Oncogenic KRAS Induces Progenitor Cell Expansion and Malignant Transformation in Zebrafish Exocrine Pancreas
2008; Elsevier BV; Volume: 134; Issue: 7 Linguagem: Inglês
10.1053/j.gastro.2008.02.084
ISSN1528-0012
AutoresSeung Woo Park, Jon M. Davison, Jerry M. Rhee, Ralph H. Hruban, Anirban Maitra, Steven D. Leach,
Tópico(s)Epigenetics and DNA Methylation
ResumoBackground & Aims: Although the cell of origin for pancreatic cancer remains unknown, prior studies have suggested that pancreatic neoplasia may be initiated in progenitor-like cells. To examine the effects of oncogene activation within the pancreatic progenitor pool, we devised a system for real-time visualization of both normal and oncogenic KRAS-expressing pancreatic progenitor cells in living zebrafish embryos. Methods: By using BAC transgenes under the regulation of ptf1a regulatory elements, we expressed either extended green fluorescent protein (eGFP) alone or eGFP fused to oncogenic KRAS in developing zebrafish pancreas. Results: After their initial specification, normal eGFP-labeled pancreatic progenitor cells were observed to actively migrate away from the forming endodermal gut tube, and subsequently underwent characteristic exocrine differentiation. In contrast, pancreatic progenitor cells expressing oncogenic KRAS underwent normal specification and migration, but failed to differentiate. This block in differentiation resulted in the abnormal persistence of an undifferentiated progenitor pool, and was associated with the subsequent formation of invasive pancreatic cancer. These tumors showed several features in common with the human disease, including evidence of abnormal Hedgehog pathway activation. Conclusions: These results provide a unique view of the tumor-initiating effects of oncogenic KRAS in a living vertebrate organism, and suggest that zebrafish models of pancreatic cancer may prove useful in advancing our understanding of the human disease. Background & Aims: Although the cell of origin for pancreatic cancer remains unknown, prior studies have suggested that pancreatic neoplasia may be initiated in progenitor-like cells. To examine the effects of oncogene activation within the pancreatic progenitor pool, we devised a system for real-time visualization of both normal and oncogenic KRAS-expressing pancreatic progenitor cells in living zebrafish embryos. Methods: By using BAC transgenes under the regulation of ptf1a regulatory elements, we expressed either extended green fluorescent protein (eGFP) alone or eGFP fused to oncogenic KRAS in developing zebrafish pancreas. Results: After their initial specification, normal eGFP-labeled pancreatic progenitor cells were observed to actively migrate away from the forming endodermal gut tube, and subsequently underwent characteristic exocrine differentiation. In contrast, pancreatic progenitor cells expressing oncogenic KRAS underwent normal specification and migration, but failed to differentiate. This block in differentiation resulted in the abnormal persistence of an undifferentiated progenitor pool, and was associated with the subsequent formation of invasive pancreatic cancer. These tumors showed several features in common with the human disease, including evidence of abnormal Hedgehog pathway activation. Conclusions: These results provide a unique view of the tumor-initiating effects of oncogenic KRAS in a living vertebrate organism, and suggest that zebrafish models of pancreatic cancer may prove useful in advancing our understanding of the human disease. In both mouse and humans, exocrine pancreatic cancer appears to be initiated by oncogenic KRAS. In humans, more than 90% of all pancreatic ductal adenocarcinomas show evidence of oncogenic KRAS mutations, and evaluation of pancreatic cancer precursor lesions suggests that these mutations represent an early event in pancreatic tumorigenesis.1Hruban R.H. van Mansfeld A.D. Offerhaus G.J. et al.K-ras oncogene activation in adenocarcinoma of the human pancreas A study of 82 carcinomas using a combination of mutant-enriched polymerase chain reaction analysis and allele-specific oligonucleotide hybridization.Am J Pathol. 1993; 143: 545-554PubMed Google Scholar, 2Hruban R.H. Wilentz R.E. Kern S.E. Genetic progression in the pancreatic ducts.Am J Pathol. 2000; 156: 1821-1825Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar In mice, expression of oncogenic KRAS is sufficient to induce pancreatic intraepithelial neoplasia, a known precursor for invasive pancreatic ductal adenocarcinoma.3Hingorani S.R. Petricoin E.F. Maitra A. et al.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 (1896) Google Scholar, 4Aguirre A.J. Bardeesy N. Sinha M. et al.Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma.Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (835) Google Scholar, 5Hruban R.H. Adsay N.V. Albores-Saavedra J. et al.Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations.Cancer Res. 2006; 66: 95-106Crossref PubMed Scopus (326) Google Scholar With low frequency and long latency, oncogenic KRAS-induced murine pancreatic intraepithelial neoplasia's progress to invasive cancer, a process that is markedly accelerated by either activation of Hedgehog signaling6Pasca di Magliano M. Sekine S. Ermilov A. et al.Hedgehog/Ras interactions regulate early stages of pancreatic cancer.Genes Dev. 2006; 20: 3161-3173Crossref PubMed Scopus (265) Google Scholar or inactivation of a variety of tumor-suppressor genes, including Trp53, Ink4a/Arf, Tgfbr2, or Smad4.4Aguirre A.J. Bardeesy N. Sinha M. et al.Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma.Genes Dev. 2003; 17: 3112-3126Crossref PubMed Scopus (835) Google Scholar, 7Ijichi H. Chytil A. Gorska A.E. et al.Aggressive pancreatic ductal adenocarcinoma in mice caused by pancreas-specific blockade of transforming growth factor-beta signaling in cooperation with active Kras expression.Genes Dev. 2006; 20: 3147-3160Crossref PubMed Scopus (312) Google Scholar, 8Hingorani S.R. Wang L. Multani A.S. et al.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 (1837) Google Scholar, 9Bardeesy N. Cheng K.H. Berger J.H. et al.Smad4 is dispensable for normal pancreas development yet critical in progression and tumor biology of pancreas cancer.Genes Dev. 2006; 20: 3130-3146Crossref PubMed Scopus (533) Google Scholar These studies have led to the view that oncogenic KRAS functions as a critical initiator of pancreatic neoplasia, with additional genetic lesions required for tumor progression. Despite the known ability of oncogenic KRAS to initiate pancreatic neoplasia, the nature of KRAS-induced initiating events remains unknown. To facilitate in vivo examination of individual cells expressing oncogenic KRAS in the context of the exocrine pancreas, we have developed technology for targeted transgene expression in developing zebrafish pancreas. By using this technology, we have generated a model of exocrine pancreatic cancer in zebrafish, and have further identified a block in progenitor cell differentiation as one of the earliest discernable effects of oncogenic KRAS expression in vertebrate exocrine pancreas. For Detailed Experimental Procedures, please see supplementary material online at www.gastrojournal.org. By using bacterial recombineering,10Cotta-de-Almeida V. Schonhoff S. Shibata T. et al.A new method for rapidly generating gene-targeting vectors by engineering BACs through homologous recombination in bacteria.Genome Res. 2003; 13: 2190-2194Crossref PubMed Scopus (45) Google Scholar we modified a genomic bacterial artificial chromosome (BAC) (CH211-142H2) spanning the zebrafish ptf1a locus to generate transgene constructs ptf1a:eGFP and ptf1a:eGFP-KRASG12V. To enable real-time visualization of oncogenic KRAS-expressing cells, the KRASG12V transgene was expressed as extended green fluorescent protein (eGFP) fusion protein.11Niv H. Gutman O. Henis Y.I. et al.Membrane interactions of a constitutively active GFP-Ki-Ras 4B and their role in signaling Evidence from lateral mobility studies.J Biol Chem. 1999; 274: 1606-1613Crossref PubMed Scopus (105) Google Scholar The purified ptf1a:eGFP and ptf1a:eGFP-KRASG12V BAC transgenes were injected into single-cell stage wild-type AB embryos, which then were raised to adulthood and outcrossed to generate F1 founders. To generate a population of fish in which to assess the time interval to visible tumor formation, transgenic adult Tg(ptf1a:eGFP-KRASG12V) were outcrossed to wild-type fish and approximately 200 heterozygous embryos expressing eGFP in the expected pattern were raised. Transcutaneous eGFP expression was evaluated in a subset of the total number of fish at 1-, 2-, 3-, 6-, and 9-month time points. Fish with and without transcutaneous eGFP fluorescence were killed at interval time points for histologic evaluation. Immunofluorescent and in situ hybridization analyses were performed either on whole embryos or on 8-μm cryosections as described previously.12Lin J.W. Biankin A.V. Horb M.E. et al.Differential requirement for ptf1a in endocrine and exocrine lineages of developing zebrafish pancreas.Dev Biol. 2004; 270: 474-486Crossref PubMed Scopus (65) Google Scholar Primary antibodies used for immunostaining, primers used to generate in situ probes, and methodology for multichannel fluorescent intensity analysis are described under Detailed Experimental Procedures in the supplementary materials. Real-time, quantitative, reverse-transcription polymerase chain reaction was performed using an ABI Prism 7700 Sequence Detector (Applied Biosystems, Foster City, CA), using the QuantiTect SYBR Green polymerase chain reaction kit (Qiagen, Valencia, CA). Primer sequences are listed under Detailed Experimental Procedures in the supplementary materials. To capture regulatory elements capable of targeting transgene expression to zebrafish pancreatic progenitor cells, we engineered a large genomic BAC spanning the ptf1a locus, so that ptf1a coding sequence was replaced with a complementary DNA encoding either eGFP alone or eGFP fused to oncogenic human KRAS 4B (Figures 1A and 2A;supplementary Figure 1; see supplementary material online at www.gastrojournal.org). By using these BAC transgenes, 7 independent Tg(ptf1a:eGFP) and 6 Tg(ptf1a:eGFP-KRASG12V) lines were established. In the absence of significant variation between lines, a single representative Tg(ptf1a:eGFP) line and a single representative Tg(ptf1a:eGFP-KRASG12V) line were selected for further analysis.Figure 2Comparison of ptf1a:eGFP and ptf1a:GFP-KRASG12V transgene expression in living zebrafish embryos. (A) Transgenes used in which ptf1a coding sequence was replaced with either eGFP or eGFP-KRASG12V coding sequence. (B–E) Confocal images of retina (B and C) and pancreas (D and E) at 48 hpf. Note nuclear and cytoplasmic localization of eGFP in Tg(ptf1a:eGFP) embryos (B and D), compared with membrane localization of eGFP-KRASG12V fusion protein (C and E), reflecting activity of KRAS C-terminal CAAX motif. (F–K) Whole-mount dark field images of transgenic embryos showing spatiotemporal expression pattern of eGFP vs eGFP-KRASG12V. (F, H, and J) ptf1a:eGFP embryos. (G, I, and K) ptf1a:eGFP-KRASG12V embryos. eGFP-KRASG12V–expressing cells undergo normal specification and initial migration, but eGFP-KRASG12V subsequently is down-regulated beginning at 48 hpf. White arrowheads indicate pancreatic domains of eGFP/ eGFP-KRASG12V expression.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Examination of living ptf1a:eGFP embryos revealed expression in retinal amacrine cells, hindbrain, spinal interneurons, and pancreas (Figure 1, Figure 2). This pattern faithfully recapitulated the previously reported pattern of endogenous ptf1a expression.12Lin J.W. Biankin A.V. Horb M.E. et al.Differential requirement for ptf1a in endocrine and exocrine lineages of developing zebrafish pancreas.Dev Biol. 2004; 270: 474-486Crossref PubMed Scopus (65) Google Scholar, 13Esni F. Ghosh B. Biankin A.V. et al.Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas.Development. 2004; 131: 4213-4224Crossref PubMed Scopus (183) Google Scholar, 14Zecchin E. Mavropoulos A. Devos N. et al.Evolutionary conserved role of ptf1a in the specification of exocrine pancreatic fates.Dev Biol. 2004; 268: 174-184Crossref PubMed Scopus (99) Google Scholar By crossing Tg(ptf1a:eGFP) fish with fish expressing mCherry fluorescent protein under the regulation of insulin promoter elements,15Pisharath H. Rhee J.M. Swanson M.A. et al.Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase.Mech Dev. 2007; 124: 218-229Crossref PubMed Scopus (294) Google Scholar we visualized the initial specification and migration of pancreatic progenitor cells with respect to the already formed principal islet (Figure 1). In addition to expression in the early pancreatic progenitor pool, eGFP expression was subsequently observed to persist in differentiated acinar cells throughout adult life (Figures 3A and 6A). We next compared patterns of eGFP fluorescence in Tg(ptf1a:eGFP) and Tg(ptf1a:eGFP-KRASG12V) embryos. Expression of eGFP-KRASG12V in the retina, hindbrain, and neural tube was highly similar to the pattern seen in Tg(ptf1a:eGFP) embryos (Figure 2F–K). However, transgene expression in developing pancreas was much more heterogeneous among Tg(ptf1a:eGFP-KRASG12V) embryos, with approximately 50% of transgenic embryos showing pancreatic expression levels that permitted serial imaging using a fluorescent stereomicroscope. Pancreatic progenitor cells expressing eGFP-KRASG12V underwent initial specification and migration in a manner identical to that observed in Tg(ptf1a:eGFP) embryos. However, when individual embryos were serially imaged between 48 and 96 hours postfertilization (hpf), we observed a noticeable decay in the intensity of eGFP-KRASG12V expression (compare Figure 2H and I, J and K). By 96 hpf, eGFP-KRASG12V expression was best detected by confocal microscopy. Gradual loss of pancreatic eGFP fluorescence in the ptf1a:eGFP-KRASG12V lines also was associated with loss of eGFP-KRASG12V transcripts as assessed by whole-mount in situ hybridization, even while transcripts for endogenous ptf1a were found to persist (Supplementary Figure 2; and data not shown). To assess the anatomic extent of pancreatic tissue in ptf1a:eGFP-KRASG12V transgenics, and also to evaluate the ability of eGFP-KRASG12V-expressing pancreatic progenitor cells to undergo normal exocrine differentiation, we performed immunofluorescent labeling for the digestive enzyme carboxypeptidase A (CPA), a marker of exocrine differentiation. When the dissected endodermal tissues of Tg(ptf1a:eGFP) larvae were examined by confocal imaging at 96 hpf, eGFP-positive cells almost uniformly co-expressed CPA in the apical cytoplasm and showed well-developed apical secretory granules (Figure 3A, C, E, and G). In contrast to uniform expression of the ptf1a:eGFP transgene, pancreatic expression of the eGFP-KRASG12V fusion protein showed a mosaic pattern characterized by the apparent random distribution of individual eGFP-positive cells and groups of cells (Figure 3B, D, F, and H). Notably, progenitor cells maintaining expression of the fluorescent eGFP-KRASG12V fusion protein showed negligible or extremely low levels of CPA expression, and none developed CPA-positive, apical secretory granules (Figure 3D and H). In the context of mosaic expression of the eGFP-KRASG12V fusion protein, adjacent and surrounding cells of the exocrine pancreas lacking detectable eGFP-KRASG12V expression showed high levels of CPA in well-formed apical secretory granules. These data suggest that oncogenic KRAS cell autonomously inhibits the differentiation of pancreatic progenitor cells. To quantify the ability of oncogenic KRAS to block pancreatic progenitor cell differentiation, we examined a series of optical sections of the pancreas from a total of 10 different embryos: 4 from Tg(ptf1a:eGFP-KRASG12V) and 6 from Tg(ptf1a:eGFP), and compared the fraction of eGFP-positive pixels that also were positive for Cy3 after immunofluorescent staining for CPA. Similar to cell-by-cell fluorescence-activated cell sorter analysis, this pixel-by-pixel analysis allowed us to determine the fraction of eGFP-positive area that was also positive for CPA, providing a quantifiable estimate of exocrine differentiation among transgene-expressing cells (Figure 3I and J). In ptf1a:eGFP larvae at 96 hpf, the fraction of eGFP-positive pixels also positive for CPA was 16.0% ± 3.1% (mean ± SD), while the corresponding fraction in Tg(ptf1a:eGFP-KRASG12V) larvae was 2.4% ± 1.4% (P < .001, unpaired t test). These observations further support the conclusion that expression of oncogenic KRAS in exocrine pancreatic progenitors prevents these cells from undergoing a normal pattern of exocrine differentiation, resulting in the abnormal persistence of a ptf1a-positive, CPA-negative undifferentiated progenitor pool. Widespread eGFP expression was sustained in the pancreas of Tg(ptf1a:eGFP) fish during all stages of development. This expression could be visualized transcutaneously in adult fish, allowing the overall anatomic distribution of the exocrine pancreas to be visualized (Figure 4A). As previously reported,16Chen S. Li C. Yuan G. et al.Anatomical and histological observation on the pancreas in adult zebrafish.Pancreas. 2007; 34: 120-125Crossref PubMed Scopus (31) Google Scholar, 17Yee N.S. Lorent K. Pack M. Exocrine pancreas development in zebrafish.Dev Biol. 2005; 284: 84-101Crossref PubMed Scopus (105) Google Scholar adult zebrafish pancreas showed a lobular configuration, with pancreatic parenchyma interposed between loops of intestine and other viscera (Figure 4B). More than 200 Tg(ptf1a:eGFP-KRASG12V) fish were raised to adulthood. By the time these fish reach the larval stage, transgene silencing has resulted in only small nests of eGFP-positive cells that are detectable by confocal microscopy, but too small to be recognized by transcutaneous fluorescence. However, as the fish aged, we observed the progressive onset of transcutaneous fluorescence, suggesting expansion of eGFP-KRAS–expressing cells. A random subset of fish was anesthetized periodically and evaluated for abnormal patterns of transcutaneous eGFP expression suggestive of tumor formation. In addition, a total of 32 adult fish (18 with transcutaneous eGFP fluorescence patterns suggesting tumor formation and 14 fish without transcutaneous eGFP fluorescence) were killed at different ages for detailed histologic evaluation. At 1 and 2 months of age, no transcutaneous fluorescence was detected among 30 adult Tg(ptf1a:eGFP-KRASG12V) fish, consistent with widespread transgene silencing observed in developing ptf1a:eGFP-KRASG12V embryos. Corresponding to this absence of detectable fluorescence, 2-month-old Tg(ptf1a:eGFP-KRASG12V) fish had histologically normal pancreas and no evidence of tumor formation in any organ. Transcutaneous abdominal eGFP fluorescence became detectable in a fraction of Tg(ptf1a:eGFP-KRASG12V) fish at 3 months of age, at which time 20% of examined fish had focal areas of abdominal eGFP expression (Figure 4C and I). The proportion of fish with transcutaneously detectable eGFP-positive lesions increased with advancing age (Figure 4I). At 6 months of age, 32% of fish had small ( 8 mm) or more diffuse/multifocal areas of eGFP fluorescence. By 9 months of age, two thirds of examined fish had detectable tumor, and almost half had widespread abdominal eGFP expression characterized by either multiple, discrete foci of eGFP expression or large masses encompassing a significant portion of the abdomen (Figure 4D and F). Detailed histologic examination of 18 fish with either focal or diffuse abdominal eGFP expression invariably revealed the presence of a pancreatic tumor (Figure 4G and H), and tumors associated with widespread abdominal eGFP expression showed overt features of malignancy (discussed in detail below). Among 14 Tg(ptf1a:eGFP-KRASG12V) fish lacking transcutaneously detectable eGFP fluorescence that were subjected to histologic analysis, 1 showed a small tumor measuring less than 1 mm in diameter, and 1 showed abnormal acinar cell hyperplasia (Table 1).Table 1Histopathologic Findings in Selectively Killed Tg(ptf1a:GFP-KRASG12V) FishNumberAge at death, moTranscutaneous fluorescenceHistologyInvasion and possible metastasis12NoneNo tumor22NoneNo tumor34NoneNo tumor44FocalSmall tumor, acinar differentiation56NoneNo tumor66NoneNo tumor76NoneNo tumor86NoneNo tumor96NoneNo tumor106FocalSmall tumor, acinar differentiation116FocalSmall tumor, acinar differentiation126FocalSmall tumor, acinar differentiation, acinar hyperplasia136FocalSmall tumor, acinar differentiation, acinar hyperplasia146DiffuseLarge tumor, mixed acinar and ductal differentiation156DiffuseMixed acinar and ductal differentiationLiver and gut invasion, ovarian metastasis166DiffusePredominantly ductal differentiationGut invasion178NoneNo tumor188NoneNo tumor198NoneNo tumor208NoneNo tumor218NoneNo tumor, acinar hyperplasia228NoneSmall tumor, acinar differentiation, acinar hyperplasia238FocalSmall tumor, acinar differentiation, acinar hyperplasia248FocalSmall tumor, ductal differentiation258FocalSmall tumor, mixed acinar and ductal differentiation268DiffuseMixed predominantly acinar differentiationInvasion of muscle and ovary, spinal metastasis278DiffuseMixed acinar and ductal differentiationOvarian metastasis288DiffuseMixed acinar and ductal differentiationLiver and gut invasion, ovarian metastasis298DiffuseMixed predominantly ductal differentiationLiver and gut invasion3011DiffuseDuctal/mucinous differentiationLiver and ovary invasion3111DiffuseMixed predominantly ductal differentiationOvary invasion3211DiffuseMixed predominantly acinar differentiationGut and liver invasionNOTE. In the absence of defined routes for tumor dissemination in fish, interpretation of metastatic disease is viewed as less than definitive. Open table in a new tab NOTE. In the absence of defined routes for tumor dissemination in fish, interpretation of metastatic disease is viewed as less than definitive. Histologically normal exocrine pancreas from Tg(ptf1a:eGFP) fish is characterized by delicately arborized clusters of pancreatic acini surrounded by adipose tissue, anatomically insinuated among loops of bowel and surrounded by other visceral organs such as the liver, spleen, and gonads (Figure 5A and B). Islet and ductal elements also are discernable (Figure 5C). Histologic examination of abdominal eGFP-positive lesions in Tg(ptf1a:eGFP-KRASG12V) fish revealed tumor in each case (Table 1). Focal eGFP-positive lesions corresponded to small pancreatic tumors comprised of disorganized proliferations of cells with recognizable acinar morphology (Figures 4G, 5E, and 5F). Several fish with focal eGFP-positive tumors also showed acinar cell hyperplasia characterized by well-organized, but abnormally abundant acinar tissue (Figure 5D). This feature was reminiscent of the enlarged but histologically normal pancreas previously observed before pancreatic tumor formation in mice.3Hingorani S.R. Petricoin E.F. Maitra A. et al.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 (1896) Google Scholar When examined histologically, all fish with large, widespread abdominal eGFP-positive tumors showed defining features of malignancy: invasion of the pancreas and surrounding organs and/or evidence of apparent metastasis (summarized in Table 1). These tumors displayed considerable heterogeneity with respect to histologic patterns of differentiation, and included acinar cell carcinoma, ductal adenocarcinoma, adenocarcinomas with mixed acinar and ductal features, and mucinous (colloid) adenocarcinoma (Figure 5E–L and Table 1). Among 19 tumors subjected to detailed histologic analysis, 9 were classified as displaying acinar or predominantly acinar differentiation (47%), 5 were classified as ductal or predominantly ductal (26%), and 5 were classified as mixed without acinar or ductal predominance (26%). Tumors with predominantly ductal differentiation also displayed dramatic stromal expansion, similar to that observed in human pancreatic cancer (Figure 5G, and Supplementary Figure 3). Tumors frequently were observed to invade adjacent gut, liver, and ovary, and often were associated with considerable tissue destruction (Figure 5M–P). Four examined fish had discrete tumor nodules in the ovary and 1 fish was found to have tumor foci in a spinal vertebra and adjacent spinal cord, suggesting possible hematogenous dissemination of tumor cells (Supplementary Figure 4). As in the case of primary tumors, metastatic lesions continued to show strong GFP fluorescence. Morphologic assessment of tumor differentiation was confirmed by additional staining for markers of acinar and ductal differentiation. In tumors with ductal features, invasive glandular structures expressed cytokeratin 18 and contained intracytoplasmic mucin, as revealed by immunofluorescent labeling and mucicarmine staining (Figure 6A–D). To evaluate areas of apparent acinar differentiation, additional immunohistochemistry was performed using the acinar cell–specific markers: amylase and CPA (Figure 6E–J). Acinar-like elements from eGFP-KRASG12V–induced pancreatic tumors showed only low-level or focal labeling, indicating a significant degree of dedifferentiation. Histologic findings are summarized in Table 1. To determine the status of downstream signaling pathways known to be activated by oncogenic KRAS, we assessed levels of phospho-AKT and phospho-ERK using immunohistochemistry (Figure 7). In contrast to infrequent ERK and AKT phosphorylation in normal zebrafish pancreas, eGFP-KRASG12V–induced pancreatic tumors showed widespread labeling for both phospho-ERK and phospho-AKT (Figure 7A–D). Associated with activation of these pathways, tumor cells expressing oncogenic KRAS fusion protein also showed an increased mitotic rate, as assessed by immunofluorescent staining for phospho-histone H3 (Figure 7E–G). Ligand-dependent activation of the hedgehog signaling pathway has been observed in invasive human pancreatic cancer as well as in pancreatic intraepithelial neoplasia lesions, and forced hedgehog activation accelerates progression of KRAS-induced tumors in mice.6Pasca di Magliano M. Sekine S. Ermilov A. et al.Hedgehog/Ras interactions regulate early stages of pancreatic cancer.Genes Dev. 2006; 20: 3161-3173Crossref PubMed Scopus (265) Google Scholar, 18Berman D.M. Karhadkar S.S. Maitra A. et al.Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours.Nature. 2003; 425: 846-851Crossref PubMed Scopus (1141) Google Scholar, 19Thayer S.P. Di Magliano M.P. Heiser P.W. et al.Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis.Nature. 2003; 425: 851-856Crossref PubMed Scopus (1331) Google Scholar, 20Prasad N.B. Biankin A.V. Fukushima N. et al.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 (208) Google Scholar We therefore evaluated tumors for evidence of hedgehog pathway activation by using immunohistochemistry, in situ hybridization, and quantitative reverse-transcription polymerase chain reaction to assess expression of hedgehog pathway components (Figure 8). These experiments revealed up-regulated expression of shh, dhh, ihha, ihhb, ptc1, gli1, and smo at the RNA level (Figure 8G–K), and Ptc2 at the protein level (Figure 8C and D), in tumor epithelium compared with normal epithelium from ptf1a:eGFP transgenics. Among these up-regulated markers, ptc1 and gli1 represent known hedgehog target genes, thereby representing surrogate markers of hedgehog pathway activation. These findings suggest that, similar to the human disease, zebrafish pancreatic cancer also is characterized by activation of hedgehog signaling. The successful modeling of human disease in experimental organisms often generates unique insights with respect to initiating molecular and cellular events, and also provides a novel platform for preclinical studies of early detection, chemoprevention, and treatment. In the current study, we have taken advantage of BAC recombineering technology to target fluorescent transgene expression to ptf1a-expressing pancreatic progenitor cells, allowing us to image the earliest events in zebrafish pancreas development, and to create a novel model of zebrafish pancreatic cancer. In addition to providing new insights regarding the influence of KRAS on the differentiation of pancreatic progenitor cells, these results also provide an important proof-of-principle regarding the ability to generate pancreatic cancer in the zebrafish species, and set the stage for future identification of genetic and small-molecule modifiers of pancreatic cancer initiation and progression. As an example of the utility of the zebrafish system, our studies have revealed a block in progenitor differentiation and the attendant accumulation of undifferentiated progenitors as the earliest discernable effects of oncogenic KRAS expression in vertebrate exocrine pancreas. Although the effects of oncogenic Ras proteins on cellular differentiation have been examined frequently in the context of mammalian tissue culture,21Crespo P. Leon J. Ras proteins in the control of the cell cycle and cell differentiation.Cell Mol Life Sci. 2000; 57: 1613-1636Crossref PubMed Scopus (157) Google Scholar previous in vivo studies typically have been limited by an inability to examine defined progenitor populations. However, a similar blockade in progenitor cell differentiation recently was reported after expression of oncogenic KRAS in either hematopoietic or bronchoalveolar stem cells.22Kim C.F. Jackson E.L. Woolfenden A.E. et al.Identification of bronchioalveolar stem cells in normal lung and lung cancer.Cell. 2005; 121: 823-835Abstract Full Text Full Text PDF PubMed Scopus (1895) Google Scholar, 23Braun B.S. Archard J.A. Van Ziffle J.A. et al.Somatic activation of a conditional KrasG12D allele causes ineffective erythropoiesis in vivo.Blood. 2006; 108: 2041-2044Crossref PubMed Scopus (34) Google Scholar In both of these settings, inhibition of differentiation was associated with expansion of an undifferentiated progenitor pool, similar to the expansion of ptf1a-positive, CPA-negative cells observed in our Tg(ptf1a:eGFP-KRASG12V) embryos. EGFP-KRASG12V–induced zebrafish pancreatic cancers displayed specific similarities and differences with respect to mouse and human pancreatic cancer. Similar to the aggressive behavior of human pancreatic cancer, zebrafish pancreatic cancers were highly invasive, and also showed a propensity for apparent metastatic spread. Also similar to human pancreatic cancer, many zebrafish tumors displayed areas of mucinous metaplasia and the classic appearance of pancreatic ductal adenocarcinoma. In addition, the observed up-regulation of hedgehog pathway components suggests additional analogy between human, mouse, and zebrafish pancreatic cancer.3Hingorani S.R. Petricoin E.F. Maitra A. et al.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 (1896) Google Scholar, 18Berman D.M. Karhadkar S.S. Maitra A. et al.Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours.Nature. 2003; 425: 846-851Crossref PubMed Scopus (1141) Google Scholar, 19Thayer S.P. Di Magliano M.P. Heiser P.W. et al.Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis.Nature. 2003; 425: 851-856Crossref PubMed Scopus (1331) Google Scholar, 20Prasad N.B. Biankin A.V. Fukushima N. et al.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 (208) Google Scholar Unlike most human pancreatic cancer, however, we frequently observed features of nonductal differentiation, especially in early lesions. Further study will be required to determine what additional genetic and epigenetic changes may occur in both ductal and nonductal tumors, and to what degree these genetic changes are required to support widespread expression of EGFP-KRASG12V. The Tg(ptf1a:eGFP-KRASG12V) transgenic model of pancreatic cancer adds to a growing list of zebrafish tumor models, including transgenic models of T- and B-cell leukemia,24Langenau D.M. Feng H. Berghmans S. et al.Cre/lox-regulated transgenic zebrafish model with conditional myc-induced T cell acute lymphoblastic leukemia.Proc Natl Acad Sci U S A. 2005; 102: 6068-6073Crossref PubMed Scopus (231) Google Scholar, 25Sabaawy H.E. Azuma M. Embree L.J. et al.TEL-AML1 transgenic zebrafish model of precursor B cell acute lymphoblastic leukemia.Proc Natl Acad Sci U S A. 2006; 103: 15166-15171Crossref PubMed Scopus (152) Google Scholar, 26Langenau D.M. Traver D. Ferrando A.A. et al.Myc-induced T cell leukemia in transgenic zebrafish.Science. 2003; 299: 887-890Crossref PubMed Scopus (459) Google Scholar pancreatic endocrine neoplasms,27Yang H.W. Kutok J.L. Lee N.H. et al.Targeted expression of human MYCN selectively causes pancreatic neuroendocrine tumors in transgenic zebrafish.Cancer Res. 2004; 64: 7256-7262Crossref PubMed Scopus (77) Google Scholar and melanoma.28Patton E.E. Widlund H.R. Kutok J.L. et al.BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.Curr Biol. 2005; 15: 249-254Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar These zebrafish cancer models provide a unique opportunity for studying interactions between known oncogenes and tumor-suppressor genes,28Patton E.E. Widlund H.R. Kutok J.L. et al.BRAF mutations are sufficient to promote nevi formation and cooperate with p53 in the genesis of melanoma.Curr Biol. 2005; 15: 249-254Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar or between genetic lesions and environmental carcinogens.29Haramis A.P. Hurlstone A. van der Velden Y. et al.Adenomatous polyposis coli-deficient zebrafish are susceptible to digestive tract neoplasia.EMBO Rep. 2006; 7: 444-449Crossref PubMed Scopus (135) Google Scholar In addition, these models also provide novel platforms with which to conduct high-throughput screens for both genetic and small-molecule modifiers of tumor progression.30Stern H.M. Zon L.I. Cancer genetics and drug discovery in the zebrafish.Nat Rev Cancer. 2003; 3: 533-539Crossref PubMed Scopus (236) Google Scholar In addition to providing novel insights regarding initiating events in pancreatic tumorigenesis, the current zebrafish model will enable these approaches to be applied to the study of pancreatic cancer. The authors thank Dr Yoel Kloog for kindly providing us with the pGFP-KRASG12V plasmid. Download .pdf (.12 MB) Help with pdf files Supplementary material Download .tif (1.13 MB) Help with tif files Supplementary figure Download .tif (1.5 MB) Help with tif files Supplementary figure Download .tif (1.4 MB) Help with tif files Supplementary material Download .tif (3.13 MB) Help with tif files Supplementary figure Download .tif (4.83 MB) Help with tif files Supplementary figure
Referência(s)