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Activating mutations in the KRAS proto-oncogene occur almost ubiquitously in pancreatic ductal adenocarcinoma (PDAC) and in its putative precursor lesions, pancreatic intraepithelial neoplasia (PanIN). Conditional expression of an activated Kras allele in the mouse pancreas produces a model that faithfully recapitulates PanIN formation and progression to PDAC. Importantly, although nearly every cell in the pancreata of these mice express activated Kras, only a very small minority of cells give rise to PanINs. How the transforming activity of Kras is constrained in the pancreas remains unknown, and the cell types from which PanINs and PDAC arise are similarly unknown. Here, we describe our recent results demonstrating that acinar cells are competent to form Kras-induced PanINs, and that active Notch signaling can synergize with Kras in PanIN initiation and progression. Further efforts to understand how Notch and Kras synergize, as well as experiments to determine how other pancreatic cell types contribute to PDAC development, should aid in the development of new therapies and early detection techniques that are desperately needed for this cancer.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth-leading cause of cancer deaths in the United States, with over 37,000 diagnoses projected for 2008 and nearly as many deaths.1 Activating mutations in the KRAS proto-oncogene are found in almost all cases of PDAC, as well as in early precursor lesions termed pancreatic intraepithelial neoplasia (PanIN), suggesting that KRAS activation may represent an initiating genetic lesion (Figure 1). Additional mutations are found in full-blown PDAC, such as loss of CDKN2A and TP53, and their increasing prevalence in increasingly dysplastic PanIN lesions provides the basis for a PanIN-to-PDAC progression model.2 It remains unclear, however, precisely what pancreatic cell type gives rise to PanINs and PDAC. Furthermore, while a number of signaling pathways appear to be hyperactive in PanINs and PDAC, their contributions to tumorigenesis remain uncertain. Addressing these questions should hopefully aid in developing new therapies and diagnostic techniques for PDAC, and thus improve currently dismal patient outcomes.
Epithelial cancers are often proposed to arise from stem cells, as the long life and continuous cycling of these cells would permit them to accumulate the numerous mutations required for carcinoma formation. Stem cells have been sought in the pancreas for many years, given their potential utility in replacing β-cells lost to diabetes, but their existence remains hypothetical.3 At present, the search for the cell of origin of PDAC focuses on the three cell types of the exocrine pancreas: acinar, duct, and centroacinar cells (CACs) (Figure 1). Acinar cells secrete digestive enzymes into a network of ductal cells, which channels them into the small intestine. CACs reside at the acinar-ductal junction, and apart from their position and small size are not obviously different from other duct cells.4 Nonetheless, interest in CACs has been kindled by the realization that, unlike most other adult pancreatic cells, CACs express the Notch target gene Hes1.5,6 As we will discuss, Notch signaling inhibits progenitor cell differentiation in the embryonic pancreas,7–9 and the apparent maintenance of this activity in CACs suggests that they may possess some stem-like properties.6
From a clinical standpoint, does it matter what cell type gives rise to PDAC? We suggest that efforts to develop early detection and treatment methods would benefit from PDAC models that reflect the cellular context in which tumors normally arise. Furthermore, genetic lesions that promote tumor formation in one cell type may be ineffectual in another, highlighting possible endogenous mechanisms of tumor resistance. Indeed, a recent study of human mammary epithelial cells demonstrated that their differentiation status, in primary culture, dictates their potential malignancy following oncogene misexpression.10 Compared to the mammary gland, primary culture of pancreatic cells is in its infancy, but recent work suggests that KRAS-induced transformation can be modeled in such cells.11 In the meantime, much effort has focused on developing genetically-engineered mouse models that develop pancreatic adenocarcinomas similar to those of human patients, and which might therefore shed light on the cell-of-origin question.
As PanINs and PDAC resemble duct cells both in morphology and marker gene expression, it is reasonable to hypothesize that PDAC arises from the ductal lineage. For historical reasons, however, GEM models have focused more on the tumor-forming potential of acinar cells. More than 20 years ago, the promoter of the Elastase1 gene was found to confer pancreatic acinar-specific expression in transgenic mice,12 and shortly thereafter several studies demonstrated that acinar cells could be transformed in vivo by a variety of oncogenes.13–15 One of these, cMyc, induced carcinomas of mixed acinar-ductal character, suggesting that transformed acinar cells could assume a duct-like phenotype.15 More recently, acinar-specific expression of an activated KrasG12D allele, in Elastase1-KrasG12D transgenic or Mist1KrasG12D knock-in mice, also yielded tumors of mixed acinar-ductal phenotype.16,17 Similarly, retroviral targeting of the polyoma middle-T oncogene to acinar cells produced mixed acinar/ductal tumors.18 Experiments to directly target duct cells have been rarer, possibly due to a lack of suitable promoters. The most informative to date involves expression of an activated KrasG12V allele from the duct specific Cytokeratin-19 promoter, which produced essentially no phenotype in vivo.19,20
It is certainly premature to draw, from the one study cited above, negative conclusions regarding the tumorigenic potential of duct cells. Nonetheless, the results of targeting Kras and other oncogenes to acinar cells do support the idea that this compartment can give rise to ductal tumors. One complication of these results, both positive and negative, is that they rely on over-or mis-expression of activated Kras, which can have unexpected consequences.21 A breakthrough in mouse cancer modeling was achieved in 2003 with the creation of a conditionally-activatable allele of the endogenous Kras gene, loxP-STOP-loxP-KrasG12D (henceforth, KrasLSLG12D). This allele is silent in the germline configuration, but can be activated in somatic tissues by Cre recombinase. When KrasLSLG12D is activated via expression of Cre in pancreatic progenitor cells (using a Pdx1Cre transgene or a Ptf1aCre knock-in allele), mice develop PanIN lesions similar to those of humans that, with advanced age and/or loss of tumor suppressor genes, progress to invasive carcinoma.20,22,23 While this model for the first time recapitulates human PDAC progression, it cannot identify the cell type from which tumors arise, as recombination occurs in pancreatic progenitor cells that give rise to all endocrine and exocrine cell types. Interestingly, despite widespread KrasG12D expression in this model, beginning in utero, PanINs are infrequent and focal while the majority of the organ is completely normal. The cells that do initiate PanIN formation may have sustained another genetic “hit”, or may be subject to epigenetic influences that affects their response to Kras.
Several further studies of KrasLSLG12D mice, or mice carrying a similar Cre-dependent KrasLSLG12V allele, suggest that their tumors may arise from acinar cells. First, the PanINs that arise when KrasLSLG12D is activated in pancreatic progenitors are found to be associated with acinar-ductal metaplasia, or the apparent conversion of acinar cells to a duct-like phenotype.24 Second, activating KrasLSLG12D specifically in acinar cell precursors, using a NestinCre transgene, yields PanIN lesions indistinguishable from those induced by Pdx1Cre or Ptf1aCre activation.25 Finally, activation of KrasLSLG12V in immature acinar and centroacinar cells also results in PanIN and PDAC.26 Of note, all of these models involve Kras activation in utero, rather than in the adult organ; as PDAC most commonly strikes the elderly, its origin in humans is unlikely to be in embryonic progenitor cells. In fact, restricting activation of KrasG12V to adult acinar cells was found to prevent PanIN formation entirely unless the mice were subjected to experimentally-induced chronic pancreatitis.26
Chronic pancreatitis is a significant risk factor for human PDAC,27 and experimental pancreatitis in mouse is associated with acinar-ductal metaplasia (Figure 2) and upregulation of Notch signaling.28 Notch signaling, in turn, is necessary and sufficient for ductal metaplasia of cultured acinar cells,5 and acts in the embryonic pancreas to suppress acinar and islet differentiation.9 Furthermore, Notch pathway components (including the downstream target gene, Hes1) are overexpressed in human and mouse PanINs and PDAC.5,22 We hypothesized that Notch activation might facilitate the Kras-driven conversion of acinar cells to PanINs, providing both an explanation for the effects of experimental pancreatitis in the KrasLSLG12V model26 and a mechanism by which a classic ductal tumor could arise from non-ductal cells.
In order to test the competence of mature acinar cells to undergo Kras-induced PanIN formation, we used ElaCreERT transgenic mice, expressing a tamoxifen (TM)-inducible form of Cre recombinase under the Elastase1 promoter, to activate the KrasLSLG12D allele specifically in adult acinar cells. Importantly, this Cre line does not induce recombination in duct or centroacinar cells29,30 (Figure 2), and its TM-dependence allowed us to delay KrasG12D expression until adulthood. In contrast to the earlier study described above, in which adult acinar cells were found to be refractory to KrasG12V,26 we found that KrasLSLG12D activation in adult acinar cells resulted in focal PanIN formation.31 This finding was corroborated by an independent study, in which KrasLSLG12D was activated in adult acinar cells using two different TM-dependent Cre lines, ElaCreERT2 and Mist1CreERT2.32 The earlier study, in which lesions did not arise from mature acinar cells, used a different Kras allele, KrasLSLG12V, which appears to have less transforming activity in other tissues.33,34 Nonetheless, pancreatitis does sensitize acinar cells to KrasG12D-induced dysplasia in our model as well (Figure 3), suggesting that injury-activated pathways, such as Notch, can override whatever mechanisms normally exist to dampen acinar responsiveness to Kras.
Our work31 and that of our colleagues32 provide further evidence that acinar cells represent a cell of origin for PDAC, but cannot exclude a ductal origin. In fact, the ductal compartment, specifically centroacinar cells, appears to be the source of invasive adenocarcinomas that form in mice with a pancreas-specific deletion of the Pten tumor suppressor gene.29 The tumors of this model have a papillary morphology reminiscent of human intraductal papillary mucinous neoplasm (IPMN), a rare lesion thought to arise within large ducts and considered an alternate precursor to PDAC.2 Pten encodes a negative regulator of phosphoinositide-3-kinase (PI3K) signaling, and although mutations in this pathway have not been described in human PanINs or PDAC, a recent study has found that a subset of IPMNs harbor activating mutations in the PI3K-alpha gene (PIK3CA).35 Together, these studies suggest that Kras activation might drive PanIN formation from acinar cells, while ductal cells give rise to an alternative lesion, IPMN, via activation of the PI3K pathway (Figure 1). Testing this model in mice will require development of new tools, not least a duct-specific Cre recombinase.
Notch inhibits progenitor cell differentiation in the embryonic pancreas,9 and its upregulation in human and mouse PanINs and PDAC suggests that it might play a role in pancreatic tumorigenesis.5,22 This is substantiated by in vitro studies in which Notch activation was found to be necessary and sufficient for acinar-ductal metaplasia.5 We and others have found metaplastic lesions in association with mouse and human PanINs,24,31 leading to the hypothesis that Notch-driven metaplasia may represent a critical step in Kras-induced PanIN formation.
Using mice in which Notch activity can be induced by Cre recombinase8 (Rosa26loxP-STOP-loxP-Notch1IC, henceforth Rosa26LSLNIC), we asked whether Notch can cooperate with Kras to induce PanIN formation.31 In initial experiments, we used a tamoxifen-inducible Pdx1CreERT transgene36 to produce pancreata mosaic in all lineages for expression of activated Notch and Kras. As expected, when Kras was activated alone, focal PanIN formation occurred, while Notch activation alone prevented islet and acinar differentiation without inducing dysplasia. Co-activation of Kras and Notch, however, resulted in pancreata that were almost completely overrun by PanINs within one month of birth. This dramatic synergy was also apparent when mice did not receive tamoxifen, under which circumstances Pdx1CreERT induces a low level of TM-independent recombination. Without TM, Kras alone produced minimal PanINs while large numbers were induced by Kras and Notch together.
As these results could have reflected effects on embryonic progenitor cell differentiation, we proceeded to test whether Notch could synergize with Kras in adult acinar cells. In parallel with the KrasLSLG12D;ElaCreERT experiments described above, we analyzed littermates also inheriting the Rosa26LSLNIC allele. Acinar-specific activation of Rosa26LSLNIC alone had no visible effect, despite robust activation of Notch target genes. This was somewhat surprising, as activation of Notch in cultured acinar cells readily causes ductal metaplasia.5 In vivo, therefore, Notch is apparently not sufficient to drive acinar-ductal metaplasia. Coactivation of Rosa26LSLNIC and KrasLSLG12D, by contrast, resulted in widespread PanIN formation greatly exceeding that seen with KrasLSLG12D alone, indicating that Notch and Kras can synergize in mature acinar cells to promote PanIN formation.
Expression of the Notch target gene Hes1 is strongly elevated in the IPMN-like tumors of pancreas-specific Pten knockouts, which appear to arise directly from ducts.29 This suggests that Notch activation may have important effects beyond acinar metaplasia. Indeed, in addition to observing an increase in PanIN incidence in mice co-expressing activated Notch and Kras, we also found that pancreata of these mice more frequently contained higher-grade PanIN-2 and PanIN-3 lesions. Previous studies in KrasLSLG12D;Pdx1Cre mice indicated that such higher grade lesions were much rarer than PanIN-1 lesions, although they became more common with advanced age.22 The more rapid appearance of advanced lesions in our model suggests that Notch promotes not only initiation of PanIN lesions but their dysplastic progression as well.31
Our work and that of others shows that mouse PanINs can arise from adult acinar cells expressing activated Kras,31,32 which indicates that there must be a switch from an acinar to a duct-like differentiation program. As activation of Rosa26LSLNIC is marked by GFP expression,8 we could follow the pathological reprogramming of acinar cells by monitoring the differentiation program of GFP-positive cells. Activating Rosa26LSLNIC alone did not inhibit expression of normal acinar differentiation markers, nor did it induce duct markers. By contrast, when KrasLSLG12D was co-activated with Rosa26LSLNIC, we found that GFP-positive cells rapidly downregulated acinar markers and upregulated duct-specific markers. We are taking advantage of the speed and synchrony of this reprogramming (complete within two weeks), and the lack of effect from either Notch or Kras alone, to begin to look for possible downstream mediators of their synergy.
Similar reprogramming has recently been documented in mice in which KrasLSLG12D has been activated in acinar precursor cells, via NestinCre.37 These mice also develop acinar-ductal metaplasia, an important hallmark of which is synthesis of primary cilia, found normally on ducts but not acini. Intriguingly, PanINs and PDAC in these mice are devoid of cilia, as are their corresponding human lesions, suggesting that inhibition of ciliogenesis represents an additional transition point from normal cell to ductal tumor.37
We set out to answer fundamental questions in the pancreatic cancer field, regarding the tumor cell of origin and the endogenous mechanisms that limit or facilitate Kras-induced transformation. Our study implicates adult acinar cells as a potential major source for pancreatic neoplasia, although we cannot exclude a ductal cell of origin. We have also demonstrated that Notch signaling can synergize with Kras, promoting PanIN initiation and progression, and that this process involves acinar cell reprogramming to a duct-like phenotype.31
A recent study has shown that Notch is required for progression of PanINs to invasive PDAC, and thus could represent a therapeutic target.38 Further studies of how Notch acts in PDAC are therefore essential, and several avenues of research are suggested by our results and those of others. First, despite abundant evidence that Notch is active in pancreatic cancer, it remains unclear how that activation is achieved. Related questions are raised by the fact that pancreatitis, like Notch activation, enhances transformation by Kras (Figure 3)26: does PDAC formation in the absence of pancreatitis reflect the occurrence of subclinical inflammation, and does such inflammation act as a trigger for Notch? Finally, the mechanism of synergy between Notch and Kras is unknown, and may involve additional therapeutic targets. Answering these questions will certainly involve further use of mouse modeling, but will also require identifying or developing suitable human cells with which these discoveries can be translated “from bench to bedside”.
JPDLO is supported by NIH Developmental Biology Training Grant 5T32-HD07491. This work is supported by NIH grant R21-CA123066.