Preinvasive Duct-Derived Neoplasms in Pancreas of Keratin 5–Promoter Cyclooxygenase-2 Transgenic Mice
2006; Elsevier BV; Volume: 130; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2006.03.053
ISSN1528-0012
AutoresKarin Müller‐Decker, Gerhard Fürstenberger, Nadine Annan, Dagmar Kucher, Andrea Pohl–Arnold, Brigitte Steinbauer, Iréne Esposito, Sara Chiblak, Helmut Friess, Peter Schirmacher, Irina Berger,
Tópico(s)Fibroblast Growth Factor Research
ResumoBackground & Aims: Basic research aimed at a better understanding of pancreatic carcinogenesis and improving the treatment of this disease is crucial because the majority of pancreatic cancers are highly aggressive and therapeutically nonaccessible. Cyclooxygenase (COX)-2, which is a key enzyme of prostaglandin (PG) biosynthesis, is overexpressed in around 75% of human carcinomas including those of the pancreas. Methods: The pathologic changes of transgenic mouse pancreas with keratin 5–promoter-driven expression and activity of COX-2 were characterized. Results: Aberrant expression of COX-2 in a few ductal cells and COX-2–mediated PG synthesis in the transgenic mice resulted in keratin 19– and mucin-positive intraductal papillary mucinous neoplasm- and pancreatic intraepithelial neoplasia-like structures, characterized by an increased proliferation index and serous cystadenomas. Moreover, Ras activation was enhanced and the HER-2/Neu receptor was overexpressed. Loss of acini, fibrosis, and inflammation were pronounced. Feeding a COX-2–selective inhibitor to the transgenic mice suppressed the accumulation of PG and the phenotype. The changes resemble the human disease in which COX-2 was overexpressed consistently. Conclusions: We present strong evidence for a causal relationship between aberrant COX-2 overexpression and COX-2–mediated PG synthesis and the development of serous cystadenoma, intraductal papillary mucinous, and pancreatic intraepithelial neoplasms. This model offers the unique possibility of identifying molecular pathways leading to the formation and malignant progression of the various types of preinvasive lesions of pancreatic adenocarcinomas that show different dismal outcomes. Background & Aims: Basic research aimed at a better understanding of pancreatic carcinogenesis and improving the treatment of this disease is crucial because the majority of pancreatic cancers are highly aggressive and therapeutically nonaccessible. Cyclooxygenase (COX)-2, which is a key enzyme of prostaglandin (PG) biosynthesis, is overexpressed in around 75% of human carcinomas including those of the pancreas. Methods: The pathologic changes of transgenic mouse pancreas with keratin 5–promoter-driven expression and activity of COX-2 were characterized. Results: Aberrant expression of COX-2 in a few ductal cells and COX-2–mediated PG synthesis in the transgenic mice resulted in keratin 19– and mucin-positive intraductal papillary mucinous neoplasm- and pancreatic intraepithelial neoplasia-like structures, characterized by an increased proliferation index and serous cystadenomas. Moreover, Ras activation was enhanced and the HER-2/Neu receptor was overexpressed. Loss of acini, fibrosis, and inflammation were pronounced. Feeding a COX-2–selective inhibitor to the transgenic mice suppressed the accumulation of PG and the phenotype. The changes resemble the human disease in which COX-2 was overexpressed consistently. Conclusions: We present strong evidence for a causal relationship between aberrant COX-2 overexpression and COX-2–mediated PG synthesis and the development of serous cystadenoma, intraductal papillary mucinous, and pancreatic intraepithelial neoplasms. This model offers the unique possibility of identifying molecular pathways leading to the formation and malignant progression of the various types of preinvasive lesions of pancreatic adenocarcinomas that show different dismal outcomes. Pancreatic ductal adenocarcinoma (PDAC) represents the major type of primary solid neoplasms of the exocrine pancreas. Cystic lesions of the exocrine pancreas are rare but increasingly recognized entities.1Adsay N.V. Klimstra D.S. Cystic forms of typically solid pancreatic tumors.Semin Diagn Pathol. 2000; 17: 81-88PubMed Google Scholar, 2Brugge W.R. Lauwers G.Y. Sahani D. Fernandez-del Castillo C. Warshaw A.L. Cystic neoplasms of the pancreas.N Engl J Med. 2004; 351: 1218-1226Crossref PubMed Scopus (591) Google Scholar A new classification of the cystic pancreatic lesions was proposed3Kosmahl M. Pauser U. Peters K. Sipos B. Lüttges J. Kremer B. Klöppel G. Cystic neoplasms of the pancreas and tumor-like lesions with cystic features a review of 418 cases and a classification proposal.Virchows Arch. 2004; 445: 168-178Crossref PubMed Scopus (290) Google Scholar that provides a distinction based on biology (neoplastic/nonneoplastic) and clinical behavior (benign/borderline/malignant). According to these criteria and excluding the cystic variant of PDAC, intraductal papillary mucinous neoplasms (IPMNs), followed by serous cystic neoplasms, represent the most common cystic epithelial tumors of the exocrine pancreas. Serous cystic neoplasms, which almost always are benign, are lined by a flat to cuboidal, glycogen-rich epithelium that expresses cytokeratins 7, 8, 18, and 19 (ie, a profile that is typical of the ductal cells).4Kosmahl M. Wagner J. Peters K. Sipos B. Kloppel G. Serous cystic neoplasms of the pancreas an immunohistochemical analysis revealing alpha-inhibin neuron-specific enolase and MUC 6 as new markers.Am J Surg Pathol. 2004; 28: 339-346Crossref PubMed Scopus (122) Google Scholar The immunohistologic profile and ultrastructural features suggest that serous cystic neoplasms originate from centroacinar cells of the pancreas.5Compagno J. Oertel J.E. Microcystic adenomas of the pancreas (glycogen-rich cystadenomas) a clinicopathologic study of 34 cases.Am J Clin Pathol. 1978; 69: 289-298Crossref PubMed Scopus (480) Google Scholar, 6Alpert L.C. Truong L.D. Bossart M.I. Spjut H.J. Microcystic adenoma (serous cystadenoma) of the pancreas. A study of 14 cases with immunohistochemical and electron-microscopic correlation.Am J Surg Pathol. 1988; 12: 251-263Crossref PubMed Scopus (99) Google Scholar, 7Compton C.C. Serous cystic tumors of the pancreas.Semin Diagn Pathol. 2000; 17: 43-55PubMed Google Scholar At the genetic level, serous cystic neoplasms are distinguished clearly from other pancreatic cystic tumors.8Gerdes B. Wild A. Wittenberg J. Barth P. Ramaswamy A. Kersting M. Luttges J. Kloppel G. Bartsch D.K. Tumor-suppressing pathways in cystic pancreatic tumors.Pancreas. 2003; 26: 42-48Crossref PubMed Scopus (41) Google Scholar IPMNs are defined as “grossly visible, noninvasive, mucin-producing, predominantly papillary, or rarely flat, epithelial neoplasms arising from the main pancreatic duct or branch ducts, with varying degrees of duct dilatation.”9Hruban R.H. Takaori K. Klimstra S.S. Adsay V. Albores-Saavedrs J. Biankin A.V. Biankin S.A. Compton C. Fukushima N. Goggins M. Kato Y. Kloppel G. Longnecker D.S. Luttges J. Maitra A. Offerhaus G.J. Shimizu M. Yonezawa S. An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms.Am J Surg Pathol. 2004; 28: 977-987Crossref PubMed Scopus (836) Google Scholar IPMNs can display a wide spectrum of cytologic and architectural atypia and therefore are classified further as benign (adenoma), borderline, and malignant (intraductal papillary mucinous carcinoma). Genetic studies have shown an accumulation of alterations in oncogenes (eg, K-ras, HER-2/Neu) and tumor-suppressor genes (eg, Ink4/Arf, Trp53) in the progression from IPMN adenoma to intraductal papillary mucinous carcinoma and invasive cancer10Z’graggen K. Rivera J.A. Compton C.C. Pins M. Werner J. Fernandez-del Castillo C. Rattner D.W. Lewandrowski K.B. Rustgi A.K. Warshaw A.L. Prevalence of activating K-ras mutations in the evolutionary stages of neoplasia in intraductal papillary mucinous tumors of the pancreas.Ann Surg. 1997; 226: 491-498Crossref PubMed Scopus (193) Google Scholar, 11Iacobuzio-Donahue C.A. Wilentz R.E. Argani P. Yeo C.J. Cameron J.L. Kern S.E. Hruban R.H. Dpc4 protein in mucinous cystic neoplasms of the pancreas frequent loss of expression in invasive carcinomas suggests a role in genetic progression.Am J Surg Pathol. 2000; 24: 1544-1548Crossref PubMed Scopus (131) Google Scholar, 12Wada K. P16 and p53 gene alterations and accumulations in the malignant evolution of intraductal papillary-mucinous tumors of the pancreas.J Hepatobiliary Pancreat Surg. 2002; 9: 76-85Crossref PubMed Scopus (57) Google Scholar, 13Sasaki S. Yamamoto H. Kaneto H. Ozeki I. Adachi Y. Takagi H. Matsumoto T. Itoh H. Nagakawa T. Miyakawa H. Muraoka S. Fujinaga A. Suga T. Satoh M. Itoh F. Endo T. Imai K. Differential roles of alterations of p53 p16 and SMAD4 expression in the progression of intraductal papillary-mucinous tumors of the pancreas.Oncol Rep. 2003; 10: 21-25PubMed Google Scholar that only partially recapitulate the molecular alterations that characterize the progression from pancreatic intraepithelial neoplasia (PanIN) to invasive PDAC.14Bardeesy N. DePinho R.A. Pancreatic cancer biology and genetics.Nature Rev. 2002; 2: 897-902Google Scholar The microscopic papillary or flat PanIN that arise from the small pancreatic ducts share common features with IPMNs, such as the intraductal neoplastic proliferations of columnar, mucin-containing cells with a variable degree of papillary growth. The columnar to cuboidal cells vary in the amount of mucin production and the degree of cytologic and architectural atypia.9Hruban R.H. Takaori K. Klimstra S.S. Adsay V. Albores-Saavedrs J. Biankin A.V. Biankin S.A. Compton C. Fukushima N. Goggins M. Kato Y. Kloppel G. Longnecker D.S. Luttges J. Maitra A. Offerhaus G.J. Shimizu M. Yonezawa S. An illustrated consensus on the classification of pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasms.Am J Surg Pathol. 2004; 28: 977-987Crossref PubMed Scopus (836) Google Scholar, 15Hruban R.H. Goggins M. Parsons J. Kern S.E. Progression model for pancreatic cancer.Clin Cancer Res. 2000; 6: 2969-2972PubMed Google Scholar Recently, transcriptomes of IPMN, PanIN, and PDAC were analyzed, showing the overexpression of distinct growth factors, cytokines, and proinflammatory proteins, including cyclooxygenase (COX)-2.16Yip-Schneider M.T. Barnard D.S. Billings S.D. Cheng L. Heilman D.K. Lin A. Marshall S.J. Crowell P.L. Marshall M.S. Sweeney C.J. Cyclooxygenase-2 expression in human pancreatic adenocarcinomas.Carcinogenesis. 2000; 21: 139-146Crossref PubMed Scopus (266) Google Scholar, 17Maitra A. Ashfaq R. Gunn C.R. Rahman A. Yeo C.J. Sohn T.A. Cameron J.L. Hruban R.H. Wilentz R.E. Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia an immunohistochemical analysis with automated cellular imaging.Am J Clin Pathol. 2002; 118: 194-201Crossref PubMed Scopus (148) Google Scholar, 18Niijima M. Yamaguchi T. Ishihara T. Hara T. Kato K. Kondo F. Saisho H. Immunohistochemical analysis and in situ hybridization of cyclooxygenase-2 expression in intraductal papillary-mucinous tumors of the pancreas.Cancer. 2002; 94: 1565-1573Crossref PubMed Scopus (56) Google Scholar, 19Iacobuzio-Donahue C.A. Ashfaq R. Maitra A. Adsay N.V. Shen-Ong G.L. Berg K. Hollingsworth M.A. Cameron J.L. Yeo C.J. Kern S.E. Goggins M. Hruban R.H. Highly expressed genes in pancreatic ductal adenocarcinomas a comprehensive characterization and comparison of the transcription profiles obtained from three major technologies.Cancer Res. 2003; 63: 8614-8622PubMed Google Scholar, 20Friess H. Ding J. Kleeff J. Fenkell L. Rosinski J.A. Guweidhi A. Reidhaar-Olson J.F. Korc M. Hammer J. Buchler M.W. Microarray-based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer.Cell Mol Life Sci. 2003; 60: 1180-1199PubMed Google Scholar, 21Sato N. Fukushima N. Maitra A. Iacobuzio-Donahue C.A. Tarda van Heek N. Cameron J.L. Yeo C.J. Hruban R.H. Goggins M. Gene expression profiling identifies genes associated with invasive intraductal papillary mucinous neoplasms of the pancreas.Am J Pathol. 2004; 164: 903-914Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 22Prasad N.B. Biankin A.V. Fukushima N. Aitra 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.Cancer Res. 2005; 65: 1619-1626Crossref PubMed Scopus (198) Google Scholar Similar to COX-2, the second known active COX isoform in mouse and human beings, COX-1 converts arachidonic acid to prostaglandin (PG) H2, which is metabolized by prostaglandin synthases into the bioactive PG.23Simmons D.L. Botting R.M. Hla T. Cyclooxygenase isozymes the biology of prostaglandin synthesis and inhibition.Pharmacol Rev. 2004; 56: 387-433Crossref PubMed Scopus (1310) Google Scholar, 24Helliwell R.J.A. Adams L.F. Mitchell M.D. Prostaglandin synthases recent developments and a novel hypothesis.Prostaglandins Leukot Essent Fatty Acids. 2004; 70: 101-113Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 25Hata A.N. Breyer R.M. Pharmacology and signaling of prostaglandin receptors multiple roles in inflammation and immune modulation.Pharmacol Ther. 2004; 103: 147-166Crossref PubMed Scopus (664) Google Scholar A large body of evidence from epidemiologic,26Thun M.J. Henley S.J. Patrono C. Nonsteroidal anti-inflammatory drugs as anticancer agents mechanistic pharmacologic and clinical issues.J Natl Cancer Inst. 2002; 94: 252-266Crossref PubMed Scopus (1260) Google Scholar genetic,27Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trazaskos J.M. Evans J.F. Taketo M.M. Suppression of intestinal polyposis in APC716 knockout mice by inhibition of cyclooxygenase-2 (COX-2).Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2268) Google Scholar, 28Chulada P.C. Thompson M.B. Mahler J.F. Doyle C.M. Gaul B.W. Lee C. Tiano H.F. Morham S.G. Smithies O. Langenbach R. Genetic disruption of Ptgs-1 as well as of Ptgs-2 reduces intestinal tumorigenesis in min mice.Cancer Res. 2000; 60: 4705-4708PubMed Google Scholar, 29Neufang G. Fürstenberger G. Heidt M. Marks F. Müller-Decker K. Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin.Proc Natl Acad Sci U S A. 2001; 98: 7629-7634Crossref PubMed Scopus (182) Google Scholar, 30Liu C.H. Chang S.H. Narko K. Trifan O.C. Wu M.T. Smith E. Haudenschild C. Lane T.F. Hla T. Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice.J Biol Chem. 2001; 276: 18563-18569Crossref PubMed Scopus (733) Google Scholar, 31Müller-Decker K. Neufang G. Berger I. Neumann M. Marks F. Fürstenberger G. Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis.Proc Natl Acad Sci U S A. 2002; 99: 12483-12488Crossref PubMed Scopus (230) Google Scholar, 32Tiano H.F. Loftin C.D. Akunda J. Lee C.A. Spalding J. Sessoms A. Dunson D.B. Rogan E.G. Morham S.G. Smart R.C. Langenbach R. Deficiency of either cyclooxygenase (COX)-1 or COX-2 alters epidermal differentiation and reduces mouse skin tumorigenesis.Cancer Res. 2002; 62: 3395-3401PubMed Google Scholar pharmacologic,31Müller-Decker K. Neufang G. Berger I. Neumann M. Marks F. Fürstenberger G. Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis.Proc Natl Acad Sci U S A. 2002; 99: 12483-12488Crossref PubMed Scopus (230) Google Scholar, 33Fischer S.M. Conti C.J. Viner J. Aldaz C.M. Lubert R.A. Celecoxib and difluoromethylornithine in combination have strong therapeutic activity against UV-induced skin tumors in mice.Carcinogenesis. 2003; 49: 709-713Google Scholar, 34Subbaramaiah K. Dannenberg A.J. Cyclooxygenase 2 a molecular target for cancer prevention and treatment.Trends Pharmacol Sci. 2003; 24: 96-102Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar and clinical studies35Steinbach G. Lynch P.M. Phillips R.K.S. Wallace M.H. Hawk E. Gordon G.B. Wakabayashi N. Saunders B. Shen Y. Fujimura T. et al.The effect of celecoxib, a cyclooxygenase-2 inhibitor, in familial adenomatous polyposis.N Engl J Med. 2000; 342: 1946-1952Crossref PubMed Scopus (2247) Google Scholar, 36Gasparini G. Longo R. Sarmiento R. Morabito A. Inhibitors of cyclo-oxygenase 2 a new class of anticancer agents?.Lancet Oncol. 2003; 4: 605-615Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 37Dannenberg A.J. Lippmann S.M. Mann J.R. Subbaramaiah K. DuBois R.N. Cyclooxygenase-2 and epidermal growth factor receptor pharmacologic targets for chemoprevention.J Clin Oncol. 2005; 23: 254-266Crossref PubMed Scopus (338) Google Scholar show that COX-2 is involved causally in the neoplastic progression of epithelial cells in colon, breast, and skin. Moreover, COX-2 may play an important role in pancreatic tumorigenesis. In human beings a large portion of PanIN lesions17Maitra A. Ashfaq R. Gunn C.R. Rahman A. Yeo C.J. Sohn T.A. Cameron J.L. Hruban R.H. Wilentz R.E. Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia an immunohistochemical analysis with automated cellular imaging.Am J Clin Pathol. 2002; 118: 194-201Crossref PubMed Scopus (148) Google Scholar and the majority of PDACs,17Maitra A. Ashfaq R. Gunn C.R. Rahman A. Yeo C.J. Sohn T.A. Cameron J.L. Hruban R.H. Wilentz R.E. Cyclooxygenase 2 expression in pancreatic adenocarcinoma and pancreatic intraepithelial neoplasia an immunohistochemical analysis with automated cellular imaging.Am J Clin Pathol. 2002; 118: 194-201Crossref PubMed Scopus (148) Google Scholar, 38Molina M.A. Sitja-Arnau M. Le Moine M.G. Frazier M.L. Sinicrope F.A. Increased cyclooxygenase-2 expression in human pancreatic carcinomas and cell lines growth inhibition by nonsteroidal anti-inflammatory drugs.Cancer Res. 1999; 59: 4356-4362PubMed Google Scholar, 39Okami J. Yamamoto H. Fujiwara Y. Tsujie M. Nagano H. Dono K. Umeshita K. Ishikawa O. Sakon M. et al.Overexpression of cyclooxygenase-2 in carcinoma of the pancreas.Clin Cancer Res. 1999; 5: 2018-2024PubMed Google Scholar, 40Tucker O.N. Dannenberg A.J. Yang E.K. Zhang F.Y. Teng L. Daly J.M. Soslow R.A. Masferrer J.L. Woerner B.M. Koki A.T. Fahey 3rd, T.J. Cyclooxygenase-2 expression is up-regulated in human pancreatic cancer.Cancer Res. 1999; 59: 987-990PubMed Google Scholar, 41Merati K. Said Siadaty M. Andrea A. Sarkar F. Ben-Josef E. Mohammed R. Philip P. Shields A.F. Vaitkevicius V. Grignon D.J. Adsay N.V. Expression of inflammatory modulator COX-2 in pancreatic ductal adenocarcinoma and its relationship to pathologic and clinical parameters.Am J Clin Oncol. 2001; 24: 447-452Crossref PubMed Scopus (70) Google Scholar serous cystadenomas (SCAs),39Okami J. Yamamoto H. Fujiwara Y. Tsujie M. Nagano H. Dono K. Umeshita K. Ishikawa O. Sakon M. et al.Overexpression of cyclooxygenase-2 in carcinoma of the pancreas.Clin Cancer Res. 1999; 5: 2018-2024PubMed Google Scholar and IPMNs18Niijima M. Yamaguchi T. Ishihara T. Hara T. Kato K. Kondo F. Saisho H. Immunohistochemical analysis and in situ hybridization of cyclooxygenase-2 expression in intraductal papillary-mucinous tumors of the pancreas.Cancer. 2002; 94: 1565-1573Crossref PubMed Scopus (56) Google Scholar, 42Kokawa A. Kondo H. Gotoda T. Ono H. Saito D. Nakadaira S. Kosuge T. Yoshida S. Increased expression of cyclooxygenase-2 in human pancreatic neoplasms and potential for chemoprevention by cyclooxygenase inhibitors.Cancer. 2001; 15: 333-338Crossref Scopus (179) Google Scholar were found to overexpress COX-2 as compared with normal duct epithelium of the pancreas. The constitutive expression of COX-1 was not modulated in the course of pancreatic tumor formation.43O’Neill G.P. Ford-Hutchinson A.W. Expression of mRNA for cyclooxygenase-1 and cyclooxygenase-2 in human tissues.FEBS Lett. 1993; 330: 156-160Abstract Full Text PDF PubMed Scopus (732) Google Scholar, 44Franco L. Doria D. Bertazzoni E. Benini A. Bassi C. Increased expression of inducible nitric oxide synthase and cyclooxygenase-2 in pancreatic cancer.Prostaglandins Other Lipid Mediat. 2004; 73: 51-58Crossref PubMed Scopus (35) Google Scholar Moreover, selective COX-245Nishikawa A. Furukawa F. Lee I.S. Tanaka T. Hirose M. Potent chemopreventive agents against pancreatic cancer.Curr Cancer Drug Targets. 2004; 4: 363-373Crossref Scopus (25) Google Scholar and nonselective COX inhibitors46Takahashi M. Furukawa F. Toyoda K. Sato H. Hasegawa R. Imaida K. Hayashi Y. Effects of various prostaglandin synthesis inhibitors on pancreatic carcinogenesis in hamsters after initiation with N-nitrosobis(2-oxopropyl)amine.Carcinogenesis. 1990; 11: 393-395Crossref PubMed Scopus (118) Google Scholar repressed tumor growth in the hamster model of N-nitrosobis(2-oxopropyl)amine–induced pancreatic cancer and in an orthotopic pancreatic tumor xenograft model of the mouse.47Raut C.P. Nawrocki S. Lashinger L.M. Davis D.W. Khanbolooki S. Xiong H. Ellis L.M. McConkey D.L. Celecoxib inhibits angiogenesis by inducing endothelial cell apoptosis in human pancreatic tumor xenografts.Cancer Biol Ther. 2004; 3: 1217-1224Crossref PubMed Scopus (56) Google Scholar We previously reported on heterozygous keratin 5 promoter (K5) COX-2 transgenic mouse lines that developed a pancreatic phenotype.29Neufang G. Fürstenberger G. Heidt M. Marks F. Müller-Decker K. Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin.Proc Natl Acad Sci U S A. 2001; 98: 7629-7634Crossref PubMed Scopus (182) Google Scholar The pancreatic phenotype of 4 heterozygous mouse lines (675+/−, 667+/−, 663+/−, and 4+/−) and 1 homozygous mouse line (675+/+) was diagnosed by pathologists and found to be similar, substantiating that the phenotype is transgene dependent and not caused by a positional effect. Here, we characterize in detail the pancreatic phenotype of the homozygous transgenic mouse line with keratin 5-driven COX-2 expression. We found a causal relationship between aberrant expression of COX-2 and COX-2–mediated PG synthesis in the pancreas and the development of dysplastic lesions characterized as SCA-like tumors and intraductal papillary mucinous lesions in large and small ducts that phenocopy human IPMN and PanIN. These changes were associated regularly with reactive changes in the surrounding parenchyma such as loss of acini and a fibroinflammatory reaction. The following materials were used and purchased from sources as indicated. Enzyme-linked immunosorbent assay–bovine serum albumin (Sigma, Deisenhofen, Germany); polyclonal rabbit anti-mouse Ki67 (Novacastra, Neuss, Germany); alkaline phosphatase–conjugated anti-rabbit IgG, peroxidase-conjugated anti-rat IgG, Cy3-conjugated anti-rabbit IgG, and Cy3-conjugated anti-rat IgG (Dianova, Hamburg, Germany); goat polyclonal anti-β-actin (SC-1616), anti-human COX-2 (SC-1745), affinity-purified rabbit polyclonal anti-HER-2/Neu-antibody (SC-284), and peroxidase-conjugated anti-rabbit IgG (SC-2020) and anti-mouse IgG (SC-2005) (Santa Cruz, Heidelberg, Germany); polyclonal rabbit anti-keratin 5 (Convance, Berkeley, CA); purified rat anti-mouse CD45R/B220 and CD4 (L3T4) monoclonal antibodies (BD Biosciences, Heidelberg, Germany); purified rat anti-mouse CD8α/Lyt-2 monoclonal antibodies (Southern Biotech, Birmingham, AL); rat anti-mouse F4/80 antiserum (Serotec, Düsseldorf, Germany); Alexa Fluor 488–conjugated anti-goat IgG (MobiTeC, Göttingen, Germany); PGE2- and PGF2α-enzyme immunoassay kits (Cayman, Ann Arbor, MI); aprotinin, leupeptin, and α2-macroglobulin (Roche Applied Sciences, Mannheim, Germany). Rat monoclonal anti-keratin 19 kindly was provided by R. Kemmler (Max-Planck-Institute, Freiburg, Germany). K5 COX-2 transgenic lines 675+/+ and 667+/− were generated and kept as described.29Neufang G. Fürstenberger G. Heidt M. Marks F. Müller-Decker K. Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin.Proc Natl Acad Sci U S A. 2001; 98: 7629-7634Crossref PubMed Scopus (182) Google Scholar, 31Müller-Decker K. Neufang G. Berger I. Neumann M. Marks F. Fürstenberger G. Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis.Proc Natl Acad Sci U S A. 2002; 99: 12483-12488Crossref PubMed Scopus (230) Google Scholar Age- and sex-matched wild-type (wt) Naval Medical Research Institute (NMRI) mice (outbred strain from RCC, Füllinsdorf, Switzerland) served as controls. Experiments were approved by the Governmental Committee for Animal Experimentation (license 053/00). Biopsy specimens were frozen immediately in liquid nitrogen or processed for histology or immunohistochemistry. To study the effect of a COX-2–selective inhibitor on the transgene-induced phenotype, a rodent diet 5010 (Purina Mills, St Louis, MO) containing 1500 ppm Celebrex (kindly provided by J. Masferrer, Pfizer, St Louis, MO) was fed to transgenic mice starting on day 1 after birth for 6–12 months. The diet was delivered to nursing mice for 4 weeks and later as regular chow. Paraffin-embedded samples of normal pancreas (n = 5), IPMN borderline (n = 4), IPMN adenomas (n = 6), PanIN lesions (n = 10), SCAs (n = 5), and mucinous cystadenomas (n = 5) were selected from the archives of the Institute of Pathology at the University of Heidelberg and used for immunohistochemistry. Immunoprecipitation of COX isozymes and immunoblot analysis were performed as described using isozyme-specific antisera.29Neufang G. Fürstenberger G. Heidt M. Marks F. Müller-Decker K. Abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 in skin.Proc Natl Acad Sci U S A. 2001; 98: 7629-7634Crossref PubMed Scopus (182) Google Scholar, 31Müller-Decker K. Neufang G. Berger I. Neumann M. Marks F. Fürstenberger G. Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis.Proc Natl Acad Sci U S A. 2002; 99: 12483-12488Crossref PubMed Scopus (230) Google Scholar For immunodetection of Neu gp 185, frozen pancreas powder was homogenized in a HP buffer (50 mmol/L Tris/HCl, pH 7.4, 1% Tween 20, 2 mmol/L ethylenediaminetetraacetic acid, 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 2 mmol/L α2-macroglobulin) and processed as described.48Müller-Decker K. Berger I. Ackermann K. Ehemann V. Zoubova S. Aulmann S. Pyerin W. Fürstenberger G. Cystic duct dilatations and proliferative epithelial lesions in mouse mammary glands upon keratin 5 promoter-driven overexpression of cyclooxygenase-2.Am J Pathol. 2005; 166: 575-584Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar The specificity of the immunosignals was confirmed by complete quenching of the immunosignal on preadsorption of the antiserum with a 500-fold molar excess of the respective peptide antigen. Ras activity was measured using a Ras Activation Assay Kit (Upstate, Lake Placid, NY) following the manufacturer’s instructions. Briefly, frozen pancreas powder was homogenized in 1 × Mg2+ lysis/wash buffer supplemented with 10 μg/mL each of aprotinin and leupeptin, 1 mmol/L sodium orthovanadate, and 25 mmol/L sodium fluoride, and kept on ice for 60 minutes. Lysates were centrifuged at 14,000g for 5 minutes. From the supernatant, 2 mg of protein each were used for a positive control (guanosine 5′-triphosphate [GTP]-γ-S–loaded at 30°C for 30 min), a negative control (guanosine 5′-diphosphate [GDP]-loaded at 30°C for 30 min), and a mock-treated sample. After termination of loading, Ras GTP was affinity precipitated from each sample using 20 μg of Ras assay reagent (Raf-1 Ras binding domain agarose/glutathione agarose beads) for 60 minutes at 4°C. The precipitates were washed 3 times with 1 × Mg2+ lysis/wash buffer and denatured in 40 μL 1 × Laemmli sample buffer and 1 mmol/L phenylmethylsulfonyl fluoride. Together with a recombinant p21 H-RasGly-12 as positive control (Oncogene, Boston, MA), the proteins were separated on a 15% sodium dodecyl sulfate–polyacrylamide gel. Ras protein was immunodetected with purified mouse monoclonal anti-Ras IgG2ακ (clone RAS 10, diluted 1:20,000) in blocking reagent (5% milk powder in phosphate-buffered saline, 0.1% Tween 20). Proteins were visualized with a goat anti-mouse secondary antibody horseradish-peroxidase conjugate and enhanced chemiluminescence detection (GE Healthcare BioSciences, Freiburg, Germany). To normalize the amount of GTP-bound Ras to the total amount of Ras, 120 μg/lane of ethanol-precipitated lysate protein were co-analyzed by immunoblotting as described earlier. As additional loading controls, 60 μg of protein of each sample were separated by 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. A quantitation of protein density (integrated density value) in each lane was performed by means of the spot denso module of the AlphaEase software of the ChemiImager (Biozym, Hess, Oldendorf, Germany). As a positive control for activated Ras we used a skin papilloma gained from a 2-stage carcinogenesis experiment.31Müller-Decker K. Neufang G. Berger I. Neumann M. Marks F. Fürstenberger G. Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis.Proc Natl Acad Sci U S A. 2002; 99: 12483-12488Crossref PubMed Scopus (230) Google Scholar Cryosections of mouse pancreas (5 μm) were used for H&E staining. Paraffin-embedded tissue sections (5 μm) served for staining of acidic mucopolysaccharides with Alcian blue (Sigma, Deisenhofen, Germany) and collagen fibers with Masson Goldner according to a routine protocol. Immunohistochemical (CD4, CD8, CD45R/B220, and COX-2) and immunofluorescence analysis (COX-2, keratin 5, keratin 19, Ki67, and F4/80) of mouse tissue were performed with 5-μm cryosections fixed in acetone for 10 minutes and processed as published.31Müller-Decker K. Neufang G. Berger I. Neumann M. Marks F. Fürstenberger G. Transgenic cyclooxygenase-2 overexpression sensitizes mouse skin for carcinogenesis.Proc Natl Acad Sci U S A. 2002; 99: 12483-12488Crossref PubMed
Referência(s)