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GASTROENTEROLOGY Vol. 133, No. 6
Metaplastic Metamorphoses in the Mammalian Pancreas
See “In vivo lineage tracing defines the role of acinar-to-ductal transdifferentiation in inflammatory ductal metaplasia” Strobel O, Dor Y, Alsina J, et al on page 1999.
When Gregor Samsa woke up one morning from unsettling dreams, he found himself changed in his bed into a monstrous vermin. . . . —Franz Kafka
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n Franz Kafka’s The Metamorphosis, Gregor Samsa finds to his horror that he has been transformed from a once reliable employee into a giant cockroach. Could pancreatic acinar cells sometimes feel the same way? A series of recent studies, including the manuscript by Strobel et al1 in this issue of GASTROENTEROLOGY, suggest that these highly specialized cells can, under certain conditions, act not only as progenitor cells capable of participating in pancreatic renewal, but also as cells of origin for pancreatic intraepithelial neoplasia (PanIN), a precancerous condition traditionally considered to involve only ductal epithelial cells. The capacity for large expanses of pancreatic epithelium (as opposed to individual cells) to undergo phenotypic conversion has been recognized for some time. Metaplastic conversion, in which one predominant cell type in a tissue is replaced by another, has been observed in both the endocrine and exocrine pancreas in response to a variety of environmental events, ranging from growth factor stimulation to the induction of epithelial injury. In vitro and in vivo examples of pancreatic epithelial metaplasia include ductal-to-islet metaplasia (islet neogenesis),2–7 islet-to-ductal metaplasia,8 –12 acinar-to-islet metaplasia2,3 and acinar-to-ductal metaplasia.14 –18 It is important to note that, in each of these examples, use of terms such as “acinar-to-ductal” and “islet-toductal” does not imply the direct metamorphosis of one cell type to another, but rather to a metaplastic change in the balance between predominant cell types.19 As in other tissues, at least 3 possible mechanisms for pancreatic epithelial metaplasia can be proposed. These include the selective proliferation or loss of mature cell types, reprogramming of undifferentiated epithelial progenitor cells in a manner that alters the balance of differentiated progeny (trans-specification), or the direct conversion of 1 cell type to an alternate phenotype (transdifferentiation). While not ruling out other mechanisms, a series of recent studies document the capacity of pancreatic acinar cells to undergo transdifferentiation and provide evidence that changes in acinar cell differentiation may
contribute to the initiation of pancreatic “ductal” neoplasia. The transdifferentiation potential of acinar cells appears to be especially robust in vitro. During the 1990s, a series of seminal investigations by DeLisle, Logsdon, Bouwens, Githens, Real, and colleagues defined the ability of acinar cell cultures to undergo in vitro conversion to a duct-like population.20 –23 More recently, the role of Notch signaling in driving this process has been realized, with either activated Notch or the Notch target gene, Hes1, shown to be sufficient to induce acinar-to-ductal metaplasia in vitro.24 Although these early investigators all realized the likelihood of direct transdifferentiation, rigorous confirmation of an acinar cell origin for metaplastic cell types awaited the development of Cre/lox-based lineage tracing technologies, in which Cre recombinase expressed in specific cell types induces recombination of a reporter gene such as -galactosidase or alkaline phosphatase. Because Cre activity results in a stable genomic recombination event, this technique effectively labels Cre-expressing cells as well as their subsequent progeny, providing a heritable mark of cell lineage. Using this technique, Means et al25 used 2 different acinar cell-specific Cre lines (Elastase:CreERT2 and Villin: Cre) to demonstrate an acinar cell origin for cytokeratinpositive duct-like cells emerging from cultures of collagenase digested mouse pancreas, and further demonstrated that this duct-like epithelium was generated by way of a dedifferentiated nestin-positive intermediate. Similarly, Minami et al13 used an acinar cell-specific Amylase:Cre transgene to show that fully differentiated acinar cells are capable of transdifferentiating to glucose-sensing, insulin-secreting cells. Similar Cre/lox-based lineage tracing techniques have also been applied in vivo to demonstrate that acinar cells have the potential to act as lineage-restricted progenitors, actively contributing to regeneration of acinar cell mass following partial pancreatectomy.26 In this issue of GASTROENTEROLOGY, Strobel et al1 now significantly extend these findings by documenting the phenomenon of acinar cell transdifferentiation in vivo. Using administration of the cholecystokinin (CCK) agonist caerulein to induce a form of chronic pancreatitis, they report the generation of 3 different types of metaplastic lesions originating from distinct cellular origins, suggesting that the term “ductal metaplasia” may represent a dramatic oversimplification. Resurrecting terminology previously established to describe lesions observed in human pancreatitis, the authors describe 2 types of “tubular complexes” (TC), and distinguish these lesions from mucinous metaplastic lesions (MML). Under this nomenclature, TC type 1 (TC1) appear in cross-section as circular lesions with a wide lumen typically lined by a
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small number of flattened epithelial cells lacking expression of either Hes1 or ductal cytokeratins. In contrast, TC2 lesions are complex branched tubules lined by cytokeratin-positive and Hes1-positive cuboidal epithelial cells. TC1 and TC2 lesions differ from MML that consist of cuboidal or columnar cells expressing PAS- and Alcian blue-positive mucins, features that are also observed in precancerous PanIN lesions. Strobel et al1 confirm the validity of this classification by demonstrating that lesions comprising these 3 entities arise from distinct cellular origins. Using a tamoxifeninducible Elastase:CreER line to selectively label acinar cells and their derivatives, the authors show that TC1 are predominantly acinar cell derived, while TC2 originate from a non-acinar source, presumably terminal ductal epithelial cells with which they share a number of phenotypic characteristics. With respect to mucinous “ductal” metaplasia, the authors further show that a fraction of MML are derived from an acinar cell population, thereby confirming the ability of acinar cells to undergo in vivo transdifferentiation. Although acknowledging that the specific cell type(s) responsible for the nonacinar origins of all TC2 and most MML remain to be demonstrated, the authors infer a ductal and/or centroacinar origin for these processes, based on common features such as Hes1 expression. Whereas a proposed ductal/centroacinar origin for these lesions certainly seems plausible, rigorous confirmation awaits the development of Cre lines for activating lineage labels in these compartments. These issues are summarized schematically in Figure 1. In addition to providing direct in vivo evidence for the potential of acinar cells to undergo metaplastic change, these findings may also have considerable relevance for identifying the cellular origins of pancreatic cancer. Given the histologic similarity between caerulein-induced MML and PanIN, as well as the apparent causal link between chronic pancreatitis and pancreatic cancer,27,28 it is entirely plausible that caerulein-induced lesions are the equivalent of low-grade mouse and human PanIN. Based on the fact that Strobel et al1 implicate both acinar and non-acinar cells as the source of MML, it remains possible that PanINs arise from similar disparate cellular origins. The ability of nonductal cells to act as cells-of-origin for pancreatic “ductal” cancer has additionally been suggested by several other new studies, as recently reviewed.29 Zhu et al30 reported the frequent onset of epithelial metaplasia as the earliest histologic alteration following activation of oncogenic K-ras in the murine pancreas, and have also documented that early PanIN lesions frequently include cells which phenotypically resemble acinar cells. An acinar cell contribution to murine PanIN is further supported by a report from Carriere et al,31 in which progressive PanIN lesions were demonstrated following Cre-mediated activation of oncogenic
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Figure 1. Cellular origins of metaplastic and neoplastic pancreatic lesions induced by either chronic inflammation or activation of oncogenic K-ras. Chronic inflammation induced by caerulein administration leads to formation of type 1 and type 2 tubular complexes, referred to as TC1 and TC2, as well as MML. Histologically, MML appear identical to murine PanIN, although the ability of these MML to progress to invasive cancer has not yet been established. TC1 and a fraction of MML arise from acinar cells. Asterisks indicate presumed but as yet unproven origin of TC2 and majority of MML from terminal ductal and/or centroacinar cells. For oncogenic K-ras, induction of progressive PanINs occurs after activation in acinar and/or centroacinar cells. Whether similar PanIN induction can occur following selective activation in terminal duct cells remains unknown. As noted, interactions may also occur between chronic inflammation and oncogenic K-ras, generating additional phenotypic complexity.
K-ras in cells expressing Nestin, an intermediate filament protein previously shown to mark an exocrine-specific progenitor pool.32,33 Similarly, Guerra et al27 used an elastase promoter-based system to activate oncogenic Kras in acinar and/or centroacinar cells, resulting in the generation of both PanINs and invasive pancreatic “ductal” adenocarcinoma. In this system, PanIN induction following oncogenic K-ras activation in older mice required the concomitant induction of chronic pancreatitis using caerulein. When considered along with the findings of Strobel et al,1 these findings suggest that mature acinar cells may only become responsive to oncogenic K-ras following inflammation-mediated transdifferentiation, suggesting a fascinating interaction between epigenetic determinants of cell identity and genetic changes leading to tumor formation. Although K-ras mutations are generally considered to be an early event in pancreatic tumorigenesis, it is important to note that some 50% of low-grade human PanINs appear to retain exclusively wild-type K-ras alleles, underscoring the fact that the earliest initiating events in this disease may involve nonmutational changes in epithelial differentiation. Ultimately, one hopes that the study of cellular origins for pancreatic epithelial metaplasia and neoplasia in the mouse will provide valid insights regarding similar cells of origin in human pancreas. In this regard, it is important to consider the advantages and disadvantages of
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various approaches, including the activation of oncogenic K-ras in various cellular compartments using different pancreatic cell type-specific Cre lines.27,30,31,34,35 It should be noted this approach essentially interrogates the competence of different cell lineages to generate PanIN in response to widespread activation of oncogenic K-ras, which differs from an interrogation of the initiating compartment in which spontaneous K-ras mutations actually occur. This distinction essentially boils down to the difference between determining what can happen as opposed to determining what actually does happen. In this regard, the approach taken by Strobel et al1 may more closely mimic pathogenic events leading to pancreatic neoplasia, because the diffuse inflammatory process induced by caerulein may mimic events that actually occur in human disease, whereas pancreas-wide activation of oncogenic K-ras seems to represent a less likely event. However, it must also be acknowledged that the ability of PanIN-like lesions induced by caerulein to progress toward more advanced lesions has not yet been documented, and the degree to which caerulein-induced events may be biased toward an influence on specific pancreatic cell types remains to be determined. Although the study by Strobel et al1 represents a seminal advance in providing rigorous evidence of acinar cell transdifferentiation in vivo, a number of questions remain. First, what determines the balance between the majority of non-acinar MML, presumably derived from ductal epithelium, and the minority of acinar-derived lesions? In this regard, finer temporal mapping of metaplastic events induced by caerulein is required. For example, it is possible that at earlier or later time points, a different balance between non-acinar and acinar-derived lesions might be observed, with the initial acinar cell contribution subsequently altered by simultaneous transspecification of exocrine progenitor cells, resulting in progressive dilution of the original acinar-derived component. Alternatively, it may be that factors specific to the caerulein model, which appear to uniquely target CCK receptor-bearing acinar cells, leads to such widespread acinar cell destruction that only a limited population remains available to contribute to the emerging metaplastic epithelium. Ultimately, it may simply be that acinar cells are faced with a higher “activation energy” than non-acinar cells in terms of the ability to generate MML; for example, acinar cells may require activation of Notch signaling in order to lose their acinar cell identity, whereas other cell types may not. In addition to providing insights regarding pancreatic epithelial plasticity, understanding the molecular pathways that instruct and permit cellular metamorphosis offers great potential to engineer cells to adopt different cell fates. In addition to raising many fascinating questions regarding the origins of metaplastic and neoplastic pancreatic epithelium, these studies cumulatively suggest the need for formal changes in the terminology applied to
GASTROENTEROLOGY Vol. 133, No. 6
these lesions. Although the entire spectrum of lesions characterized by Strobel et al1 was once referred to under the broad category of “ductal metaplasia,” it now appears necessary to use more specific terminology reflecting the diverse biology and distinct cellular origins of TC1, TC2, and MML. Given growing awareness of the fundamental difference between neoplastic pancreatic epithelium and normal ductal epithelium, we predict that the term “ductal neoplasia” may similarly require modification over time. At the very least, it appears necessary to revise the criteria for murine PanIN, defined by a recent consensus conference.36 The consensus report limits use of the term “PanIN” to describe proliferative lesions arising in native pancreatic ducts, reflecting the shared view of a panel of expert pancreatic pathologists convened in 2004. The authors of this consensus report sagely predicted that their classification system might require modification based on newly developed models, a prediction borne out by multiple recent demonstrations of nonductal origins of murine PanIN.
STEPHEN F. KONIECZNY Department of Biological Sciences and the Purdue Cancer Center Purdue University West Lafayette, Indiana STEVEN D. LEACH Departments of Surgery, Oncology, and Cell Biology Johns Hopkins University Baltimore, Maryland References 1. Strobel O, Dor Y, Alsina J, et al. In vivo lineage tracing defines the role of acinar-to-ductal transdifferentiation in inflammatory ductal metaplasia. Gastroenterology 2007;133:1999 –2009. 2. Fernandes A, King LC, Guz Y, et al. Differentiation of new insulinproducing cells is induced by injury in adult pancreatic islets. Endocrinology 1997;138:1750 –1762. 3. Gu D, Sarvetnick N. Epithelial cell proliferation and islet neogenesis in IFN-␥ transgenic mice. Development 1993;118:33– 46. 4. O’Reilly LA, Gu D, Sarvetnick N, et al. Alpha-Cell neogenesis in an animal model of IDDM. Diabetes 1997;46:599 – 606. 5. Sharma A, Zangen DH, Reitz P, et al. The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 1999;48:507–513. 6. Song SY, Gannon M, Washington MK, et al. Expansion of Pdx1expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpressing transforming growth factor alpha. Gastroenterology 1999;117:1416 –1426. 7. Wang RN, Kloppel G, Bouwens L. Duct- to islet-cell differentiation and islet growth in the pancreas of duct-ligated adult rats. Diabetologia 1995;38:1405–1411. 8. Krakowski ML, Kritzik MR, Jones EM, et al. Pancreatic expression of keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells. Am J Pathol 1999; 154:683– 691. 9. Means AL, Ray KC, Singh AB, et al. Overexpression of heparinbinding EGF-like growth factor in mouse pancreas results in fibro-
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10. 11.
12. 13.
14.
15.
16.
17.
18.
19. 20.
21.
22.
23.
24.
sis and epithelial metaplasia. Gastroenterology 2003;124: 1020 –1036. Schmied BM, Liu G, Matsuzaki H, et al. Differentiation of islet cells in long-term culture. Pancreas 2000;20:337–347. Yuan S, Rosenberg L, Paraskevas S, et al. Transdifferentiation of human islets to pancreatic ductal cells in collagen matrix culture. Differentiation 1996;61:67–75. Pour PM, Pandey KK, Batra SK. What is the origin of pancreatic adenocarcinoma? Mol Cancer 2003;2:13. Minami K, Okuno M, Miyawaki K, et al. Lineage tracing and characterization of insulin-secreting cells generated from adult pancreatic acinar cells. Proc Natl Acad Sci U S A 2005;102: 15116 –15121. Rooman I, Heremans Y, Heimberg H, et al. Modulation of rat pancreatic acinoductal transdifferentiation and expression of PDX-1 in vitro. Diabetologia 2000;43:907–914. Jhappan C, Stahle C, Harkins RN, et al. TGF alpha overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 1990;61: 1137–1146. Sandgren EP, Luetteke NC, Palmiter RD, et al. Overexpression of TGF alpha in transgenic mice: induction of epithelial hyperplasia, pancreatic metaplasia, and carcinoma of the breast. Cell 1990; 61:1121–1135. Scoggins CR, Meszoely IM, Wada M, et al. p53-dependent acinar cell apoptosis triggers epithelial proliferation in duct-ligated murine pancreas. Am J Physiol Gastrointest Liver Physiol 2000;279: G827– 836. Yuan S, Duguid WP, Agapitos D, et al. Phenotypic modulation of hamster acinar cells by culture in collagen matrix. Exp Cell Res 1997;237:247–258. Slack JM, Tosh D. Transdifferentiation and metaplasia—switching cell types. Curr Opin Genet Dev 2001;11:581–586. Bouwens L. Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 1998;43:332–336. De Lisle RC, Logsdon CD. Pancreatic acinar cells in culture: expression of acinar and ductal antigens in a growth-related manner. Eur J Cell Biol 1990;51:64 –75. Githens S, Schexnayder JA, Moses RL, et al. Mouse pancreatic acinar/ductular tissue gives rise to epithelial cultures that are morphologically, biochemically, and functionally indistinguishable from interlobular duct cell cultures. In Vitro Cell Dev Biol Anim 1994;30A:622– 635. Vila MR, Nakamura T, Real FX. Hepatocyte growth factor is a potent mitogen for normal human pancreas cells in vitro. Lab Invest 1995;73:409 – 418. Miyamoto Y, Maitra A, Ghosh B, et al. Notch mediates TGFalphainduced changes in epithelial differentiation during pancreatic tumorigenesis. Cancer Cell 2003;3:565–576.
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25. Means AL, Meszoely IM, Suzuki K, et al. Pancreatic epithelial plasticity mediated by acinar cell transdifferentiation and generation of nestin-positive intermediates. Development 2005;132: 3767–3776. 26. Desai BM, Oliver-Krasinski J, De Leon DD, et al. Preexisting pancreatic acinar cells contribute to acinar cell, but not islet beta cell, regeneration. J Clin Invest 2007;117:971–977. 27. Guerra C, Schuhmacher AJ, Canamero M, et al. Chronic pancreatitis is essential for induction of pancreatic ductal adenocarcinoma by K-Ras oncogenes in adult mice. Cancer Cell 2007;11: 291–302. 28. Lowenfels AB, Maisonneuve P, Cavallini G, et al. Pancreatitis and the risk of pancreatic cancer. International Pancreatitis Study Group. N Engl J Med 1993;328:1433–1437. 29. Murtaugh LC, Leach SD. A case of mistaken identity? Nonductal origins of pancreatic ”ductal” cancers. Cancer Cell 2007;11: 211–213. 30. Zhu L, Shi G, Schmidt CM, et al. Acinar cells contribute to the molecular heterogeneity of pancreatic intraepithelial neoplasia. Am J Pathol 2007;171:263–273. 31. Carriere C, Seeley ES, Goetze T, et al. The Nestin progenitor lineage is the compartment of origin for pancreatic intraepithelial neoplasia. Proc Natl Acad Sci U S A 2007;104:4437– 4442. 32. Delacour A, Nepote V, Trumpp A, et al. Nestin expression in pancreatic exocrine cell lineages. Mech Dev 2004;121:3–14. 33. Esni F, Stoffers DA, Takeuchi T, et al. Origin of exocrine pancreatic cells from nestin-positive precursors in developing mouse pancreas. Mech Dev 2004;121:15–25. 34. Aguirre AJ, Bardeesy N, Sinha M, et al. Activated Kras and Ink4a/Arf deficiency cooperate to produce metastatic pancreatic ductal adenocarcinoma. Genes Dev 2003;17:3112–3126. 35. Hingorani SR, Petricoin EF, Maitra A, et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 2003;4:437– 450. 36. Hruban RH, Adsay NV, Albores-Saavedra J, et al. Pathology of genetically engineered mouse models of pancreatic exocrine cancer: consensus report and recommendations. Cancer Res 2006;66:95–106.
Address requests for reprints to: Steven D. Leach, MD, Professor of Surgery and Cell Biology, Johns Hopkins University, Broadway Research Bldg, Rm. 471, 733 N. Broadway, Baltimore, Maryland 21205. e-mail:
[email protected]; fax: (410) 614-2913. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.10.061
