubMed Scopus (6) Google Scholar, 9Gardner-Thorpe J Ito H Ashley SW Whang EE Differential display of expressed genes in pancreatic cancer cells.Biochem Biophys Res Commun. 2002; 293: 391-395Crossref PubMed Scopus (34) Google Scholar, 13Geng MM Ellenrieder V Wallrapp C Muller-Pillasch F Sommer G Adler G Gress TM Use of representational difference analysis to study the effect of TGFB on the expression profile of a pancreatic cancer cell line.Genes Chromosom Cancer. 1999; 26: 70-79Crossref PubMed Scopus (29) Google Scholar to the newer strategies of serial analysis of gene expression,14Ryu B Jones J Hollingsworth MA Hruban RH Kern SE Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers.Cancer Res. 2001; 61: 1833-1838PubMed Google Scholar, 15Ryu B Jones J Blades NJ Parmigiani G Hollingsworth MA Hruban RH Kern SE Relationships and differentially expressed genes among pancreatic cancers examined by large-scale serial analysis of gene expression.Cancer Res. 2002; 62: 819-826PubMed Google Scholar oligonucleotide microarrays,11Iacobuzio-Donahue CA Maitra A Shen-Ong GL van Heek T Ashfaq R Meyer R Walter K Berg K Hollingsworth MA Cameron JL Yeo CJ Kern SE Goggins M Hruban RH Discovery of novel tumor markers of pancreatic cancer using global gene expression technology.Am J Pathol. 2002; 160: 1239-1249Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar and cDNA microarrays.8Han H Bearss DJ Browne LW Calaluce R Nagle RB Von Hoff DD Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray.Cancer Res. 2002; 62: 2890-2896PubMed Google Scholar To differentiate gene expression patterns arising from the primary cancer from those arising in the surrounding stroma investigators have used several strategies including limiting their analysis to comparison of pancreatic cancer cell lines with normal pancreas,8Han H Bearss DJ Browne LW Calaluce R Nagle RB Von Hoff DD Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray.Cancer Res. 2002; 62: 2890-2896PubMed Google Scholar comparing pancreatic cancer cell lines with normal pancreatic ductal epithelium,15Ryu B Jones J Blades NJ Parmigiani G Hollingsworth MA Hruban RH Kern SE Relationships and differentially expressed genes among pancreatic cancers examined by large-scale serial analysis of gene expression.Cancer Res. 2002; 62: 819-826PubMed Google Scholar whereas other investigators have used laser capture microdissection.10Crnogorac-Jurcevic T Efthimiou E Nielsen T Loader J Terris B Stamp G Baron A Scarpa A Lemoine NR Expression profiling of microdissected pancreatic adenocarcinomas.Oncogene. 2002; 21: 4587-4594Crossref PubMed Scopus (192) Google Scholar Overexpressed genes now recognized as potentially important in pancreatic cancer include, but are not limited to, mesothelin,16Argani P Iacobuzio-Donahue C Ryu B Goggins M Rosty C Wilentz RE Murugesan S Kaushal M Leach SD Jaffee E Yeo CJ Cameron JL Kern SE Hruban RH Mesothelin is expressed in the vast majority of adenocarcinomas of the pancreas: identification of a new cancer marker by serial analysis of gene expression (SAGE).Clin Cancer Res. 2001; 7: 3862-3868PubMed Google Scholar prostate stem cell antigen,17Argani P Rosty C Reiter RE Wilentz RE Murugesan SD Leach SD Ryu B Skinner HG Goggins M Jaffee EM Yeo CJ Cameron JL Kern SE Hruban RH Discovery of new markers of cancer through serial analysis of gene expression (SAGE): prostate stem cell antigen (PSCA) is overexpressed in pancreatic adenocarcinoma.Cancer Res. 2001; 61: 4320-4324PubMed Google Scholar claudin-4,18Michl P Buchholz M Rolke M Kunsch S Lohr M McClane B Tsukita S Leder G Adler G Gress TM Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin.Gastroenterology. 2001; 121: 678-684Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar biglycan,19Weber CK Sommer G Michl P Fensterer H Weimer M Gansauge F Leder G Adler G Gress TM Biglycan is overexpressed in pancreatic cancer and induces G1-arrest in pancreatic cancer cell lines.Gastroenterology. 2001; 121: 657-667Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar S100A4,20Rosty C Ueki T Argani P Jansen M Yeo CJ Cameron JL Hruban RH Goggins M Overexpression of S100A4 in pancreatic ductal adenocarcinomas is associated with poor differentiation and DNA hypomethylation.Am J Pathol. 2002; 160: 45-50Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar TMPRSS3,21Wallrapp C Hahnel S Muller-Pillasch F Burghardt B Iwamura T Ruthenburger M Lerch MM Adler G Gress TM A novel transmembrane serine protease (TMPRSS3) overexpressed in pancreatic cancer.Cancer Res. 2000; 60: 2602-2606PubMed Google Scholar transglutaminase II,22Nishimura T Horino K Nishiura H Shibuya Y Hiraoka T Tanase S Yamamoto T Apoptotic cells of an epithelial cell line, AsPC-1, release monocyte chemotactic S19 ribosomal protein dimer.J Biochem (Tokyo). 2001; 129: 445-454Crossref PubMed Scopus (55) Google Scholar fascin, and hsp47.23Maitra A Iacobuzio-Donahue C Rahman A Sohn TA Argani P Meyer R Yeo CJ Cameron JL Goggins M Kern SE Ashfaq R Hruban RH Wilentz RE Immunohistochemical validation of a novel epithelial and a novel stromal marker of pancreatic ductal adenocarcinoma identified by global expression microarrays: sea urchin fascin homolog and heat shock protein 47.Am J Clin Pathol. 2002; 118: 52-59Crossref PubMed Scopus (151) Google Scholar The identification of these genes provides new opportunities for drug and therapeutic development aimed at targeting pancreatic cancers.18Michl P Buchholz M Rolke M Kunsch S Lohr M McClane B Tsukita S Leder G Adler G Gress TM Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin.Gastroenterology. 2001; 121: 678-684Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 24Hassan R Viner JL Wang QC Margulies I Kreitman RJ Pastan I Anti-tumor activity of K1-LysPE38QQR, an immunotoxin targeting mesothelin, a cell-surface antigen overexpressed in ovarian cancer and malignant mesothelioma.J Immunother. 2000; 23: 473-479Crossref PubMed Scopus (61) Google Scholar In an effort to further our efforts to identify novel genes highly expressed in pancreatic cancers with the potential for development into serological markers or therapeutic targets, we analyzed a large set of surgically resected pancreatic cancer tissues, pancreatic cancer cell lines, and normal pancreas tissues using a 45,000 cDNA microarray. The data presented not only confirm other earlier reports of highly expressed genes in pancreas cancer identified through a variety of approaches, but also provides new information regarding the genes and cellular pathways that play a role in this tumor type. Samples of primary invasive pancreatic ductal adenocarcinoma from pancreaticoduodenectomy specimens were collected from patients undergoing Whipple resections at the Johns Hopkins Hospital or the Stanford University School of Medicine. In each case, specimens of bulk tumor were harvested within 10 minutes of resection and snap-frozen in liquid nitrogen before storage at −80°C. Hematoxylin and eosin-stained sections of the adjacent tissue were prepared before snap-freezing to confirm the presence of infiltrating adenocarcinoma within the section. Human pancreatic cancer cell lines AsPc1, BxPc3,CAPAN1, CAPAN2, CFPAC1, Hs766T, MiaPaca2, Panc-1, and Su86.86 cell lines were obtained from the American Type Culture Collection, Rockville, MD. COLO357 was obtained from the European Collection of Animal Cell Cultures, Salisbury, UK. The pancreas cancer line (PL) cell lines used are low-passage pancreatic carcinoma cell lines established in our laboratories.25Jaffee EM Hruban RH Biedrzycki B Laheru D Schepers K Sauter PR Goemann M Coleman J Grochow L Donehower RC Lillemoe KD O'Reilly S Abrams RA Pardoll DM Cameron JL Yeo CJ Novel allogeneic granulocyte-macrophage colony-stimulating factor-secreting tumor vaccine for pancreatic cancer: a phase I trial of safety and immune activation.J Clin Oncol. 2001; 19: 145-156Crossref PubMed Scopus (517) Google Scholar, 26Jaffee EM Schutte M Gossett J Morsberger LA Adler AJ Thomas M Greten TF Hruban RH Yeo CJ Griffin CA Development and characterization of a cytokine-secreting pancreatic adenocarcinoma vaccine from primary tumors for use in clinical trials.Cancer J Sci Am. 1998; 4: 194-203PubMed Google Scholar Cell lines were grown in their recommended media. Use of different media minimized the variance in growth rates but presumably introduced other variations in gene expression patterns. Total RNA was obtained from homogenized frozen tissues and cell lines grown at 70 to 90% confluence were using TRIzol reagent (Life Technologies, Inc., Grand Island, NY). Polyadenylated mRNAs were purified from total RNA using the Fast Track 2.0 mRNA isolation kit (InVitrogen, Carlsbad, CA). cDNA microarrays were printed and used as previously described in detail27Iyer VR Eisen MB Ross DT Schuler G Moore T Lee JC Trent JM Staudt LM Hudson Jr, J Boguski MS Lashkari D Shalon D Botstein D Brown PO The transcriptional program in the response of human fibroblasts to serum.Science. 1999; 283: 83-87Crossref PubMed Scopus (1738) Google Scholar (detailed protocols are available at http://cmgm.Stanford.EDU/pbrown/). Briefly, mRNA from 11 different normal cultured cell lines were pooled and used as a reference control sample to prepare cDNA labeled with Cy3-deoxyuridine triphosphate (dUTP), and mRNA harvested from the 14 individual pancreatic cancer cell lines or 22 resected pancreas tissues (5 normal pancreas, 12 ductal adenocarcinomas, 2 ampullary carcinomas, 1 islet cell tumor, and 2 carcinomas arising in intraductal papillary mucinous neoplasms of the pancreas) was used to prepare cDNA labeled with Cy5-dUTP. The two differentially labeled cDNA probes were mixed and simultaneously hybridized to cDNA microarrays. Microarrays were scanned with a Genepix 4000B microarray scanner (Axon Instruments) using Genepix 5.0 software, and analyzed using the Cluster and TreeView programs (http://www.microarrays.org/software.html).28Eisen MB Spellman PT Brown PO Botstein D Cluster analysis and display of genome-wide expression patterns.Proc Natl Acad Sci USA. 1998; 95: 14863-14868Crossref PubMed Scopus (13338) Google Scholar The complete data for each sample described in this report are available through the Stanford Microarray Database site (http://genome-www4.stanford.edu/MicroArray/SMD/). Significance analysis of microarrays (SAM) v1.13 (http://www-stat.stanford.edu/∼tibs/SAM/)29Tusher VG Tibshirani R Chu G Significance analysis of microarrays applied to the ionizing radiation response.Proc Natl Acad Sci USA. 2001; 98: 5116-5121Crossref PubMed Scopus (9842) Google Scholar was used to perform the two-class comparison for differentially expressed genes between the 31 samples with pancreatic cancer (cell lines and resected cancer tissues) and the 5 samples of normal pancreas. The log-transformed and filtered gene expression data used for the original hierarchical cluster analysis described above was exported into an Excel 5.0 spreadsheet, and reformatted according to specifications outlined by the SAM v1.13 program. The K-nearest neighbor imputation was used to account for missing data within the dataset, and output criteria selected for SAM included at least threefold greater expression in the pancreatic cancers as compared to normal tissues, and a significance threshold expected to produce fewer than five false-positive genes. Complete data are available at http://genome-www.stanford.edu/pancreatic 1. Immunohistochemical analysis was performed to validate the translation and differential expression of selected genes in archival tissue sections of infiltrating pancreatic ductal adenocarcinomas. Adjacent sections of the infiltrating primary adenocarcinoma and normal nonneoplastic pancreatic tissue were formalin-fixed and paraffin-embedded. The proteins analyzed were S100A10, RON, Trop-2, cytokeratin 19, transglutaminase II, cdc-2, gamma synuclein, 14-3-3σ, and fibronectin. A detailed description of the methods involved in the immunolabeling of these proteins is available from the authors. Staining was evaluated by two of the authors (AM and RHH). Total RNA was isolated from 20 pancreatic cancer cell lines and an aliquot of 1 μg of total RNA from each sample was reverse-transcribed to cDNA using the SuperScript II kit (Life Technologies, Inc.) with oligo(dT)12-18 primer. Gene expression was compared against the simultaneous PCR of glyceraldehyde-3-phosphate dehydrogenase cDNA. Methylation status of the 5′ region of 14-3-3σ was determined by MSP as described previously.30Herman JG Graff JR Myohanen S Nelkin BD Baylin SB Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5261) Google Scholar PCR primers and conditions are provided in supplemental data. MiaPaCa2 cells were treated with a demethylating agent, 5-aza-2′-deoxycitidine (5-aza-dC; Sigma Chemical Co., St. Louis, MO) at a final concentration of 1 μmol/L for 5 days. Total RNA was isolated from the untreated and treated cells using Trizol and was subjected to RT-PCR for 14-3-3σ expression. Human cDNA microarrays containing 45,000 individual cDNAs were hybridized with cDNAs prepared from 14 pancreatic cancer cell lines, 5 samples of normal pancreatic tissue, and 17 samples of primary pancreatic cancer tumor tissue. The complete dataset is freely available at http://genome-www.stanford.edu/pancreatic1. Samples of normal pancreas were analyzed to provide a basis for assessing the contributions of acinar and islet cells to the gene expression profiles detected in primary tumors. Pancreas cancer cell lines were similarly analyzed to identify the gene expression patterns in the neoplastic cells. Those cDNAs with the greatest variation in expression among these samples were retained for the analysis. For each sample, the R/G ratio was normalized to the mean across all samples for each cDNA and log2-transformed. A filter was applied to remove those cDNAs whose expression did not vary by at least two standard deviations from the mean in this sample set in at least two of the samples. As a result, 1492 cDNAs were selected for use in the analyses described below. We analyzed the global gene expression patterns of pancreatic cancer to search for features that might provide insights into the biology of this tumor type. We first organized the data using hierarchical clustering of the cDNAs, the cell lines, and the tissue samples based on their global gene expression profiles (Figure 1, A and B). As expected, normal and tumor tissue samples clustered separately from the cell lines, primarily on the basis of differential expression of proliferation-related genes, which were much more highly expressed in the rapidly dividing cell lines, or the presence of nonneoplastic stromal and inflammatory cell gene expression within the tissue specimens. Among the tissues, normal pancreas was distinguished from invasive carcinomas, predominantly because of the presence of acinar and islet cell gene expression in the former (Figure 1A). Hierarchical clustering of the 1492 cDNAs based on the similarity in the patterns of gene expression revealed systematic features of the gene expression programs in these samples, which could be related to biological or histological features of the pancreatic samples (Figure 1B). For example, gene expression patterns related to acinar and islet cells were identified in normal pancreas, a gene expression pattern that appeared to be related to the desmoplastic response was noted in pancreas cancer tissues, and genes related to rates of cellular proliferation in cancer cell lines could be recognized. Two major clusters of genes were differentially expressed in the pancreatic cancer cell lines and primary pancreatic cancer tissues as compared to normal pancreas tissues. These pancreas cancer-specific clusters together contained 424 cDNAs. A detailed account of all of the cDNAs included within these various clusters are presented on our web page (http://pathology.jhu.edu/pancreas/microarray). The genes represented in the pancreas cancer-specific clusters appeared to reflect a diversity of functions, including cell-cell junctions (annexins A4 and A11; claudins 3, 4, and 7), cell/matrix interactions (integrin-α3 and -α6), cytoskeletal assembly (cytokeratins 7, 17, and 19; pleckstrin), cell-cycle regulation (Cdc42 effector protein 3), transcription factors (TCF7), tissue invasion (S100A4, S100P, S100A10, and S100A11), proteolytic processing (urokinase plasminogen activator; matrix metalloproteinases 7, 14, and 24), and interferon- or retinoic acid-induced functions (interferon gamma-induced protein 16; interferon α-induced protein 27; interferon-induced transmembrane proteins 1, 2, and 3; retinoic acid receptor responder 3; retinoic acid receptor gamma). To confirm the expression of genes identified by hierarchical clustering, the expression patterns of four of the genes represented in the pancreas cancer-specific clusters (S100A10, Trop-2, RON, and cytokeratin 19)15Ryu B Jones J Blades NJ Parmigiani G Hollingsworth MA Hruban RH Kern SE Relationships and differentially expressed genes among pancreatic cancers examined by large-scale serial analysis of gene expression.Cancer Res. 2002; 62: 819-826PubMed Google Scholar, 31Schussler MH Skoudy A Ramaekers F Real FX Intermediate filaments as differentiation markers of normal pancreas and pancreas cancer.Am J Surg Pathol. 1992; 140: 559-568Google Scholar were analyzed by immunohistochemical labeling of paraffin-embedded sections of 6 of the 17 pancreas ductal adenocarcinomas analyzed by cDNA microarray. Strong labeling of the neoplastic epithelium was seen with each marker, in contrast to the weak to negative labeling of the normal duct epithelium present in the same tissue sections. However, three of the four markers (S100A10, Trop-2, and RON) also showed variable amounts of labeling of the surrounding stromal tissues, whereas cytokeratin 19 labeled the neoplastic epithelium of the infiltrating carcinomas only (see supplementary figure at http://pathology.jhu.edu/pancreas/microarray/supplementfigure1.cfm). Two additional and informative clusters were found among the 1492 cDNAs analyzed by hierarchical cluster analysis. The first cluster associated with cellular proliferation has been well described. This proliferation cluster included chromosome-remodeling genes (ie, SMC4-like 1), cell cycle-regulating genes (ie, cyclin A2), and genes associated with cytoskeletal remodeling (ie, myosin heavy polypeptide 1). Proliferating cell nuclear antigen was also present in this cluster. Pancreatic cancer cell lines showed high levels of expression of these genes, in contrast to primary tumor tissues that had low levels of expression. Pancreatic cancers are well recognized for their exuberant host stromal response to invasive carcinoma, known as desmoplasia, which often accounts for the majority of the cellularity of the actual mass produced by the carcinoma.14Ryu B Jones J Hollingsworth MA Hruban RH Kern SE Invasion-specific genes in malignancy: serial analysis of gene expression comparisons of primary and passaged cancers.Cancer Res. 2001; 61: 1833-1838PubMed Google Scholar, 32Iacobuzio-Donahue CA Ryu B Hruban RH Kern SE Exploring the host desmoplastic response to pancreatic carcinoma: gene expression of stromal and neoplastic cells at the site of primary invasion.Am J Pathol. 2002; 160: 91-99Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 33Crnogorac-Jurcevic T Efthimiou E Capelli P Blaveri E Baron A Terris B Jones M Tyson K Bassi C Scarpa A Lemoine NR Gene expression profiles of pancreatic cancer and stromal desmoplasia.Oncogene. 2001; 20: 7437-7446Crossref PubMed Scopus (176) Google Scholar A cluster of genes that appeared to be related to this prominent desmoplastic response was also identified by hierarchical cluster analysis. This cluster of several genes highly expressed in the invasive pancreatic tumor tissues as compared to pancreas cancer cell lines or normal pancreas included collagen 1α1 and 1α2, matrix metalloproteinases and their inhibitors, apolipoprotein C-1 and C-II, hevin, osteonectin and biglycan. Some of these genes (biglycan, MMPs, TIMP1) have been previously identified as overexpressed in pancreatic cancer.10Crnogorac-Jurcevic T Efthimiou E Nielsen T Loader J Terris B Stamp G Baron A Scarpa A Lemoine NR Expression profiling of microdissected pancreatic adenocarcinomas.Oncogene. 2002; 21: 4587-4594Crossref PubMed Scopus (192) Google Scholar, 19Weber CK Sommer G Michl P Fensterer H Weimer M Gansauge F Leder G Adler G Gress TM Biglycan is overexpressed in pancreatic cancer and induces G1-arrest in pancreatic cancer cell lines.Gastroenterology. 2001; 121: 657-667Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 34Ellenrieder V Hendler SF Boeck W Seufferlein T Menke A Ruhland C Adler G Gress TM Transforming growth factor beta1 treatment leads to an epithelial-mesenchymal transdifferentiation of pancreatic cancer cells requiring extracellular signal-regulated kinase 2 activation.Cancer Res. 2001; 61: 4222-4228PubMed Google Scholar To determine those genes with statistically significant differences in expression in pancreatic cancer cell lines and pancreatic cancer tissues compared to normal pancreas, we used Significance Analysis of Microarray (SAM) as described in the Materials and Methods section. Using a threefold differential cutoff, we found 216 cDNAs expressed at higher levels and 236 cDNAs expressed at lower levels in pancreatic cancers compared to normal pancreatic tissues at a rate of five false-po
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