Cellular Plasticity and Heterogeneity in Pancreatic Regeneration and Malignancy

REVIEW

Cellular plasticity and heterogeneity in pancreatic regeneration and malignancy

C. Benedikt Westphalen1 Bernhard W. Renz2, Maximilian Reichert3,6, Anil K. Rustgi6, Timothy C. Wang4,5

1Department of Internal Medicine III, Hospital of the University of Munich, Munich, 81377, Germany 2Department of General, Visceral, and Transplantation Surgery, Hospital of the University of Munich, Munich, 81377, Germany 3Department of Internal Medicine II, Klinikum rechts der Isar, Technische Universität Munich, Munich, 81675, Germany 4Divison of Digestive and Liver Disease, Columbia University Medical Center, New York, NY 10032, USA 5Herberg Irving Comprehensive Cancer Center, Columbia University Medical Center, New York, NY 10032, USA 6Division of Gastroenterology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

Correspondence: Timothy C. Wang or C. Benedikt Westphalen E-mail: [email protected] or [email protected] Received: November 07, 2016 Published online: December 19, 2016

Pancreatic regeneration in response to tissue injury is a complex process. Recent progress in the understanding of this process has underscored the need for cellular plasticity as a prerequisite in the course of regeneration. A number of different pancreatic cell types have been proposed as progenitors, stem cells or differentiated cells with a high degree of plasticity. Moreover, in the setting of an oncogenic mutation, similar mechanisms appear to drive pancreatic tumorigenesis. Here, we aim to summarize the recent advances in our understanding of cellular plasticity in the setting of regeneration and tumorigenesis. Keywords: Dclk1; Prrx1; pancreatic cancer; plasticity; pancreatic stem cells; pancreatic regeneration To cite this article: C. Benedikt Westphalen, et al. Cellular plasticity and heterogeneity in pancreatic regeneration and malignancy. Can Cell Microenviron 2016; 3: e1472. doi: 10.14800/ccm.1472. Copyright: © 2016 The Authors. Licensed under a Creative Commons Attribution 4.0 International License which allows users including authors of articles to copy and redistribute the material in any medium or format, in addition to remix, transform, and build upon the material for any purpose, even commercially, as long as the author and original source are properly cited or credited.

Introduction therapies for diabetic patients. Potential strategies for regenerating pancreatic islet cells have been extensively The pancreas possesses a remarkable capacity to respond reviewed previously and are beyond the scope of this review to injurious insults and regenerates after cessation of tissue [4]. Only in the last decade, the cellular mechanisms for injury. This ability has generated considerable interest in the regeneration within the pancreatic exocrine compartment underlying mechanisms for this regenerative response. In have gained increasing interest. Despite major scientific general, two possibilities for tissue regeneration in the adult efforts, true stem cells in the adult pancreas have not been exist: (1) the presence of bona fide or facultative stem cells found and their existence appears unlikely. Nevertheless, an or (2) differentiated cells with the ability to de- or understanding of the mechanisms underlying regeneration of transdifferentiate and then proliferate in response to injury the pancreas might provide insights into the process of [1-3]. The scientific interest in adult pancreatic stem cells has malignant transformation of the pancreas. As pancreatic largely been fueled by the interest in developing regenerative ductal adenocarcinoma is a major health burden, a deeper

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Cancer Cell & Microenvironment 2016; 3: e1472. doi: 10.14800/ccm.1472; © 2016 by C. Benedikt Westphalen, et al. http://www.smartscitech.com/index.php/ccm understanding of its core biology is imperative for future metaplasia (ADM) was subsequently uncovered [13-17]. In diagnostic and therapeutic measures. Here, we will outline response to injury, acinar cells reactivate an embryonic the recent progress in the understanding of cellular plasticity program that involves the re-expression of PDX1, Sox9 and in the process of exocrine pancreatic regeneration and Hes1, and the emergence of ductal markers such as CK19 tumorigenesis. [18-21]. The process of ADM is reversible, and after cessation of injury, regeneration of acinar cells appears to be driven Cellular Plasticity in the Pancreas from the acinar compartment.

Under resting conditions, there is a steady but slow Since early studies applied rather broad Cre-drivers to turnover of pancreatic endocrine and exocrine cells. After study the process of acinar regeneration, it remained unclear injury, cell division rates increase markedly and the tissue if there are subsets of acinar cells that possess more plasticity mass is restored to a certain extent but not completely, given than others. Bmi1 is a marker of reserve stem cells in the the limited regenerative capacity of the pancreas compared to intestine [22]. Using Bmi1-CreERT mice, Capecchi and say, the liver. The drivers of this cellular response in the colleagues defined a subset of long-lived acinar cells in the pancreas have been a point of considerable debate, with some adult pancreas. They proposed a role for these cells in investigators favoring the existence of dedicated pancreatic pancreatic regeneration, as they found an expansion of this stem cells, while others proposing that differentiated cells lineage after pancreatic injury. However, since labeling of drive pancreatic regeneration through an intermediate stage the cells was induced only after injurious insult to the involving dedifferentiation [1, 3, 5, 6]. pancreas, it remained unclear if the regeneration occurred from existing Bmi1+ cells, or from newly generated Bmi1+ In the search for potential pancreatic stem cells, cells secondary to upregulation of the Bmi1 transcript [23]. investigators have turned to clonogenic or sphere forming assays [7]. These experiments yielded the finding that the Dclk1 has been proposed as a marker of quiescent stem most efficient clonogenic cells seem to reside within a subset cells in the intestine and the pancreas [24-26]. Using Dclk1 of ductal cells and/or in centroacinar cells [8, 9]. However, in BAC-CreERT mice, we were able to show that Dclk1 labels contrast to studies in other organ systems, there are rare and long-lived cells in the adult pancreas [27]. While insufficient experimental data supporting the concept that quiescent under homeostatic conditions, these cells became these clonogenic cells serve as pancreatic stem cells in vivo. activated following diverse forms of pancreatic injury and While ductal cells acquire some features of embryonic contribute to organ regeneration. Loss of Dclk1+ cells progenitor cells in response to pancreatic injury [10], (through diphtheria toxin-mediated cell ablation) prevented lineage-tracing experiments using a variety of ductal pancreatic repair and caused significant mortality after Cre-markers did not show convincing evidence of cerulein-induced pancreatitis. Finally, we were able to differentiation of ductal cells into other pancreatic lineages demonstrate that Dclk1+ cells had a proliferative advantage after pancreatic injury [1]. However, these findings do not rule over the Dclk1- compartment, arguing against the hypothesis out the possible existence of a subset of ductal cells with a that all acinar cells are equal and that a stochastic model higher degree of plasticity. In fact, recent findings from our underlies the regenerative processes in the pancreas [27]. labs have provided evidence for a rare subset of Prrx1+ ductal cells with a remarkable degree of sphere forming Centroacinar cells have been proposed as yet another ability and features of progenitor cells [11]. In fact, Prrx1 source of pancreatic stem cells due to their location at the tip ductal cells expanded markedly after cerulein-induced of the ductal tree, and their transcriptional profile [2, 6, 9, 28]. pancreatitis, consistent with a role in pancreatic regeneration While these cells express embryonic markers and form after injury [11]. Of note, Prrx1+ serve as stem cells in bone pancreatic organoids with higher frequency than other cell and cartilage formation [12]. types, subsequent lineage tracing studies did not reveal a major contribution of centroacinar cells to the regenerating Acinar to Ductal Metaplasia pancreas [29, 30].

In response to pancreatic tissue damage, complex ductal In summary, cellular plasticity may be an important driver structures arise. Because of their resemblance to the ductal of exocrine pancreatic regeneration. In particular, the epithelium, investigators thought initially that these existence of specialized subsets of acinar and ductal cells structures developed from pancreatic ducts. However, with a higher degree of plasticity has emerged in recent seminal lineage tracing studies revealed that differentiated studies. Consequently, it is tempting to speculate that these acinar cells are the predominant source of these ductal cells are potential candidates for facultative progenitor cells structures and the molecular framework of acinar-to-ductal involved in pancreatic repair after injury.

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Cellular Plasticity in Pancreatic Tumorigenesis potent cancer initiating cells in transplantation experiments [40]. Using our Dclk1 BAC-CreERT transgenic mice, we As outlined above, cellular plasticity within the exocrine investigated the role of Dclk1+ cells in the initiation of compartment plays an important role in pancreatic pancreatic cancer. Targeting mutant Kras expression regeneration. Furthermore, cellular plasticity seems to be an specifically to adult pancreatic Dclk1+ cells had no effect on underlying mechanism for malignant progression [31]. their quiescence and longevity. Similar to the behavior of Pancreatic ductal adenocarcinoma (PDAC) was originally other acinar cells, the combination of an oncogenic mutation thought to arise from cells of the ductal epithelium because in Dckl1+ cells with an injurious insult (cerulein-induced of its histological appearance [32]. However, in genetically pancreatitis) led to the emergence of PanINs from Dclk1+ engineered mouse models of PDAC, a variety of different lineage traced cells. Given their exceptional role in pancreatic cell types possess the ability to give rise to pancreatic regeneration, we investigated if these cells were precursors of pancreatic cancer [33, 34]. Acinar-to-ductal more efficient to give rise to PanINs than the bulk of the metaplasia, similar to that seen in response to pancreatic acinar compartment. The basic helix-loop-helix transcription injury, can be observed when an oncogenic Kras mutation is factor, Mist1, labels the majority of adult acinar cells. targeted to the acinar compartment [35]. In contrast to the Accordingly, cerulein-induced pancreatic leads to transient nature of injury-induced ADM, Kras-induced ADM widespread PanIN development in mice with mutant Kras persists and is also termed acinar-to-ductal reprogramming targeted to the Mist1 compartment. To estimate the degree of (ADR) [19, 36]. Importantly, expression of mutant Kras in transformation of pancreatic Dclk1+ cells we compared the adult acinar cells not only causes ADM/ADR but is sufficient efficiency of Dclk1+ and Mist1+ lineage traced cells to give to induce formation of pancreatic intraepithelial neoplasia rise to PanINs after pancreatitis. We found that Dclk1+ cells (PanIN), a precursor of pancreatic cancer [35]. were up to 12x more efficient in the induction of PanINs when compared to the Mist1+ acinar cells alone. These PanINs resemble ductal structures, and like PDAC were findings are consistent with the plasticity of Dclk1+ cells originally thought to be derived from the ductal epithelium. observed in pancreatic sphere cultures and pancreatic Lineage tracing studies, however established firmly the regeneration [27]. acinar compartment as the predominant source of murine PanINs [35]. Targeting mutant Kras to the adult acinar While there is compelling evidence that acinar cells serve compartment was shown to be sufficient to induce PanIN as the major source of murine PanINs, a number of formation. However, progression to PDAC is infrequently observations have also pointed to ductal cells as an origin for observed in these models, particularly when Kras is targeted PanIN lesions. Targeted expression of mutant Kras to CK19+ to adult pancreatic acinar cells. By contrast, Kras mutant ductal cells was sufficient to induce low frequency PanIN acinar cells readily give rise to PDAC when subjected to development [41]. Recently, Leach and co-workers were able experimental pancreatitis [37, 38]. Given the fact that to show that pancreatic cancer can also be derived from the pancreatitis reactivates an embryonic program in acinar cells ductal epithelium using Hnf1bCreERT mice, where the duct and increases their plasticity, it is not surprising that the specific transcription factor Hnf1b drives CreERT combination of an oncogenic mutation and an inflammatory expression. In their experiments, mutant Kras targeted to the insult converts pancreatic acinar cells into potent cancer ductal epithelium was not sufficient to initiate pancreatic initiating cells. In fact, Sander and co-workers were able to tumorigenesis. However, combined expression of mutant show that pancreatic acinar cells are >100 times more Kras and two mutant p53 alleles (R172H) led to rapid efficient in their ability to give rise to murine PanINs when development of PDAC from Hnf1b+ lineage traced cells. compared to duct cells [39]. Importantly, no other stimulus such as pancreatic injury was needed to induce malignant transformation. Of note, PanIN While these studies clearly pointed towards an important lesions were only infrequently observed when PDAC was role of acinar cells as an origin of pancreatic neoplasia, they driven from Krasmt/wt/p53mt/mt ductal cells [42]. Thus, in failed to answer the question if indeed all acinar cells were contrast to the acinar model where pancreatitis provides a alike or if some acinar cells existed that were more prone to second hit to induce pancreatic cancer, in the ductal model, give rise to PanINs. Bailey and co-workers were the first to the mutation (and loss of heterozygosity) of a tumor investigate the role of Dclk1+ cells in murine PanINs and suppressor gene (p53) suffices as the “second hit” to drive pancreatic cancer cell lines. They found that Dclk1 labeled a progression to mPDAC [43]. Taken together, these findings morphologically and functionally distinct subset of cells show that both ductal and acinar cells harbor the ability to within PanINs and pancreatic cancer cell lines. Functionally, initiate pancreatic cancer in mice. these cells displayed a high degree of sphere forming capacity and stemness. Furthermore, they were found to be Interestingly, pancreatic organoids, a long-term

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Cancer Cell & Microenvironment 2016; 3: e1472. doi: 10.14800/ccm.1472; © 2016 by C. Benedikt Westphalen, et al. http://www.smartscitech.com/index.php/ccm three-dimensional culture system of pancreatic epithelial relevance of Dclk1 expression was provided by in silico and cells, appear to be derived from pancreatic ductal epithelium in vitro studies from our group, which suggest a direct and [44]. While primary culture of acinar cells is possible as well functionally important interaction between Dclk1 and Kras and has been used to study acinar to ductal metaplasia [13, 45], [27, 48-50]. long-term primary cultures of acinar cells have not been established to date. Even if one takes into account, that acinar The Notch pathway is critically involved in cell fate cells represent the vast majority of pancreatic epithelial cells, decisions and regulates pancreatic development [51, 52]. In the experiments with forced expression of mutant Kras suggest adult, expression of Notch pathway components is restricted that a subset of acinar cells have a much higher degree of to centroacinar and terminal duct cells [29]. In response to plasticity. This higher degree of plasticity may translate into pancreatic injury, Notch signaling is reactivated and critical a higher degree of susceptibility to the deleterious effects of for pancreatic regeneration [53]. During tissue repair Notch mutant Kras and/or injury. While more resistant to Kras and Wnt/beta-catenin signaling cooperate to allow for acinar mutation and injury, ductal cells still retain the ability to give cell regeneration [53]. Furthermore, ongoing beta-catenin rise to pancreatic cancer under the appropriate conditions [42]. signaling is sufficient to block Kras-mediated acinar Our experimental data also suggest that not all acinar and transformation underscoring [19] the importance of tightly ductal cells are alike. Morphology alone is insufficient to regulated cellular identity in response to injurious stimuli. discriminate between subsets within a cellular compartment and evidence points to considerable heterogeneity within Additionally, cellular plasticity is regulated on an pancreatic lineages that remains to be explored. Considering epigenetic level. In zebrafish, loss of the pivotal DNA the remarkable heterogeneity of pancreatic ductal methyltransferase 1 (Dnmt1) leads to loss of the pancreas 84 adenocarcinoma - in terms of genetic alterations, histologic hours post fertilization. While duct and endocrine cells subtype and response to treatment - the concept of a develop normally, the acinar compartment undergoes complex, hierarchically organized pancreatic epithelium with complete apoptosis. Interestingly, loss of Dnmt1 leads to variable susceptibility towards oncogenic insults seems enhanced regeneration of pancreatic beta cells after targeted plausible. ablation. These early studies underscored the complex roles of epigenetic regulation in development and regeneration [54]. Mechanisms of Cellular Plasticity In injury, the epigenetic regulator Bmi1, a member of the polycomb repressor complex 1 (Prc1), is upregulated in Above we have aimed to outline, that cellular plasticity is acinar cells and coordinates regeneration in the exocrine an important mechanism in pancreatic regeneration and compartment. Moreover, loss of Bmi1 greatly impairs cancer development. The loss of cellular identity is exocrine regeneration after pancreatic injury. This is in part orchestrated on a genetic and epigenetic level. due to upregulation of Cdkn2a and p53-regulated Mechanistically, Sox9 seems to play a pivotal role in the proapoptotic genes [55]. Ezh2 is polycomb group protein and reprogramming of acinar cells towards dedifferentiation and part of the polycomb repressor complex 2 (Prc2). Following neoplasia. Sander and co-workers were able to show that loss pancreatic injury, Ezh2 mediates pancreatic regeneration by of the Sox9 protein prevented conversion of acinar cells into suppression of p16Ink4a. Suppression of p16Ink4a leads to PanINs; however Sox9 is not absolutely necessary for acinar enhanced proliferation of progenitor cells driving tissue to ductal metaplasia [39]. In contrast, forced overexpression of repair. Consequently, loss of Ezh2 causes failure of Sox9 in the setting of mutant Kras significantly enhanced pancreatic regeneration and accelerates pancreatic pancreatic tumorigenesis through downregulation of acinar tumorigenesis. The complex interplay of genetic and markers such as Mist1 [39]. Mist1 is a transcription factor epigenetic mechanisms mediating pancreatic plasticity and critically involved in acinar cell identity. Mist1 expression is repair have recently been excellently elsewhere [31, 56]. regulated by the transcription factor Gata6 and loss of Gata6 causes degeneration of the acinar compartment [46]. More Finally, the microenvironment plays a pivotal role in importantly, Gata6 controls multiple differentiation programs pancreatic regeneration and tumorigenesis by influencing the in the adult pancreas and counteracts Kras-driven epithelial compartment [57-59]. In response to an injurious tumorigenesis [47]. Furthermore, we were able to show that stimulus, acinar cells release proinflammatory factors such as the transcription factor Prrx1b binds to the Sox9 promoter Interleukin 6 (IL-6), tumor necrosis factor (TNF) and positively regulating Sox9 expression and thereby promoting transforming growth factor-beta (TGF-beta), which in turn cellular plasticity. Importantly, Dclk1+ cells express high activate the immune system but also otherwise quiescent levels of Sox9 under resting conditions (unpublished pancreatic stellate cells (PSC) [60]. Activated stellate cells observations), offering a potential explanation for their high secrete a variety of factors such as insulin-like growth factor, degree of plasticity and transformability. Insight into the epidermal growth factor, hepatocyte growth factor,

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Nodal/Activin and platelet derived growth factor. These compartment are collectively mutated to induce pancreatic mediators in turn lead to increased aggressiveness of tumorigenesis. A major advance in the field would be the surrounding cancer cells [61-65]. The importance of this generation of spontaneous models that do not rely on broad crosstalk between PSC and pancreatic cancer cells is Cre-drivers to achieve malignant transformation. Interesting demonstrated by the fact, that restoration of PSC quiescence new in vivo model systems are on the rise that combine profoundly affects cancer cells in proximity [66]. Furthermore, genomic engineering and at the same time recapitulate the we were able to show that an epithelial-stromal crosstalk stochastic nature of the disease [74]. Given the fact that some mediated by the transcription factor ETV1 regulates stromal forms of chronic pancreatitis pose an increased risk for the expansion, epithelial to mesenchymal transition and development of pancreatic cancer in humans we can only metastatic spread underscoring the importance of the stromal speculate that the same mechanisms driving malignancy in compartment in regulating epithelial plasticity [67]. mice also dominate the process in humans. Further studies Immunosurveillance plays as major role to eliminate mutant and novel technologies such as organoid cultures and cells before their expansion [68]. However, ongoing activation humanized animal models will be instrumental in translating of the immune system is an important driver of early these fundamental questions from mice into humans and pancreatic tumorigenesis, in part through the might then help to design future preventive and therapeutic NFATc1-STAT3 transcription complex [69]. Furthermore, approaches for pancreatic cancer. mutant Kras causes expression of GM-CSF which in turn leads to recruitment of immunosuppressive myeloid and Conflicting interests exclusion of cytotoxic T-cells [70, 71]. The authors have declared that no conflict of interests Conclusions and Outlook exists.

It is widely acknowledged that the most efficient cellular Acknowledgements origin of PDAC precursors lies within the acinar epithelium. Only recently, it was proposed that specific subsets of acinar This work was supported by the National Pancreas cells could exist that drive pancreatic regeneration (and Foundation (MR), the KKF Program and the TUM Junior tumorigenesis) [23]. Indeed, we were able to show that Dclk1 Fellow Program of the Technical University of Munich labels such a progenitor population involved in repair and (MR), the German Cancer Aid Foundation (Max-Eder cancer initiation [27]. Future studies are needed to establish if Program, Deutsche Krebshilfe 111273 to MR), and the the “plastic pancreas” [5] contains more than just one of these AGA-Actavis Research Award in Pancreatic Disorders progenitor/stem cell populations or if multiple different pools (MR). of cell types with different properties coexist in the pancreas. Of note, coexistence of different stem cell pools has been Abbrevations demonstrated in other organs such as the skin and the intestine [72, 73]. Additionally work from our groups has CK19: Cytokeratin 19; Dclk1: Doublecortin like kinase clearly demonstrated that a distinct subset of Prrx1+ ductal protein 1; ADM: acinar to ductal metaplasia; (m)PanIN: cells exists and that these are functionally different from the murine pancreatic intraepithelial neoplasia; PDAC: bulk of the ductal epithelium [11]. It is tempting to speculate pancreatic ductal adenocarcinoma; Sox9: SRY-box 9; Prxx1: that cancers arising in the setting of mutant Kras/p53 targeted Paired related homeobox 1; Hnf1b: HNF1 homeobox B; to the duct compartment (see above) originate from this IPMN: intraductal papillary mucinous neoplasia; ADR: specific population. acinar-to ductal reprogramming.

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