Pancreatic HIF2a Stabilization Leads to Chronic Pancreatitis and Predisposes to Mucinous Cystic Neoplasm

BACKGROUND & AIMS: Tissue hypoxia controls cell differen- tiation in the embryonic pancreas, and promotes tumor growth in pancreatic cancer. The cellular response to hypoxia is controlled by the hypoxia-inducible factor (HIF) proteins, including HIF2a. Previous studies of HIF action in the pancreas have relied on loss-of-function mouse models, and the effects of HIF2a expression in the pancreas have remained undefined.
METHODS: We developed several transgenic mouse models based on the expression of an oxygen-stable form of HIF2a, or indirect stabilization of HIF proteins though deletion of von Hippel-Lindau, thus preventing HIF degradation. Furthermore, we crossed both sets of animals into mice expressing oncogenic KrasG12D in the pancreas.RESULTS: We show that HIF2a is not expressed in the normal human pancreas, however, it is up-regulated in human chronic pancreatitis. Deletion of von Hippel-Lindau or stabilization of HIF2a in mouse pancreata led to the development of chronic pancreatitis. Importantly, pancreatic HIF1a stabilization did not disrupt the pancreatic parenchyma, indicating that the chronic pancreatitis phenotype is specific to HIF2a. In the presence of oncogenic Kras, HIF2a stabilization drove the formation of cysts resembling mucinous cystic neoplasm (MCN) in humans. Mechanistically, we show that the pancreatitis phenotype is linked to expression of multiple inflammatory cytokines and activation of the unfolded protein response. Conversely, MCN formation is linked to activation of Wnt signaling, a feature of human MCN.CONCLUSIONS: We show that pancreatic HIF2a stabilization disrupts pancreatic homeostasis, leading to chronic pancreatitis, and, in the context of oncogenic Kras, MCN formation. These findings provide new mouse models of both chronic pancrea- titis and MCN, as well as illustrate the importance of hypoxia signaling in the pancreas. (Cell Mol Gastroenterol Hepatol 2017; https://doi.org/10.1016/j.jcmgh.2017.10.008)

During pancreas development, oxygen levels posi- tively control b-cell differentiation.1 Pancreatic cancer, the third leading cause of cancer-related death,2 is extensively hypoxic.3 Hypoxia has been shown to promote pancreatic tumor growth, invasion, and metastasis.4,5 At the cellular level, the response and adaptation to hypoxia is controlled by hypoxia-inducible factors (HIFs). In vertebrates, the HIF family contains 3 isoforms: HIF1a, HIF2a, and HIF3a. The HIF proteins are transcription factors, activating genes containing a hypoxia response element in response to low levels of cellular oxygen.6,7 In normal oxygen conditions, HIF proteins are hydroxylated post-translationally, allowing association with the von Hippel-Lindau (VHL) tumor suppressor and tagging for proteosomal degradation.8 In hypoxia, VHL is unable to tag the HIF proteins for degradation and the HIFs accumulate intracellularly, translocate to the nucleus, and activate target genes. Hypoxia induces HIF1a and HIF2a expression in the pancreas.9 Furthermore, a number of studies have associated perturbation of the HIF factors with pancreatic abnormalities/disease. HIF1a is expressed during the development of pancreatic cancer, and its deletion promotes pancreatic tumorigenesis in a Kras-driven model of pancreatic cancer.10 HIF2a expression is required for the embryonic development of the pancreas, and a lack of HIF2a expression in developing mice leads to smaller pancreata and decreased branching.11

In the presence of oncogenic Kras, HIF2a inactivation inhibits the progression of precancerous lesions.12Here, we show that pancreas-specific inactivation of VHL, or stabilization of HIF2a (but not HIF1a), induces chronic pancreatitis in mice. Although many mouse models of pancreatitis recover over time (for review, see Lerch and Gorelick13), mice overexpressing HIF2a have bona fide chronic pancreatitis that persists until most of the pancreatic parenchyma is substituted by fibrotic or fatty tissue. The link between HIF2a expression and pancreatitis is strengthened further by the observation that this protein accumulates in a subset of human chronic pancreatitis samples.Different precursor lesions are described for human pancreatic cancer. Although pancreatic intraepithelial neoplasia (PanIN) is the most common, intraductal papillary mucinous neoplasms and mucinous cystic neoplasms (MCNs) can progress to malignancy (for review see Hezel et al14 and Ying et al15). The molecular drivers underpinning progression to each of these specific precursor lesions are only partially understood. Here, we show that stabilization of HIF2a in the presence of oncogenic Kras specifically drives formation of MCN, through activation of Wnt signaling. Notably, Wnt signaling is a common feature of human MCN.Mice were housed in the specific pathogen-free facility at the University of Michigan Comprehensive Cancer Center. This study was approved by the University of MichiganCommittee on the Use and Care of Animals, and the University of Pittsburgh Institutional Animal Care and Use Committee. Pdx1-Cre, Ptf1a-Cre, LSL-KrasG12D, R26-LSLHif2a/+, and VHL floxed mice have been described previously.16–18Glucose tolerance testing was performed as previously described.19 Before testing, animals were fasted for 4 hours during the light cycle. Initial blood glucose levels were measured using tail blood samples. Then, animals were administered glucose at a dose of 2 g glucose per kilogram of body weight by intraperitoneal injection. Tail blood samples then were measured for blood glucose levels at 15, 30, 60, 90, and 120 minutes after glucose injection.

Blood glucose level was measured using the Accu-Chek Aviva diabetes monitoring kit and Accu-Chek (Roche Diabetes Care, Indianapolis, IN) Aviva Plus testing strips.Overnight fasted mice were anesthetized by an intra- peritoneal injection of Avertin (Sigma-Aldrich, St. Louis, MO). Anesthetized mice then were injected intraperitoneally with glucose at 3 g/kg body weight and blood was collected retro-orbitally at 0, 2, 7, 15, and 30 minutes. Serum was separated by centrifuging the blood at 8000 rpm for 8 mi-nutes at 4◦C. Serum insulin was measured using the Ultra- sensitive mouse insulin ELISA kit (CrystalChem, DownersGrove, IL), following the manufacturer’s recommendation.Histology and immunohistochemistry studies, as well as periodic acid–Schiff and Gomori trichrome staining, were performed as previously described.20 To prepare for stain- ing, tissue was collected and fixed overnight in 10% neutral buffered formalin. Tissue then was embedded in paraffin and sectioned. The University of Michigan Cancer Center Histopathology Core performed embedding and sectioning. Sections were imaged using an Olympus (Olympus, Center Valley, PA) BX-51 microscope, Olympus DP71 digital cam- era, and CellSens (Olympus) Standard software. Primary antibodies used are included in Supplementary Table 1.Tissue for RNA extraction was collected in lysis buffer (Ambion, Foster City, CA) and RNA was isolated using the PureLink RNA Mini Kit (Ambion). Reverse transcription wasperformed using the High-Capacity Complementary DNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). Primers were optimized for amplification con-ditions of 95◦C for 10 minutes, then 40 cycles of 95◦C for 15 seconds, and 60◦C for 1 minute.

Melt curve analysis was performed for all samples. Cyclophilin A was used asthe control housekeeping gene for normalization. The primer sequences for genes analyzed are included in Supplementary Table 2. Quantitative polymerase chain reaction (qPCR) array was performed using the Mouse Th17 Response PCR Array (Qiagen, Frederick, MD) ac- cording to the manufacturer’s instructions.Tissue for protein extraction was collected in radioimmunoprecipitation assay buffer with protease inhibitor. Equal amounts of protein were added per lane, run by electrophoresis in sodium dodecyl sulfate– polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Each membrane was blocked with 5% milk. Primary antibody incubationwas performed overnight at 4◦C. Secondary antibody incubation was performed for 2 hours at room tempera-ture. Protein bands were detected using Western Light- ning Plus Enhanced Chemiluminescence (NEL103001EA; PerkinElmer, Waltham, MA) and film.Statistical significance for reverse-transcription qPCR results was determined by an unpaired 2-tailed t test, with the threshold for statistical significance determined to be P < .05.The University of Michigan Institutional Review Board approved all human studies. Informed consent was received from all human patients before inclusion in the studies detailed in this work. All animal studies were approved by the University of Michigan Committee on the Use and Care of Animals.

Results
HIF2a protein expression is not detectable in the normal human pancreas. However, we observed abundant HIF2a expression in human samples of chronic pancreatitis (n = 5 chronic pancreatitis samples) (Figure 1A). Overexpression of HIF2a was observed in 3 of 5 human chronic pancreatitissamples, with lower levels of expression seen in the other2 samples, but still higher than observed in the normal pancreas. This may indicate variability in HIF2a expression in chronic pancreatitis, or suggest that high HIF2a expres- sion levels represent a subset of human chronic pancreatitis. Because a functional role for HIF2a had not been described in this disease, we generated Pdx1-Cre;Rosa26LSL-HIF2a/+mice. In these animals, an oxygen-stable form of HIF2a16 is expressed in the pancreas upon Cre recombination (Figure 1B). We observed that pancreata with stabilized HIF2a were smaller than in their littermate controls (shown at 9 weeks of age) (Figure 1C). However, there was no difference in total body weight between the controls and HIF2a stabilized mice at all ages analyzed. From 7 weeks to 1 year of age HIF2a stabilized animals showed similar totalbody weight to age-matched littermate control animals (n = 6 pairs of age-matched littermates, 1 control, and 1 HIF2a stabilized) (Figure 1D).At 2 weeks of age (n = 2), animals expressing HIF2a had atrophic pancreatic parenchyma resembling end-stage chronic pancreatitis (Figure 1E). By 4 weeks of age,shortly after weaning, HIF2a stabilized animals had developed further signs of chronic pancreatitis (n = 2) (Figure 1F). These changes progressed over time, with few residual acini and significant inflammatory infiltrates at 9 weeks of age (n = 5) (Figure 1G).

In older mice (n = 4analyzed at 1 year of age), pancreata were mostly replaced by adipose tissue with small remnant clusters of acinar cells, dilated ducts, and intermixed inflammatory infiltration (Figure 1G), indicating chronic disease and lack of recovery from pancreatitis. This feature distinguishes this model from most other mouse models of pancreatitis, in which repair is observed over time.13,21 To analyze the molecular changes underlying the onset of pancreatitis, were extracted RNA from 2-week-old mouse pancreata and performedqPCR analysis (n = 3 mice per genotype). By 2 weeks of age, HIF2a stabilized animals were not expressing amylase(Amy2b), indicating loss of acinar differentiation. At the same age, cytokeratin 19 (CK19) expression was unchanged compared with littermate controls. Finally, smooth muscle actin (SMA) expression was increased in HIF2a stabilized mice, indicating an increase in activated fibroblasts (Figure 1I).To validate HIF2a stabilization in experimental animals, we performed a Western Blot. As expected, HIF2a accu-mulated in Pdx1-Cre;Rosa26LSL-HIF2a/+ pancreata but not in littermate controls (n = 2 pancreata per genotype) (Figure 2A). Although immunostaining for HIF2a is techni-cally challenging, our results were in line with an increase in HIF2a protein in the experimental mice (Figure 2B). Gene expression analysis by qPCR showed up-regulation of HIF targets, including Pdk1,22 indicating functional up-regulation of the HIF signaling pathway in HIF2a stabi- lized animals (Figure 2C).

To expand our analysis, we generated Ptf1a+/Cre; Rosa26LSL-HIF1a/+ mice in which a different Cre recombinase was used to activate the same stabilized HIF2a in thepancreas (Figure 3A). Similar to our previous results, Ptf1a+/Cre;Rosa26LSL-HIF2a/+ animals showed inflammatory infiltration and pancreatic atrophy typical of chronic pancreatitis (Figure 3B). Possibly owing to differences inCre expression, the phenotype developed more slowly, with small pockets of inflammation at 1 month, progressing to full pancreatic involvement by 7 months of age (n = 1 at 1 month, n = 3 at 3 months, and n = 2 at 7 months)(Figure 3B).To determine whether the phenotype observed upon HIF2a stabilization was indeed owing to activation of the hypoxia pathway, we generated mice lacking VHL expression in the pancreas. Under normoxic conditions, VHL acts to inhibit HIF function by tagging HIF proteins for degradation in the proteasome.8 Thus, deletion of VHL prevents the degradation of HIF proteins, leading to stabi- lization of the endogenous HIF proteins. We crossed micebearing Ptf1a+/Cre with mice carrying a floxed allele of VHL17 (Figure 3C) to generate Ptf1a+/Cre;VHLfl/fl (termed VHLPanKO) animals. Histologic analysis of VHLPanKO miceshowed that HIF stabilization by this method phenocopied the chronic pancreatitis observed in HIF2a stabilization, observable from as early as 3 months of age (n = 5 at 3 months, n = 2 at 5 months, and n = 2 at 7 months)(Figure 3D). Taken together, these data further show thatactivation of the HIF pathway in the pancreas through sta- bilization of HIF2a leads to the development of a sponta- neous fibroinflammatory response resembling human chronic pancreatitis.To determine whether HIF1a, a factor related to HIF2a, was similarly implicated in human pancreatitis, we probed HIF1a protein expression in the same human chronic pancreatitis samples in which we observed HIF2a up-regulation. In human chronic pancreatitis HIF1a was up-regulated dramatically in only 1 of 5 samples byWestern blot (Figure 4A).

We then generated Pdx1-Cre;Rosa26LSL-HIF1a/+ and Ptf1a+/Cre;Rosa26LSL-HIF1a/+ mice, in which HIF1a stabilization is stabilized in a tissue- specific manner. Consistent with the human findings indicating no strong correlation between HIF1a and pancreatitis, animals with pancreatic HIF1a stabilization, independently from the Cre driver used, had normal pan-creata (Pdx1-Cre;Rosa26LSL-HIF1a/+; n = 10 mice, histology analyzed at ages 6 wk to 1 y; Ptf1a+/Cre;Rosa26LSL-HIF1a/+; n = 2) (Figure 4B and C). This indicates that the chronic pancreatitis phenotype is specific to HIF2a expression, andnot HIF pathway activation in general. We next sought to determine whether the pancreatitis phenotype caused by HIF2a expression was caused by impaired embryonic development, thus analyzed pancreata from newly born animals. At 1 day of age, we observed nodiscernible differences in histology between HIF2a stabi- lized animals and their control littermates (n = 2) (Figure 5A) via H&E staining. HIF2a stabilization was confirmed by immunohistochemistry, in which both control and HIF2a stabilized animals showed positive HIF2aexpression in the developing islets, as expected,11 and only HIF2a stabilized pancreata showed positive HIF2a staining throughout the pancreas (Figure 5B). Control and HIF2a stabilized animals showed high levels of proliferation, as evidenced by Ki67 staining, with no differences between the 2 groups (Figure 5C). Similarly, apoptotic cells, as measured by immunostaining for cleaved caspase 3, were equally infrequent in both groups (Figure 5D).

Thus, mice express- ing stabilized HIF2a are born with a normal pancreas.In comparison, at 2 weeks of age, when the phenotype is clearly evident, changes in proliferation and apoptosisbecame apparent. At this age, the pancreas is undergoing active proliferation. Although the acinar cell population was reduced in the HIF2a animals, if acinar cells were present they were highly proliferative; furthermore, when considering proliferation across all cell types, there was an increase in the HIF2a pancreata (Figure 6A). At the same time, apoptosis was increased in HIF2a pancreata, both in areas of acinar-to- ductal metaplasia and in the remaining acini (Figure 6B). We next analyzed animals shortly after weaning, at 4 weeks of age(n = 2) (histology in Figure 1F). In these animals, proliferation was higher in HIF2a stabilized animals compared with controllittermates (Figure 6C). Similarly, apoptotic cell death was increased in HIF2a animals compared with controls (Figure 6D). Thus, in both 2- and 4-week-old mice, the process of pancreatitis is actively ongoing.At 9 weeks of age, the pancreatitis had progressed so that most of the pancreas parenchyma was severelydisrupted. However, clusters of acini persisted within the tissue: immunostaining for Mist1, an acinar lineage marker, showed reduced expression even in those areas (Figure 7A). The tubular structures common across the tissue expressed Sox9, a ductal marker also expressed in acinar-to-ductal metaplasia and a promoter of pancreatic carcinogenesis23 (Figure 7B). Even in areas of ongoing inflammation and acinar cell loss, chromogranin A staining confirmed thatislets persisted in the tissue (Figure 7C) (n = 3 animals per genotype for each immunostain). Other features of chronicpancreatitis, such as extensive fibrosis (Gomori trichrome, n = 5 pancreata per genotype) (Figure 7D) and abundant infiltration of immune cells (immunostaining for CD45, n = 3 pancreata per genotype) (Figure 7E) were similarly commonacross HIF2a pancreata.

Molecular markers of fibrosis detec- ted in human pancreatitis were similarly increased. In addi- tion, HIF2a stabilized pancreata expressed higher levels ofgenes that are associated with the fibrosis present in chronic pancreatitis, including TGFb and MMP924,25 (Figure 8A).We then sought to gather a mechanistic understanding connecting HIF2a and chronic pancreatitis. In cells exposed to a hypoxic environment, HIF pathway activation occurs in parallel to activation of other stress response pathways, such as the unfolded protein response (UPR) and its resulting endoplasmic reticulum (ER) stress. In hypoxia, HIF signaling and the UPR interact directly, cooperating to activate some of the same downstream targets, to control outcomes such as metabolism and cell survival during tumor growth.26–29 In the exocrine pancreas, disruption of the UPR via acinar-specific deletion of one of its main signaling components, Xbp1, resulted in ER stress and led to extensive apoptosis and expansion of tubules expressing Sox9, similar to the phenotype observed in HIF2a stabilizedanimals.28 In addition, ER stress is an early pathologic event in chronic pancreatitis and persists throughout the course of the disease, in both human beings and mice.30 We thus performed qPCR analysis of HIF2a or control pancreata. Consistent with activation of the unfolded protein response and subsequent ER stress, we found increased expression of 2 key components of the ER stress pathway, Bip and Chop,28in HIF2a stabilized pancreata (n = 3) (Figure 8B). There- fore, HIF2a stabilized animals show higher levels of ductalmarkers and ER stress compared with control littermates, both consistent with chronic pancreatitis.Chronic pancreatitis is associated with a specific profile of cytokine expression. To conduct an unbiased analysis of cytokine profiles we used a qPCR array to compare expression between control and HIF2a stabilized animals at6 weeks of age. The 6-week age was chosen because it corresponds to an actively developing phenotype (Figure 8C, and Supplementary Table 3, online only). Cytokines that were different among the groups in the array we then validated by qPCR analysis using different sets of 6-week-old animals for each genotype. Interestingly, HIF2astabilized animals expressed higher levels of cytokines associated with human chronic pancreatitis such as Icam1, Ccr2, and Il6r31–33 (Figure 8D). Although many mouse models of chronic pancreatitis recover over time,13 HIF2a stabilized mice recapitulate many aspects of human chronic pancreatitis, including molecular and histologic aspects, making them an exciting new experimental model.Chronic pancreatitis patients develop type 3c diabetes over time.34,35 To determine whether HIF2a stabilization recapitulated this aspect of chronic pancreatitis, we analyzed the endocrine islets in 9-week-old mice. The islets in HIF2a stabilized pancreata were morphologically normal,containing insulin-positive b-cells (n = 3 animals per genotype) (Figure 9A).

To measure islet function, we sub-jected 9-week-old animals to glucose tolerance testing. HIF2a stabilized animals had impaired glucose tolerance, with asharper initial increase and sustained increase of blood glucose levels compared with controls (n = 5 animals per genotype) (Figure 9B). To test for insulin secretion, we measured blood insulin levels in mice after a glucose chal- lenge. Unlike wild-type, HIF2a stabilized animals had no in-crease in blood insulin levels after glucose challenge (Figure 9C), indicating b-cell dysfunction (n = 3 animals per genotype). Multiple mechanisms for diabetes development in type 3c diabetes have been postulated, but one likely mech- anism is decreased insulin secretion from b-cells, whichwould mirror the state seen in the HIF2a stabilized animals.36Chronic pancreatitis is a risk factor for the development of pancreatic cancer.37 In addition, animals lacking HIF2aexpression in a mouse model of pancreatic cancer develop lesions that fail to progress to cancer, suggesting a role for HIF2a in pancreatic cancer progression.12Because HIF2a stabilization caused pancreatitis, we hypothesized that it might similarly promote carcinogenesis in the presence of oncogenic Kras. Mice that express onco- genic Kras in the pancreas, such as Pdx1-Cre;Kras+/LSL-G12Dor Ptf1a-Cre;Kras+/LSL-G12D (KC) develop PanIN, a precursor lesion to pancreatic cancer.18 We crossed both Pdx1- Cre;Kras+/LSL-G12D and Ptf1a-Cre;Kras+/LSL-G12D mice with the Rosa26LSL-Hif2a/+ mice to generate mice that express both oncogenic Kras and stabilized HIF2a in the pancreas,here named KC;HIF2a (Figures 10A and 11C). At 9–12 weeks, KC mice had, as expected, sporadic PanINsinterspersed within a largely normal pancreas. In contrast, in age-matched KC;HIF2a mice we observed large cystic lesions. These lesions developed with full penetrance (Figure 10B, arrows) (n = 7 mice), a pathologic evaluation recognized them as corresponding to human MCN(Figure 10C). We then aged mice of each genotype to obtain a time course of MCN development (Figure 11A). As ex- pected, KC animals developed PanINs, with their prevalence increasing over time from sporadic at 1 month of age to prevalent in the pancreas by 9 months (n = 14)(Figure 11C). Conversely, age-matched KC;HIF2a animalsdeveloped cystic lesions resembling human MCN. These cysts were small at 1 month of age and increased in size over time (n = 10) (Figure 11D).We then used a complementary approach to stabilizeHIF2a, by deleting VHL in KC mice (Figure 11B). Similar to KC;HIF2a, KC;VHLfl/fl mice developed MCN-like lesions that progressed over time, from small lesions at 1 month of ageto large cysts in older animals (n = 9) (Figure 11E). Thus, activation of the HIF pathway, independently from the mode of activation, cooperates with oncogenic Kras to drive for-mation of cystic lesions.We then compared the cystic lesions in our mouse model with human MCN.

Similar to human histology, in KC;HIF2a mice the cystic lesions were lined by flat cuboidal epithelial cells with no papillary architecture and sur- rounded by a fibrotic reaction. The lesions presented with apical expression of mucin (periodic acid–Schiff staining) and expression of CK19 in the epithelial cells, similar to human MCN38 (Figure 12A and B). Furthermore, we observed positive staining for ER surrounding the lesions, a characteristic of ovarian-type stroma (Figure 12C), a diag- nostic feature of human MCN. Other histologic features characteristic of human MCN in KC;HIF2a mice include expression of mesenchymal markers such as vimentin in the stroma but not the epithelium (Figure 12D). Analysisof mucin expression in the animals showed expression of Muc1 in both KC and KC;HIF2a animals, as expected in both PanIN and MCN type lesions39 (Figure 12E). In addition, both the PanINs in KC animals and the cystic lesions in KC;HIF2a mice were positive for Muc5ac expression (Figure 12F). This pattern of Muc1 and Muc5ac expression has been described previously in human MCNs.40 In addition, we used qPCR to analyze gene expression analysis of pancreatic cell types in KC and KC;HIF2a animals. We observed lower levels of amylase, and higher levels of CK19 and SMA in KC;HIF2a micecompared with controls (n = 6) (Figure 12G). Thus, stabi- lization of HIF2a in the presence of oncogenic Kras directedformation of MCN-like lesions rather than PanINs.We then investigated whether molecular underpinnings of MCN formation were found in our model. Human MCN is associated with de-regulated Wnt signaling41 and mouse experiments have shown Wnt signaling to be a driver of this disease.41 HIF2a modulates Wnt expression during the development of PanINs in the KC mouse model. Accordingly, our analysis showed increased levels of Wnt target genes (Lef1, MYC, and Axin) in KC;HIF2a animals compared with KCat 9 weeks of age (n = 3) (Figure 13A). We then performed immunohistochemistry for the Wnt target Lef1 in both human MCN (n = 2 human MCN samples) and in MCN of KC;HIF2a animals compared with KC (n = 2 per genotype) (Figure 13B). In both human and mouse MCN we observed stromalLef1 expression (Figure 13B and C). Thus, our model mimicsboth the histology and molecular features of human MCN and will be useful to study this disease in the future. Of note, human MCN is prevalent in females,40 although we observed no difference in incidence among female and male mice, a finding likely reflecting the different hormonal regulation.

Discussion
Here, we show that HIF2a protein accumulates in human chronic pancreatitis. The expression of an oxygen-stable form of HIF2a in the mouse pancreas results in chronic pancreatitis and atrophy, with loss of acini and increased chronic inflammation in the lobule. In addition, stabilization of HIF2a along with oncogenic Kras expression recapitulates human MCN. Notably, these effects were restricted to HIF2a expression and not to expression of the closely related family member HIF1a. Thus, we describe 2 new models of human disease, and provide new insight into the role of hypoxia signaling, specifically through HIF2a, in the RMC-7977 pancreas.