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Trp53 Deletion Stimulates the Formation of Metastatic Pancreatic Tumors

2008; Elsevier BV; Volume: 172; Issue: 4 Linguagem: Inglês

10.2353/ajpath.2008.070778

1525-2191

Jennifer P. Morton, David S. Klimstra, Michelle E. Mongeau, Brian C. Lewis,

Cancer Research and Treatments

The presence of distant metastases is a common finding on diagnosis of pancreatic cancer; however, the mechanisms underlying the dissemination of this tumor type remain poorly understood. Loss of the p53 tumor suppressor protein has been associated with tumor progression and metastasis in several tumor types including pancreatic ductal adenocarcinoma. Here, we describe the generation of a progressive and metastatic pancreatic cancer mouse model after the somatic and sporadic delivery of avian retroviruses encoding the mouse polyoma virus middle T antigen to elastase-tv-a transgenic mice with a pancreas-specific deletion of the Trp53 tumor suppressor locus. In this model, the tumors metastasize most frequently to the liver, consistent with human pancreatic carcinomas. Analysis of metastatic lesions demonstrated that concomitant loss of the Ink4a/Arf locus was not required for metastasis; however, pancreas-specific deletion of a single Ink4a/Arf allele cooperated with Trp53 deletion in a haploinsufficient manner to accelerate tumor development. Thus, our findings illustrate the potential role of p53 loss of function in pancreatic tumor progression, demonstrate the feasibility of modeling pancreatic cancer metastasis after somatic and sporadic oncogene activation, and indicate that our model may provide a useful experimental system for investigation of the molecular mechanisms underlying pancreatic cancer progression and metastasis. The presence of distant metastases is a common finding on diagnosis of pancreatic cancer; however, the mechanisms underlying the dissemination of this tumor type remain poorly understood. Loss of the p53 tumor suppressor protein has been associated with tumor progression and metastasis in several tumor types including pancreatic ductal adenocarcinoma. Here, we describe the generation of a progressive and metastatic pancreatic cancer mouse model after the somatic and sporadic delivery of avian retroviruses encoding the mouse polyoma virus middle T antigen to elastase-tv-a transgenic mice with a pancreas-specific deletion of the Trp53 tumor suppressor locus. In this model, the tumors metastasize most frequently to the liver, consistent with human pancreatic carcinomas. Analysis of metastatic lesions demonstrated that concomitant loss of the Ink4a/Arf locus was not required for metastasis; however, pancreas-specific deletion of a single Ink4a/Arf allele cooperated with Trp53 deletion in a haploinsufficient manner to accelerate tumor development. Thus, our findings illustrate the potential role of p53 loss of function in pancreatic tumor progression, demonstrate the feasibility of modeling pancreatic cancer metastasis after somatic and sporadic oncogene activation, and indicate that our model may provide a useful experimental system for investigation of the molecular mechanisms underlying pancreatic cancer progression and metastasis. Pancreatic cancer is the fourth-leading cause of cancer-related mortalities in the United States, with ∼32,000 deaths annually.1Jemal A Siegel R Ward E Murray T Xu J Smigal C Thun MJ Cancer statistics, 2006.CA Cancer J Clin. 2006; 56: 106-130Crossref PubMed Scopus (5549) Google Scholar The median survival after diagnosis is 6 months, and the 5-year survival rate is only 5%. These statistics reflect the advanced stage at which most pancreatic tumors are identified, exemplified by extra-pancreatic invasion and metastasis to the liver and peritoneum, and the resistance of pancreatic cancers to conventional chemotherapeutic intervention.2Hezel AF Kimmelman AC Stanger BZ Bardeesy N Depinho RA Genetics and biology of pancreatic ductal adenocarcinoma.Genes Dev. 2006; 20: 1218-1249Crossref PubMed Scopus (902) Google Scholar, 3Warshaw AL Fernandez-del Castillo C Pancreatic carcinoma.N Engl J Med. 1992; 326: 455-465Crossref PubMed Scopus (1559) Google Scholar Thus, understanding the factors that contribute to pancreatic tumor progression and metastasis is important in combating this disease. Mouse models are attractive experimental systems for exploring the genesis and behavior of human malignancies, and as a result, several mouse models for pancreatic cancer have been generated using transgenic approaches.2Hezel AF Kimmelman AC Stanger BZ Bardeesy N Depinho RA Genetics and biology of pancreatic ductal adenocarcinoma.Genes Dev. 2006; 20: 1218-1249Crossref PubMed Scopus (902) Google Scholar, 4Hruban RH Rustgi AK Brentnall TA Tempero MA Wright CV Tuveson DA Pancreatic cancer in mice and man: the Penn Workshop 2004.Cancer Res. 2006; 66: 14-17Crossref PubMed Scopus (47) Google Scholar, 5Leach SD Mouse models of pancreatic cancer: the fur is finally flying!.Cancer Cell. 2004; 5: 7-11Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar These models, although valuable, were restricted in several respects including the expression of the oncogene throughout pancreatic development. Indeed, in some of the published models mice die shortly after birth, or perinatally, with architecturally abnormal pancreata.6Quaife CJ Pinkert CA Ornitz DM Palmiter RD Brinster RL Pancreatic neoplasia induced by ras expression in acinar cells of transgenic mice.Cell. 1987; 48: 1023-1034Abstract Full Text PDF PubMed Scopus (196) Google Scholar Further, because all cells of a particular lineage within the pancreas express the transgene, tumors arise in an environment of aberrant intercellular signaling, unlike the scenario of sporadic tumor development in humans, in which tumor cells are surrounded by genetically normal cells. To address these issues, we recently generated a mouse model for pancreatic cancer using the RCAS-TVA gene delivery system.7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar This system allows the sporadic postnatal delivery of oncogene-bearing avian retroviruses to targeted cells engineered to express the avian leukosis virus subgroup A (ALV-A) receptor TVA.8Orsulic S An RCAS-TVA-based approach to designer mouse models.Mamm Genome. 2002; 13: 543-547Crossref PubMed Scopus (53) Google Scholar Delivery of ALV-A-derived RCAS viruses encoding the mouse polyoma virus middle T antigen (PyMT) led to the formation of ductal precursor lesions in transgenic mice in which the elastase promoter directs tv-a expression.7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar If the Ink4a/Arf locus was also inactivated in these mice, acinar carcinomas and ductal lesions, either precursor PanIN lesions or cystadenocarcinomas, developed. Interestingly, the tumors were positive for the progenitor cell marker Pdx1, and aberrantly expressed the neuroendocrine marker synaptophysin, whose expression within the pancreas is normally restricted to endocrine cells of the islets of Langerhans. The induction of multiple tumor types, and the expression of progenitor and endocrine markers, suggested that the tumors were derived from the transformation of pancreatic progenitor cells. Consistent with this hypothesis, introduction of RCAS viruses encoding c-Myc into elastase-tv-a transgenic mice deficient at the Ink4a/Arf locus led to the formation of pancreatic endocrine neoplasms exclusively. These findings demonstrated the influence of the initiating oncogenic lesion on the tumor phenotype. Yet, the effects of tumor suppressor gene mutations on progression to invasive and metastatic disease in our model remain unclear, in part attributable to the shortened lifespan of animals with germline tumor suppressor gene deletion.9Donehower LA Harvey M Slagle BL McArthur MJ Montgomery Jr, CA Butel JS Bradley A Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours.Nature. 1992; 356: 215-221Crossref PubMed Scopus (4078) Google Scholar, 10Jacks T Remington L Williams BO Schmitt EM Halachmi S Bronson RT Weinberg RA Tumor spectrum analysis in p53-mutant mice.Curr Biol. 1994; 4: 1-7Abstract Full Text Full Text PDF PubMed Scopus (1755) Google Scholar, 11Serrano M Lee H Chin L Cordon-Cardo C Beach D DePinho RA Role of the INK4a locus in tumor suppression and cell mortality.Cell. 1996; 85: 27-37Abstract Full Text Full Text PDF PubMed Scopus (1420) Google Scholar Recently published studies by others suggested that tumor suppressor gene mutations could indeed influence the tumor phenotype. An endogenously expressed activated Kras allele induced moderately differentiated pancreatic ductal adenocarcinomas with metastatic potential in animals with pancreas-specific deletion of Trp53, but induced undifferentiated carcinomas that invade adjacent tissues, but do not readily form distant metastases, in animals with pancreas-specific Ink4a/Arf loss.12Aguirre 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 (828) Google Scholar, 13Hingorani 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 (1797) Google Scholar Here we report that pancreas-specific deletion of Trp53 cooperates with somatic and sporadic PyMT expression to induce both acinar cell carcinomas and ductal adenocarcinomas of the pancreas, and additionally leads to the development of liver and lung metastases, similar to human pancreatic cancer. Thus, our findings demonstrate that pancreatic tumor progression and metastasis can be accurately modeled after the sporadic and somatic activation of oncogenes in vivo, and further show that our model is an attractive system in which to study factors regulating pancreatic tumor progression. The elastase-tv-a, Trp53 flox, Ink4a/Arf flox, and Ptf1a-cre knockin mouse strains have been previously described.7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar, 14Jonkers J Meuwissen R van der Gulden H Peterse H van der Valk M Berns A Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.Nat Genet. 2001; 29: 418-425Crossref PubMed Scopus (813) Google Scholar, 15Kawaguchi 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 (808) Google Scholar, 16Krimpenfort P Quon KC Mooi WJ Loonstra A Berns A Loss of p16Ink4a confers susceptibility to metastatic melanoma in mice.Nature. 2001; 413: 83-86Crossref PubMed Scopus (473) Google Scholar Animals were kept in specific pathogen-free housing with abundant food and water under guidelines approved by the University of Massachusetts Medical School Institutional Animal Care and Use Committee and Department of Animal Medicine. The RCAS-GFP and RCAS-PyMT vectors have been previously described.7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar, 17Holland EC Li Y Celestino J Dai C Schaefer L Sawaya RA Fuller GN Astrocytes give rise to oligodendrogliomas and astrocytomas after gene transfer of polyoma virus middle T antigen in vivo.Am J Pathol. 2000; 157: 1031-1037Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar DF1 chicken fibroblasts18Himly M Foster DN Bottoli I Iacovoni JS Vogt PK The DF-1 chicken fibroblast cell line: transformation induced by diverse oncogenes and cell death resulting from infection by avian leukosis viruses.Virology. 1998; 248: 295-304Crossref PubMed Scopus (348) Google Scholar, 19Schaefer-Klein J Givol I Barsov EV Whitcomb JM VanBrocklin M Foster DN Federspiel MJ Hughes SH The EV-O-derived cell line DF-1 supports the efficient replication of avian leukosis-sarcoma viruses and vectors.Virology. 1998; 248: 305-311Crossref PubMed Scopus (166) Google Scholar transfected with RCAS vectors were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in humidified 37°C incubators under 5% CO2. Cells to be injected were harvested, washed once with phosphate-buffered saline (PBS), and resuspended in PBS at a final concentration of 104 cells/μl. One hundred μl of the cell suspension was delivered intraperitoneally to 2- to 3-day-old animals using tuberculin syringes attached with 27-gauge needles. Animals were sacrificed with a lethal dose of CO2 followed by cervical dislocation as per institutional guidelines. Pancreata were removed and either fixed in 10% buffered formalin overnight at room temperature or snap-frozen in liquid nitrogen. Fixed tissues were paraffin embedded and 5-μm sections placed on sialynated slides at Histoserv Inc. (Gaithersburg, MD). Paraffin sections were deparaffinized and rehydrated by passage through Clear-Rite 3 and a graded alcohol series, and immunostaining performed as described.7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar Primary antibodies used were mouse anti-human keratin 19 (Abcam, Cambridge, UK) 1:50; rabbit anti-mouse Pdx1 (gift of C.V. Wright, Vanderbilt University, Nashville, TN) 1:5000; mouse anti-human p16 (Santa Cruz Biotechnology, Santa Cruz, CA) 1:50; mouse anti-human E-cadherin (BD Pharmingen, Franklin Lakes, NJ) 1:100; mouse anti-human β-catenin (BD Pharmingen) 1:100; and rabbit anti-human synaptophysin 1:1000 (DAKO, Carpinteria, CA). For chymotrypsin staining, instead of citric buffer incubation, slides were pretreated with 0.05 μg/ml of protease 24 (Sigma, St. Louis, MO) for 10 minutes at 37°C. Sheep anti-human chymotrypsin antibody (Biodesign International, Saco, ME) was incubated at 4°C overnight at a dilution of 1:20,000. We previously showed that delivery of RCAS viruses encoding the mouse PyMT to elastase-tv-a transgenic mice initiates pancreatic tumorigenesis in vivo, and that germline deletion of the Ink4a/Arf locus cooperates with PyMT to induce both acinar and ductal tumors. However, we failed to observe grossly visible pancreatic tumors after RCAS-PyMT delivery to elastase-tv-a, Trp53-null mice because of the early demise of these animals attributable to lymphomas and sarcomas. To circumvent this problem, we crossed elastase-tv-a mice to mice bearing conditional mutant (floxed) alleles at the Trp53 locus, and to mice expressing the cre recombinase from the Ptf1a locus.14Jonkers J Meuwissen R van der Gulden H Peterse H van der Valk M Berns A Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer.Nat Genet. 2001; 29: 418-425Crossref PubMed Scopus (813) Google Scholar, 15Kawaguchi 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 (808) Google Scholar Analysis of genomic DNA isolated from the pancreata of Trp53 flox, Ptf1a-cre mice demonstrated complete recombination of the Trp53 locus (data not shown). To determine whether PyMT and p53 deficiency cooperate in pancreatic tumorigenesis in our mouse model, we delivered DF1 fibroblasts producing RCAS-PyMT to 2- or 3-day-old elastase-tv-a, Trp53 flox litters containing cre-positive mice (resulting in pancreas-specific deletion of Trp53; hereafter referred to as p53-deficient) and cre-negative (p53 wild type) littermates. Animals were sacrificed at 6 and 8 months of age, and their pancreata harvested and analyzed by histopathology. TVA-producing p53 wild-type mice did not display any pancreatic pathology at 6 months of age. However, by 8 months of age 6 of 10 mice had pancreatic lesions, identified on sectioning of their resected pancreata (Table 1). All six of these animals had acinar lesions, some of which displayed histological features of immature squamous metaplasia (Figure 1A). The acinar character of the lesions was demonstrated by the expression of the acinar cell marker chymotrypsin. Interestingly, as was the case with PyMT-induced tumors in animals lacking Ink4a/Arf, the acinar lesions were also positive for the neuroendocrine marker synaptophysin, suggesting that the lesions might have originated from pancreas progenitor cells (Figure 1B). Consistent with this hypothesis, the acinar lesions were positive for Pdx1, a marker for pancreatic progenitor cells (Figure 1C).Table 1Tumor Development in Eight-Month-Old Elastase-tv-a Mice Injected with RCAS-PyMTMouse IDTrp53 statusTumor histologyMetastasisMM235.2WTSquamous metaplasiaNoneMM235.3WTNo pathologyNoneMM236.1WTSquamous metaplasiaNoneMM237.1WTSquamous metaplasiaNoneMM237.2WTNo pathologyNoneMM237.3WTSquamous metaplasia, PanINNoneMM238.1WTSquamous metaplasia, PanINNoneMM239.1WTNo pathologyNoneMM239.3WTNo pathology*Animal presented with pancreatitis.NoneMM240.3WTAcinar carcinoma, PanINNoneMM235.1NullAcinar carcinomaNoneMM236.2NullAcinar carcinomaDiaphragmMM236.3NullAcinar carcinoma w/ductal metaplasiaLiverMM238.2NullAcinar carcinoma, ductal carcinomaDiaphragmMM238.3NullAcinar carcinoma, ductal carcinomaNoneMM238.4NullDuctal carcinomaNoneMM239.2NullAcinar carcinomaNoneMM240.2NullAcinar, ductal, sarcomatoid carcinomasLiver, lungAll mice are elastase-tv-a, Trp53 flox/flox. Pancreas Trp53 status is determined by the presence (Trp53-null) or absence (Trp53 WT) of the Ptf1a-cre allele.* Animal presented with pancreatitis. Open table in a new tab All mice are elastase-tv-a, Trp53 flox/flox. Pancreas Trp53 status is determined by the presence (Trp53-null) or absence (Trp53 WT) of the Ptf1a-cre allele. In addition to the acinar lesions, three of the mice had pancreatic lesions with the histological features of pancreatic intraepithelial neoplasia (PanINs), the presumptive precursor lesions of pancreatic ductal adenocarcinoma (Figure 1, D and E). Importantly, these lesions appeared without any evidence for acinar to ductal metaplasia. The lesions displayed invaginated epithelia with papillae, and columnar cells with abundant cytoplasm rich in apical mucins as highlighted by Alcian blue staining (Figure 1F). In addition, there was moderate nuclear atypia, mitotic figures, and loss of polarity. The presence of these features classifies these lesions as mouse PanIN-1 and -2, as determined by the Penn working group.20Hruban 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 (321) Google Scholar Consistent with our previously published findings, the PanIN lesions were positive for Pdx1 and contained scattered synaptophysin-positive cells (Figure 1, G and H). Thus, PyMT induces early acinar and ductal lesions in the mouse pancreas. In contrast to the precursor lesions identified in wild-type animals injected with RCAS-PyMT, all Trp53-null littermates displayed multiple grossly visible tumors as early as 6 months of age. The three Trp53-null animals examined at this time had acinar cell carcinomas with similar histological features to those observed in RCAS-PyMT infected, Ink4a/Arf-null pancreata (data not shown).7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar As was observed for the acinar cell carcinomas induced in Ink4a/Arf-null mice, all acinar cell carcinomas were positive for Pdx1 and synaptophysin (data not shown). Further, all eight Trp53-null animals analyzed at 8 months of age had multiple pancreatic tumors, some of which exceeded 1 cm in diameter (Table 1). Histological examination of these pancreatic tumors demonstrated that most were moderately to poorly differentiated acinar cell carcinomas that stained positive for chymotrypsin, Pdx1, and synaptophysin, consistent with the findings observed at 6 months of age, as well as our previous findings in Ink4a/Arf-null animals (Figure 2, A–D).7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar Interestingly, half of the tumor-bearing mice sacrificed at 8 months of age also had ductal adenocarcinomas with individual tubular glands, mucin-producing cells, and stromal desmoplasia, findings not observed in mice with germline deletion of the Ink4a/Arf locus (Figure 2E). The ductal character of these lesions was confirmed by immunoreactivity with antibodies against keratin 19, which is specifically present within duct epithelial cells, and the absence of chymotrypsin labeling (Figure 2, F and G). In addition, we observed an undifferentiated carcinoma with sarcomatoid features in one animal (Figure 2H). Interestingly, although early acinar lesions were identified in some p53-deficient pancreata, PanIN lesions were not seen in any of these mice, potentially indicating the rapid progression of preinvasive lesions in these mice. Consistent with our previously published findings, animals injected with DF1 cells producing RCAS-GFP did not harbor any pancreatic lesions, demonstrating that p53 deficiency is not sufficient to initiate pancreatic tumorigenesis within this time frame (Table 1). Thus, Trp53 loss cooperates with PyMT to induce pancreatic tumors with acinar and ductal characteristics. Our earlier studies with Ink4a/Arf-null mice failed to identify metastatic lesions in the lymph nodes, liver, or lungs, common sites of spread in pancreatic cancer patients, potentially because of the sacrifice of these animals between 6 and 7 months of age. However, in four of eight tumor-bearing p53-deficient mice sacrificed at 8 months of age, we observed metastasis to the liver, lungs, and/or diaphragm (Figure 3, A–C). The metastatic lesions had undifferentiated histology, and formed discrete nests in the distant tissue without any signs of invading further within these organs. Interestingly, although the metastatic lesions frequently maintained synaptophysin expression (Figure 3D), Pdx1 was localized in the cytoplasm (Figure 3, E and F), in contrast to the majority of primary tumors in which Pdx1 was localized to the nucleus (Figure 2C). Pancreatic acinar and ductal epithelial cells display membrane localization of the adhesion molecule E-cadherin and its binding partner β-catenin (Figure 3, G and H). Loss of E-cadherin expression often correlates with tumor cell invasion and metastasis, and is frequently associated with the epithelial-to-mesenchymal transition, which is postulated to be involved in tumor metastasis.21Thiery JP Epithelial-mesenchymal transitions in tumour progression.Nat Rev Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5587) Google Scholar We therefore determined whether loss of E-cadherin expression and mislocalization of β-catenin occur in primary and metastatic lesions in our model. By immunostaining, we observed that both primary and metastatic lesions display reduced levels of membrane-associated E-cadherin and β-catenin, consistent with the loss of tight junctions that characterize epithelial cells (Figure 3, I–L). Importantly, there were no discernible differences between primary and metastatic lesions, or lesions present in mice without distant metastases. The inactivation of the Ink4a/Arf locus is a common early event in pancreatic tumorigenesis.2Hezel AF Kimmelman AC Stanger BZ Bardeesy N Depinho RA Genetics and biology of pancreatic ductal adenocarcinoma.Genes Dev. 2006; 20: 1218-1249Crossref PubMed Scopus (902) Google Scholar We therefore determined whether the tumors induced in p53-deficient pancreata retained expression of the Ink4a/Arf gene products p16 and p19. We performed PCR on DNA extracted from p53-deficient tumors and confirmed that the locus was not deleted (data not shown). We additionally performed reverse transcriptase-polymerase chain reaction analysis for p16 and p19 on RNA extracted from several primary tumors. All analyzed tumor samples retained expression of these gene products, indicating that the locus was not silenced by promoter methylation as commonly occurs in human pancreatic ductal adenocarcinomas.2Hezel AF Kimmelman AC Stanger BZ Bardeesy N Depinho RA Genetics and biology of pancreatic ductal adenocarcinoma.Genes Dev. 2006; 20: 1218-1249Crossref PubMed Scopus (902) Google Scholar We further confirmed the expression of p16 by immunostaining of primary and metastatic tumors. p16 was detected in all lesions analyzed, although one primary tumor, and its associated lung metastasis, contained some p16-negative cells (Figure 4, A–C; and data not shown). Thus, inactivation of the Ink4a/Arf locus is not required for tumor metastasis. We next asked whether the loss of a single Ink4a/Arf allele could cooperate with p53 deficiency in pancreatic tumor development in our mouse model. We crossed elastase-tv-a, Trp53 flox, Ptf1a-cre mice to mice bearing floxed alleles at the Ink4a/Arf locus to generate tv-a transgenic animals with pancreas-specific deletion of Trp53 and a single copy of the Ink4a/Arf locus. We found that delivery of RCAS-PyMT to these animals led to the rapid formation of pancreatic tumors in five of seven injected animals. Mice displayed distended abdomens and tumors exceeding 2 cm in diameter as early as 14 weeks of age, and all tumor-bearing mice had to be sacrificed by 18 weeks of age. All tumors were undifferentiated, and the tumor cells displayed sarcomatoid morphology with regions of ductal differentiation (Figure 4, D and E). Immunostaining confirmed the presence of p16 in the tumors, demonstrating retention of the wild-type Ink4a/Arf allele (Figure 4F). Injection of Trp53, Ink4a/Arf double-null mice with RCAS-GFP did not result in tumor formation through 12 months. Thus, the Ink4a/Arf locus demonstrates a haploinsufficient phenotype in collaboration with p53 loss. Pancreatic cancer is a deadly disease with a median survival after diagnosis of 6 months. This poor prognosis is attributable in large part to extra-pancreatic metastases at the time of diagnosis, which renders patients ineligible for surgical resection, the best potential curative option.3Warshaw AL Fernandez-del Castillo C Pancreatic carcinoma.N Engl J Med. 1992; 326: 455-465Crossref PubMed Scopus (1559) Google Scholar Thus, understanding the mechanisms by which pancreatic tumors metastasize to distant sites is of critical importance in combating this disease. Mouse models are attractive systems for the analysis of tumor development and progression. We have previously described a mouse model in which the somatic and sporadic delivery of oncogenes induces the formation of pancreatic tumors. In our current study, we demonstrate that metastatic pancreatic cancers can be induced after the postnatal and sporadic induction of oncogene expression using this model system. Consistent with features of the human disease, the tumors spread most frequently to the liver, abdominal surface, and lungs.22Li D Xie K Wolff R Abbruzzese JL Pancreatic cancer.Lancet. 2004; 363: 1049-1057Abstract Full Text Full Text PDF PubMed Scopus (1780) Google Scholar Notably, our tumors and metastatic lesions were induced after somatic and sporadic introduction of the PyMT oncogene. An important consequence of this strategy, absent in previous models, is that tumor progression and metastasis are not influenced by the expression of the oncogene in neighboring cells. This attribute makes it easier to identify cell autonomous factors that contribute to these processes, which is an important avenue of investigation in ongoing efforts to understand the mechanisms of tumor spread in pancreatic cancer. Thus, the findings described here demonstrate that metastatic pancreatic cancer can be induced after postnatal oncogene activation, and suggest that our model system may be useful for the elucidation of the molecular mechanisms that underlie the development and progression of pancreatic carcinomas. The loss of the Ink4a/Arf locus is an early and common event in the genesis of human pancreatic ductal tumors. We were therefore interested in identifying whether loss of this locus is required for tumor development in our model. Using amplification of genomic DNA and mRNA, we found that the locus remains intact and that the p16 and p19 tumor suppressor gene products are expressed. Thus, loss of this locus is not required for pancreatic tumor formation. Further, we demonstrated by immunostaining that p16 expression is retained in metastatic cells, and therefore its loss is not required for metastatic disease. These findings are consistent with those of the Tuveson13Hingorani 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 (1797) Google Scholar and Depinho23Bardeesy 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 (467) Google Scholar laboratories in other mouse models. However, loss of the Ink4a/Arf locus is important in pancreatic tumorigenesis. Delivery of RCAS-PyMT to tv-a transgenic mice with pancreas-specific deletion of Trp53 and a single Ink4a/Arf allele led to acceleration of pancreatic tumor formation, and conferred an undifferentiated histology on the resulting tumors. Importantly, the locus displays a haploinsufficient phenotype as the wild-type locus is retained and expressed in the developed tumors. These findings are consistent with the early loss of the locus during pancreatic adenocarcinoma formation,2Hezel AF Kimmelman AC Stanger BZ Bardeesy N Depinho RA Genetics and biology of pancreatic ductal adenocarcinoma.Genes Dev. 2006; 20: 1218-1249Crossref PubMed Scopus (902) Google Scholar and our observation that the loss of this locus, but not the loss of Trp53, enhances the proliferation of primary pancreatic duct epithelial cells in culture (J.P.M and B.C.L., unpublished observations). During immunohistochemical analysis of tumor specimens, we observed that the metastatic lesions were negative for nuclear localized Pdx1, whereas nuclear localized Pdx1 is a commonly observed feature in primary pancreatic lesions in mouse models.13Hingorani 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 (1797) Google Scholar Instead, the metastatic lesions displayed cytoplasmic localization of this transcription factor. We also observed cytoplasmic localization of Pdx1 in some of the larger primary tumors, whereas precursor PanIN lesions and smaller primary tumors contain nuclear Pdx1. Together, these findings suggest that loss of Pdx1 nuclear function, or the gain of cytoplasmic Pdx1 function, may be important for pancreatic tumor progression. Alternatively, the cytoplasmic localization of Pdx1 may reflect the activation of signaling pathways, such as those regulated by c-jun N-terminal kinase (JNK), that regulate Pdx1 activity during pancreatic development and β-cell function.24Kaneto H Xu G Fujii N Kim S Bonner-Weir S Weir GC Involvement of c-Jun N-terminal kinase in oxidative stress-mediated suppression of insulin gene expression.J Biol Chem. 2002; 277: 30010-30018Crossref PubMed Scopus (279) Google Scholar, 25Kawamori D Kajimoto Y Kaneto H Umayahara Y Fujitani Y Miyatsuka T Watada H Leibiger IB Yamasaki Y Hori M Oxidative stress induces nucleo-cytoplasmic translocation of pancreatic transcription factor PDX-1 through activation of c-Jun NH(2)-terminal kinase.Diabetes. 2003; 52: 2896-2904Crossref PubMed Scopus (186) Google Scholar Future exploration of these issues may provide important information regarding pancreatic cancer metastasis. The generation of multiple tumor types (acinar, ductal) in our model, and the expression of progenitor markers (Pdx1) and markers for multiple lineages (chymotrypsin, synaptophysin) raise the issue of the cell of origin. In our model, expression of tv-a is controlled by the elastase promoter, which is believed to be acinar-specific.26Ornitz DM Palmiter RD Hammer RE Brinster RL Swift GH MacDonald RJ Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice.Nature. 1985; 313: 600-602Crossref PubMed Scopus (115) Google Scholar However, the presence of ductal tumors, in the absence of any evidence for metaplasia, suggests that the tumors may be derived from multipotential progenitors, consistent with our previously published data.7Lewis BC Klimstra DS Varmus HE The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.Genes Dev. 2003; 17: 3127-3138Crossref PubMed Scopus (101) Google Scholar The identity of this progenitor remains unknown, although the hypothesis that the elastase-tv-a transgene may be expressed in progenitors is consistent with published findings by Chiang and Melton27Chiang MK Melton DA Single-cell transcript analysis of pancreas development.Dev Cell. 2003; 4: 383-393Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar that indicated that mRNAs for lineage-specific genes are detected in individual cells simultaneously with mRNAs associated with early pancreas development. Recent studies have suggested that centroacinar cells or nestin-positive cells may act as progenitor cells in the pancreas and may be target cells for transformation in pancreatic tumorigenesis.28Carriere C Seeley ES Goetze T Longnecker DS Korc M The Nestin progenitor lineage is the compartment of origin for pancreatic intraepithelial neoplasia.Proc Natl Acad Sci USA. 2007; 104: 4437-4442Crossref PubMed Scopus (109) Google Scholar, 29Stanger BZ Stiles B Lauwers GY Bardeesy N Mendoza M Wang Y Greenwood A Cheng KH McLaughlin M Brown D Depinho RA Wu H Melton DA Dor Y Pten constrains centroacinar cell expansion and malignant transformation in the pancreas.Cancer Cell. 2005; 8: 185-195Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar Whether either, or both, of these cell types are the cell of origin in our model is a provocative idea that remains to be elucidated. This work was initiated in the laboratory of Harold Varmus, and we thank him for his support and guidance, and we thank Michael Twarog for technical assistance, Paul Krimpenfort and Anton Berns for the Trp53 and Ink4a/Arf conditional mutant mouse strains, Chris Wright for the Ptf1a-cre mouse line, Kirsten A. Hubbard for critical review of the manuscript, and members of the Lewis Lab for helpful discussions.