Acinar Cells Contribute to the Molecular Heterogeneity of Pancreatic Intraepithelial Neoplasia
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.061176
ISSN1525-2191
AutoresLiqin Zhu, Guanglu Shi, C. Max Schmidt, Ralph H. Hruban, Stephen F. Konieczny,
Tópico(s)Pancreatitis Pathology and Treatment
ResumoA number of studies have shown that pancreatic ductal adenocarcinoma develops through precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). PanINs are thought to initiate in the small ducts of the pancreas through activating mutations in the KRAS proto-oncogene. What remains unanswered is the identification of the individual cell type(s) that contributes to pancreatic ductal adenocarcinoma formation. To follow the cellular and molecular changes that occur in acinar and duct cell properties on KrasG12D expression, we took advantage of LSL-KrasG12D/+/p48Cre/+ mice, which faithfully mimic the human disease. In young animals (4 weeks), the predominant cellular alteration in the exocrine pancreas was acinar metaplasia in which individual acini consisted of acinar cells and duct-like cells. Metaplastic acinar structures were highly proliferative, expressed Notch target genes, and exhibited mosaic expression patterns for epidermal growth factor receptor, ErbB2, and pErk. This expression pattern paralleled the expression pattern detected in mouse PanINs, suggesting that mouse PanINs and acinar-ductal metaplasia follow similar molecular pathways. Indeed, immunofluorescence studies confirmed the presence of acinar cells within mPanIN lesions, raising the possibility that KrasG12D-induced mPanINs develop from acinar cells that undergo acinar-ductal metaplasia. Identification of an acinar contribution to PanIN formation offers new directions for successful targeted therapeutic approaches to combat this disease. A number of studies have shown that pancreatic ductal adenocarcinoma develops through precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). PanINs are thought to initiate in the small ducts of the pancreas through activating mutations in the KRAS proto-oncogene. What remains unanswered is the identification of the individual cell type(s) that contributes to pancreatic ductal adenocarcinoma formation. To follow the cellular and molecular changes that occur in acinar and duct cell properties on KrasG12D expression, we took advantage of LSL-KrasG12D/+/p48Cre/+ mice, which faithfully mimic the human disease. In young animals (4 weeks), the predominant cellular alteration in the exocrine pancreas was acinar metaplasia in which individual acini consisted of acinar cells and duct-like cells. Metaplastic acinar structures were highly proliferative, expressed Notch target genes, and exhibited mosaic expression patterns for epidermal growth factor receptor, ErbB2, and pErk. This expression pattern paralleled the expression pattern detected in mouse PanINs, suggesting that mouse PanINs and acinar-ductal metaplasia follow similar molecular pathways. Indeed, immunofluorescence studies confirmed the presence of acinar cells within mPanIN lesions, raising the possibility that KrasG12D-induced mPanINs develop from acinar cells that undergo acinar-ductal metaplasia. Identification of an acinar contribution to PanIN formation offers new directions for successful targeted therapeutic approaches to combat this disease. The development of individual cell types or organ systems progresses through restricted molecular and morphological pathways that ultimately define the endpoint phenotype. In most instances, the terminal differentiation state is static with cells primed to perform a final, highly specialized biological function. However, at times of stress, injury, or genetic alterations, cells are capable of exhibiting remarkable cellular plasticity in which tissues undergo metaplasia. Metaplasia is defined as the process by which one predominant adult cell type is replaced by a different adult cell type.1Li WC Yu WY Quinlan JM Burke ZD Tosh D The molecular basis of transdifferentiation.J Cell Mol Med. 2005; 9: 569-582Crossref PubMed Scopus (57) Google Scholar, 2Slack JM Tosh D Transdifferentiation and metaplasia—switching cell types.Curr Opin Genet Dev. 2001; 11: 581-586Crossref PubMed Scopus (161) Google Scholar This process occurs through a variety of mechanisms, including activation and expansion of quiescent stem cells with the concomitant removal of the unwanted cell population.3Tosh D Slack JM How cells change their phenotype.Nat Rev Mol Cell Biol. 2002; 3: 187-194Crossref PubMed Scopus (368) Google Scholar Metaplasia can also be achieved through transdifferentiation events in which individual cells are reprogrammed to transition from one differentiated cell type into another. In instances in which this process is relatively slow, biphenotypic cells can be identified that exhibit aspects of each differentiated cell, whereas in cases of rapid conversion identification of biphenotypic cells is elusive. Metaplasia is frequently associated with an increased risk of neoplasia and is often a hallmark of many human cancers. One cancer that shows metaplastic properties is pancreatic ductal adenocarcinoma (PDA). PDA is the fourth leading cause of cancer deaths in the United States with ∼213,000 new cases diagnosed world-wide each year.4Jemal A Tiwari RC Murray T Ghafoor A Samuels A Ward E Feuer EJ Thun MJ Cancer statistics, 2004.CA Cancer J Clin. 2004; 54: 8-29Crossref PubMed Scopus (3921) Google Scholar Despite extensive clinical efforts, the mortality of PDA patients has not significantly changed, and the 5-year survival rate (3 to 5%) remains unacceptably low. Although clinical progress has been slow, our understanding of the genetic and cellular events that precede PDA formation have been extensive and a common model for the development of PDA has emerged in which clinical, histopathological, and genetic studies have identified precursor lesions and genetic alterations that lead to PDA formation. The most common precursor lesions are known as pancreatic intraepithelial neoplasia (PanIN).5Hruban RH Adsay NV Albores-Saavedra J Compton C Garrett ES Goodman SN Kern SE Klimstra DS Kloppel G Longnecker DS Luttges J Offerhaus GJ Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions.Am J Surg Pathol. 2001; 25: 579-586Crossref PubMed Scopus (984) Google Scholar, 6Hruban RH Goggins M Parsons J Kern SE Progression model for pancreatic cancer.Clin Cancer Res. 2000; 6: 2969-2972PubMed Google Scholar, 7Hruban RH Wilentz RE Maitra A Identification and analysis of precursors to invasive pancreatic cancer.Methods Mol Med. 2005; 103: 1-13PubMed Google Scholar PanINs are thought to initiate in the small ducts of the pancreas and are classified from low grade to high grade (PanIN-1, PanIN-2, PanIN-3) based on the relative degree of cellular architecture and nuclear atypia. Molecular profiles for each grade of PanIN have been defined with activating mutations in the KRAS proto-oncogene thought to be early events in the development of PanIN-1 lesions. Additional genetic alterations occur temporally and include telomere shortening (PanIN-1), inactivation of the p16INK4a locus (PanIN-2), and inactivation of Trp53, SMAD4/DPC4, and occasionally BRCA2 (PanIN-3).5Hruban RH Adsay NV Albores-Saavedra J Compton C Garrett ES Goodman SN Kern SE Klimstra DS Kloppel G Longnecker DS Luttges J Offerhaus GJ Pancreatic intraepithelial neoplasia: a new nomenclature and classification system for pancreatic duct lesions.Am J Surg Pathol. 2001; 25: 579-586Crossref PubMed Scopus (984) Google Scholar, 6Hruban RH Goggins M Parsons J Kern SE Progression model for pancreatic cancer.Clin Cancer Res. 2000; 6: 2969-2972PubMed Google Scholar, 7Hruban RH Wilentz RE Maitra A Identification and analysis of precursors to invasive pancreatic cancer.Methods Mol Med. 2005; 103: 1-13PubMed Google Scholar These alterations cause pleiotropic effects that are reflected in the deregulation of signaling pathways controlling cell proliferation, survival, adhesion, and migration.8Schneider G Siveke JT Eckel F Schmid RM Pancreatic cancer: basic and clinical aspects.Gastroenterology. 2005; 128: 1606-1625Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, 9Prasad NB Biankin AV Fukushima N Maitra A Dhara S Elkahloun AG Hruban RH Goggins M Leach SD 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 (204) Google Scholar What remains unanswered is how these pathways generate unique pathologies within the PanIN and PDA spectrums. Equally important is identifying the individual cell types that contribute to this disease. To study the initiation and progression events that are responsible for PDA promotion, the endogenous mouse Kras locus has been targeted with a KrasG12D allele (LSL-KrasG12D).10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar, 11Jackson EL Willis N Mercer K Bronson RT Crowley D Montoya R Jacks T Tuveson DA Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras.Genes Dev. 2001; 15: 3243-3248Crossref PubMed Scopus (1460) Google Scholar In this model, the LSL-KrasG12D locus remains transcriptionally inert unless activated by Cre recombinase. Pancreas-specific Cre expression is provided by crossing LSL-KrasG12D/+ mice to pdx1-Cre10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar or p48Cre/+12Kawaguchi Y Cooper B Gannon M Ray M MacDonald RJ Wright CV The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors.Nat Genet. 2002; 32: 128-134Crossref PubMed Scopus (802) Google Scholar mice, which express Cre throughout the developing and adult pancreas. LSL-KrasG12D/+/pdx1-Cre and LSL-KrasG12D/+/p48Cre/+ mice develop PanINs (called mPanIN in the mouse) with 100% penetrance.10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar In all cases, the number and grade of the mPanIN lesions increase (mPanIN-1 → mPanIN-2 → mPanIN-3) with advancing age. Animals 2 to 3 months old contain primarily normal ducts (>80%), whereas the majority of the duct structures in older animals (7 to 10 months) exhibit high-grade mPanIN-3 to rare invasive and metastatic PDA.10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar Inclusion of additional genetic defects leads to more severe and rapid PDA disease.13Aguirre AJ Bardeesy N Sinha M Lopez L Tuveson DA Horner J Redston MS DePinho RA Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma.Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (823) Google Scholar, 14Bardeesy N Aguirre AJ Chu GC Cheng KH Lopez LV Hezel AF Feng B Brennan C Weissleder R Mahmood U Hanahan D Redston MS Chin L Depinho RA Both p16(Ink4a) and the p19(Arf)-p53 pathway constrain progression of pancreatic adenocarcinoma in the mouse.Proc Natl Acad Sci USA. 2006; 103: 5947-5952Crossref PubMed Scopus (463) Google Scholar, 15Hingorani SR Wang L Multani AS Combs C Deramaudt TB Hruban RH Rustgi AK Chang S Tuveson DA Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.Cancer Cell. 2005; 7: 469-483Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar Although there is convincing evidence that PanINs progress to PDA as they accumulate additional genetic alterations, there remains uncertainty as to the contribution of individual cell lineages in this progression scheme. For instance, it is unknown if duct cells are solely responsible for PanIN development or whether additional cell types (acinar, islet, stellate, adult progenitor cells) may contribute to the formation of PanIN-1 lesions. Preliminary data from several groups13Aguirre AJ Bardeesy N Sinha M Lopez L Tuveson DA Horner J Redston MS DePinho RA Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma.Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (823) Google Scholar, 15Hingorani SR Wang L Multani AS Combs C Deramaudt TB Hruban RH Rustgi AK Chang S Tuveson DA Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.Cancer Cell. 2005; 7: 469-483Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar, 16Miyamoto Y Maitra A Ghosh B Zechner U Argani P Iacobuzio-Donahue CA Sriuranpong V Iso T Meszoely IM Wolfe MS Hruban RH Ball DW Schmid RM Leach SD Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis.Cancer Cell. 2003; 3: 565-576Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar have shown that KrasG12D- or transforming growth factor (TGF)-α-induced PanIN structures often exhibit molecular heterogeneity, suggesting that mPanIN cells are not equivalent. Indeed, these initial observations support the concept that more than one pancreatic cell lineage may participate in PanIN formation and ultimately in PDA development. To investigate the initial changes that occur in acinar and duct cell properties on KrasG12D expression, we took advantage of the LSL-KrasG12D/+/p48Cre/+ model. Acinar-ductal metaplasia was the predominant alteration observed at 4 weeks in the pancreas. Metaplastic acinar structures were highly proliferative, expressed Notch target genes, and consisted of acinar and duct cell phenotypes. These cells also exhibited mosaic expression patterns for EGF signaling components, in which cells that retained acinar characteristics were epidermal growth factor receptor (EGFR)- and cytoplasmic ErbB2-positive but pErk-negative. In contrast, cells exhibiting a duct cell phenotype expressed high levels of pErk and nuclear ErbB2 but became EGFR-negative. Interestingly, the expression of these signaling intermediates precisely mimicked expression detected in PanIN lesions, suggesting that PanINs and acinar-ductal metaplasia follow similar molecular pathways. Indeed, early mPanIN-1 lesions consisted of both duct and acinar cell types, raising the possibility that KrasG12D-induced PanINs develop from acinar cell lineages that undergo acinar-ductal metaplasia to generate the characteristic ductal phenotype observed in PDA. Conditional LSL-KrasG12D/+, pdx1-Cre, and p48Cre/+ strains (gifts of D. Tuveson, Cambridge Research Institute, Cambridge, UK; and C. Wright, Vanderbilt University, Nashville, TN; respectively) were intercrossed to generate LSL-KrasG12D/+/p48Cre/+ and LSL-KrasG12D/+/pdx1-Cre mice as previously described.10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar Individual genotypes were confirmed by standard polymerase chain reaction (PCR) conditions using gene-specific primer sets.10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar All studies were conducted in compliance with the National Institutes of Health and the Purdue University Institutional Animal Care and Use Committee guidelines. Murine tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and 4-μm serial sections prepared. Routine hematoxylin and eosin (H&E) staining was performed by standard procedures. Immunohistochemistry was accomplished with biotinylated secondary antibodies using the Elite Vectastain ABC kit and peroxidase substrate diaminobenzidine kit (Vector Laboratories, Burlingame, CA). Briefly, sections were deparaffinized, rehydrated, and antigens were retrieved using the 2100-Retriever (PickCell Laboratories, Amsterdam, The Netherlands) and antigen unmasking solution (Vector Laboratories). For keratin 19 (K19) immunolabeling, antigen retrieval was done by digesting sections with 250 μg/ml proteinase K in 2.5 mmol/L CaCl2 and 10 mmol/L Tris-HCl, pH 7.5, for 6 minutes at room temperature. Samples were blocked using the MOM blocking reagent (Vector Laboratories). Primary antibodies were incubated at 4°C overnight and included rabbit amylase (1:1000; Calbiochem, San Diego, CA), rat K19 (TROMA-3, 1:100; a gift of Rolf Kemler, Department of Molecular Embryology, Max-Planck Institute, Freiburg, Germany), rabbit EGFR (1:300; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit ErbB2 (1:200; Santa Cruz Biotechnology), rabbit phospho-p44/42 MAPK (1:100; Cell Signaling, Charlottesville, VA), rabbit Hes1 (1:2000; a gift of Tetsuo Sudo, Pharmaceutical Research Laboratories, Toray Industries, Inc., Tebiro, Kamakura, Japan), mouse Ki67 (1:100; Novocastra, Newcastle On Tyne, UK), rabbit PDX1 (1:2500; a gift of Michael Rukstalis, Department of Molecular Endocrinology, Massachusetts General Hospital, Boston, MA), and rabbit Mist1 (1:2000).17Pin CL Bonvissuto AC Konieczny SF Mist1 expression is a common link among serous exocrine cells exhibiting regulated exocytosis.Anat Rec. 2000; 259: 157-167Crossref PubMed Scopus (101) Google Scholar, 18Pin CL Rukstalis JM Johnson C Konieczny SF The bHLH transcription factor Mist1 is required to maintain exocrine pancreas cell organization and acinar cell identity.J Cell Biol. 2001; 155: 519-530Crossref PubMed Scopus (214) Google Scholar For K19/amylase costaining, the K19 and amylase signals were visualized by diaminobenzidine and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium sequential detection following the manufacturer's (Vector Laboratories) recommendations. Whole cell protein extracts (50 μg) were separated on 7.5% acrylamide gels, transferred to polyvinylidene difluoride membranes, and incubated with primary antibodies against rabbit amylase (1:5000; Calbiochem), rat K19 (1:500), sheep carbonic anhydrase II (1:500; The Binding Site, Birmingham, UK), EGFR (1:1000, no. 2232; Cell Signaling), rabbit Neu/ErbB2 (1:1000, sc-284; Santa Cruz Biotechnology), and rabbit phospho-ErbBs (1:1000; Cell Signaling) including phospho-EGFR (Tyr845) (no. 2231), phospho-EGFR (Tyr992) (no. 2235), and phospho-Her2/ErbB2 (Tyr1221/1222) (6B12, no. 2243). Rabbit Hsp 90α/β (1:2000, sc-7947; Santa Cruz Biotechnology) was used as a loading control. After secondary antibody incubation, the immunoblots were developed using an enhanced chemiluminescence kit (Pierce, Rockford, IL) as per the manufacturer's instructions. Total RNA was isolated from the pancreas using the RNeasy isolation system (Qiagen, Valencia, CA). One μg of total RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). cDNA reactions were amplified with Taq polymerase and gene-specific primers for mouse Hes1 (5′-TCTACACCAGCAACAGTG-3′, 5′-TCAAACATCTTTGGCATCAC-3′), Hey1 (5′-GCGGACGAGAATGGAAACTTG-3′, 5′-GCTCAGATAACGGGCAACTTCG-3′), and Hey2 (5′-TGAGCATTGGATTCCGAGAGTG-3′, 5′-ATACCGACAAGGGTGGGCTGATTG-3′). Target sequences were amplified within the linear range using 95°C for 40-second, 55°C for 40-second, and 72°C for 55-second conditions. PanIN in humans is often associated with lobulocentric parenchymal atrophy with three distinct cellular compartments, ducts at the center of lobules with associated PanINs, a zone of acinar-ductal metaplasia, and areas of normal, differentiated acinar cells (Figure 1A).19Detlefsen S Sipos B Feyerabend B Kloppel G Pancreatic fibrosis associated with age and ductal papillary hyperplasia.Virchows Arch. 2005; 447: 800-805Crossref PubMed Scopus (138) Google Scholar, 20Brune K Abe T Canto M O'Malley L Klein AP Maitra A Adsay N Volkan Fishman EK Cameron JL Yeo CJ Kern SE Goggins M Hruban RH Multifocal neoplastic precursor lesions associated with lobular atrophy of the pancreas in patients having a strong family history of pancreatic cancer.Am J Surg Pathol. 2006; 30: 1067-1076PubMed Google Scholar The acinar-ductal metaplastic structures contain both acinar (zymogen granules, intense eosinophilic staining) and duct-like cells with a mucinous cytoplasm (Figure 1B). Despite these clinical observations, the relationship between PanINs and acinar structures in disease progression remains unclear. To study the molecular events involved in acinar-ductal metaplasia, we examined the LSL-KrasG12D/+/pdx1-Cre and LSL-KrasG12D/+/p48Cre/+ mouse models.10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar, 15Hingorani SR Wang L Multani AS Combs C Deramaudt TB Hruban RH Rustgi AK Chang S Tuveson DA Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.Cancer Cell. 2005; 7: 469-483Abstract Full Text Full Text PDF PubMed Scopus (1763) Google Scholar These animals develop ductal lesions (mPanINs)21Hruban RH Adsay NV Albores-Saavedra J Anver MR Biankin AV Boivin GP Furth EE Furukawa T Klein A Klimstra DS Kloppel G Lauwers GY Longnecker DS Luttges J Maitra A Offerhaus GJ Perez-Gallego L Redston M Tuveson DA Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations.Cancer Res. 2006; 66: 95-106Crossref PubMed Scopus (319) Google Scholar that recapitulate the full spectrum of human PanINs, eventually progressing to rare PDA. As with the human condition, the pancreatic lobules in the KrasG12D/+/pdx1-Cre and LSL-KrasG12D/+/p48Cre/+ mice developed three distinct zones of cells consisting of PanINs, acinar-ductal metaplasia, and normal acinar cells (Figure 1C). Examination of the mouse acinar-ductal metaplastic units again revealed the presence of acinar cells and duct-like cells within a single structure (Figure 1D). Thus, LSL-KrasG12D/+ mice faithfully model the human disease and provide an opportunity to identify the earliest events that participate in acinar-ductal metaplasia and mPanIN formation. Although PanINs are considered the precursor lesions to PDA, the contribution of acinar cells and acinar-ductal metaplasia to the initial stages of disease progression has not been extensively studied, in part because of the difficulty of following early events in patients. We examined young (4 to 6 weeks) LSL-KrasG12D/+/p48Cre/+ mice to investigate the earliest changes in acinar and duct cell properties that are induced on KrasG12D expression. As expected, wild-type and control LSL-KrasG12D/+ pancreata (lacking Cre) contained normal ducts and acinar cells and showed no signs of activation of the KrasG12D allele (Figure 1E). LSL-KrasG12D/+/p48Cre/+ pancreata similarly exhibited large areas of normal acinar and duct cells that were indistinguishable from control animals (Figure 1F). Indeed, at this age most ductal epithelium remained normal, comprised solely of cuboidal cells with uniform, round nuclei and amphophilic cytoplasm. mPanIN lesions were rarely observed. However, 4-week LSL-KrasG12D/+/p48Cre/+ samples did exhibit significant acinar metaplasia in which individual acini developed distended, open lumens and contained mucin-expressing duct-like cells (Figure 1, G and H). The duct-like cells showed a loss of zymogen granules and assumed a columnar appearance with atypical nuclei. To examine the molecular events that defined the acinar-ductal structures, LSL-KrasG12D/+/p48Cre/+ tissues were immunolabeled (immunohistochemistry) for amylase (acinar-specific) and K19 (duct-specific). At 4 weeks, individual acini were identified that expressed both amylase and K19 gene products (Figure 1I). In many cases, biphenotypic cells coexpressing amylase and K19 were observed. In contrast, normal ducts from wild-type or control LSL-KrasG12D/+ mice never contained amylase-positive cells, and normal acinar cells never expressed K19 (Figure 1J). The molecular changes from an acinar to ductal phenotype was also observed in older animals (4 months) by immunoblot analysis in which acinar (amylase) gene products decreased and duct (K19, CA II) gene products increased on activation of KrasG12D expression (Figure 1K). These results demonstrate that LSL-KrasG12D/+/p48Cre/+ mice undergo a dramatic shift from acinar to ductal cell types on activation of the KrasG12D gene. The early appearance of acinar metaplasia in LSL-KrasG12D/+/p48Cre/+ pancreas samples suggested that metaplastic acinar cells participate in disease progression. If true, metaplastic structures should exhibit an elevated cellular proliferation index. To examine this, 4-week control LSL-KrasG12D/+ and experimental LSL-KrasG12D/+/p48Cre/+ pancreata were analyzed for evidence of cellular proliferation. At this age, only 3.7% of acinar cells and 9.7% of duct cells from control LSL-KrasG12D/+ mice expressed the cell proliferation marker Ki67 (Figure 2A). In LSL-KrasG12D/+/p48Cre/+ mice the proliferation rate of histologically normal acinar and of ductal cells both increased (35.1 and 37.8%) (Figure 2B). However, the proliferation indices were highest in the metaplastic acinar structures. For the earliest metaplastic events (open lumens), multiple Ki67-positive acinar cells were always identified (Figure 2, C and D), whereas only random, single Ki67-positive acinar cells were detected in age-matched control LSL-KrasG12D/+ mice. As metaplastic structures grew in size, the percentage of Ki67-positive cells also increased (Figure 2, E and F). Similar results were obtained using a phospho-(ser10) histone 3 antibody (data not shown). Interestingly, most proliferating cells within a single structure exhibited a duct cell phenotype whereas the few remaining Ki67-negative cells retained acinar cell characteristics (zymogens) (Figure 2, E and F). These results suggest that cells within a metaplastic acinus actively divide only after converting to a duct-like cell. Although KrasG12D expression is activated in all acinar cells in LSL-KrasG12D/+/p48Cre/+ mice,10Hingorani SR Petricoin EF Maitra A Rajapakse V King C Jacobetz MA Ross S Conrads TP Veenstra TD Hitt BA Kawaguchi Y Johann D Liotta LA Crawford HC Putt ME Jacks T Wright CV Hruban RH Lowy AM Tuveson DA Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse.Cancer Cell. 2003; 4: 437-450Abstract Full Text Full Text PDF PubMed Scopus (1842) Google Scholar, 12Kawaguchi Y Cooper B Gannon M Ray M MacDonald RJ Wright CV The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors.Nat Genet. 2002; 32: 128-134Crossref PubMed Scopus (802) Google Scholar only a subset of the cells undergo acinar-ductal metaplasia and exhibit an increased proliferation rate. Differences in specific signaling pathways probably account for some of the observed alterations in the acini. One pathway that has been implicated in pancreatic cancer progression is Notch signaling. Induction of Notch activity is thought to be an initiating event of PanIN formation and pancreatic tumorigenesis, and this pathway may be critical to maintaining transformed cells in an undifferentiated state.16Miyamoto Y Maitra A Ghosh B Zechner U Argani P Iacobuzio-Donahue CA Sriuranpong V Iso T Meszoely IM Wolfe MS Hruban RH Ball DW Schmid RM Leach SD Notch mediates TGF alpha-induced changes in epithelial differentiation during pancreatic tumorigenesis.Cancer Cell. 2003; 3: 565-576Abstract Full Text Full Text PDF PubMed Scopus (584) Google Scholar, 22Jensen J Pedersen EE Galante P Hald J Heller RS Ishibashi M Kageyama R Guillemot F Serup P Madsen OD Control of endodermal endocrine development by Hes-1.Nat Genet. 2000; 24: 36-44Crossref PubMed Scopus (962) Google Scholar, 23Murtaugh LC Stanger BZ Kwan KM Melton DA Notch signaling controls multiple steps of pancreatic differentiation.Proc Natl Acad Sci USA. 2003; 100: 14920-14925Crossref PubMed Scopus (629) Google Scholar, 24Apelqvist A Li H Sommer L Beatus P Anderson DJ Honjo T Hrabe de Angelis M Lendahl U Edlund H Notch signalling controls pancreatic cell differentiation.Nature. 1999; 400: 877-881Crossref PubMed Scopus (993) Google Scholar, 25Jensen JN Cameron E Garay MV Starkey TW Gianani R Jensen J Recapitulation of elements of embryonic development in adult mouse pancreatic r
Referência(s)