Pancreatic HIF2α Stabilization Leads to Chronic Pancreatitis and Predisposes to Mucinous Cystic Neoplasm

Abstract

Background & aims: Tissue hypoxia controls cell differentiation 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 HIF2α. Previous studies of HIF action in the pancreas have relied on loss-of-function mouse models, and the effects of HIF2α expression in the pancreas have remained undefined.

Methods: We developed several transgenic mouse models based on the expression of an oxygen-stable form of HIF2α, 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 Kras^G12D in the pancreas.

Results: We show that HIF2α 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 HIF2α in mouse pancreata led to the development of chronic pancreatitis. Importantly, pancreatic HIF1α stabilization did not disrupt the pancreatic parenchyma, indicating that the chronic pancreatitis phenotype is specific to HIF2α. In the presence of oncogenic Kras, HIF2α 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 HIF2α 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 pancreatitis and MCN, as well as illustrate the importance of hypoxia signaling in the pancreas.

Keywords: Chronic Pancreatitis; ER, endoplasmic reticulum; HIF2α; HIF2α, hypoxia-inducible factor 2α; Hypoxia; KC, Pdx1-Cre;LSLKrasG12D; KrasG12D; MCN, mucinous cystic neoplasm; Mucinous Cystic Neoplasm; PanIN, pancreatic intraepithelial neoplasia; Pancreas; UPR, unfolded protein response; VHL, von Hippel-Lindau; qPCR, quantitative polymerase chain reaction.

Graphical abstract

Figure 1

HIF2α expression is associated with chronic pancreatitis in human beings and mice. (A) Western blot analysis of HIF2α expression in lysates from human normal and chronic pancreatitis pancreata (n = 5 chronic pancreatitis samples). (B) Transgenic mouse scheme. (C) Gross morphology of control and HIF2α stabilized pancreata. (D) Average weight of mice with HIF2α stabilization compared with age-matched littermate controls (n = 6 pairs of age-matched littermates). H&E evaluation of (E) 2-week (n = 2), (F) 4-week (n = 2), (G) 9-week (n = 5), and (H) 1-year-old (n = 4) pancreata. (I) qPCR analysis of gene expression levels in 2-week-old pancreata. SMA, smooth muscle actin.

Figure 2

Validation of HIF2α stabilization in mouse pancreata. (A) Western blot for HIF2α in lysates from control and HIF2α stabilized pancreata, with Coomassie stain for loading control (n = 2 samples per condition). (B) Immunohistochemistry for HIF2α in control and HIF2α stabilized pancreata. (C) qPCR analysis of HIF target gene expression levels in 9-week-old pancreata.

Figure 3

HIF2α stabilization causes chronic pancreatitis in multiple mouse models. (A) Transgenic mouse scheme for Ptf1a-Cre; HIF2α-LSL/+ (HIF2α stabilized) animals. (B) H&E analysis of HIF2α stabilized animals at 1, 3, and 7 months of age (n = 1 at 1 month, n = 3 at 3 months, and n = 2 at 7 months). (C) Transgenic mouse scheme for Ptf1a-Cre; VHL^fl/fl animals (VHL^PanKO). (D) H&E analysis of VHL^PanKO animals at 3, 5, and 7 months of age (n = 5 at 3 months, n = 2 at 5 months, and n = 2 at 7 months).

Figure 4

HIF1α stabilization does not disrupt the pancreatic parenchyma. (A) Western blot for HIF1α levels in lysates from human normal and chronic pancreatitis samples, as well as mouse HIF1α stabilized pancreata as positive control. (B) Transgenic mouse scheme and H&E for Pdx1-Cre; HIF1α-LSL/+ (HIF1α stabilized) pancreata (n = 10 mice, histology analyzed from ages 6 wk to 1 y). (C) Transgenic mouse scheme and H&E for Ptf1a-Cre; HIF1α-LSL/+ pancreata (n = 2 at 6 months of age).

Figure 5

Pancreas histology in 1-day-old HIF2α stabilized pancreata. (A) H&E analysis in 1-day-old animals (n = 2 HIF2α stabilized, n = 3 control). (B) Immunohistochemistry for HIF2α in 1-day-old animals. (C) Immunohistochemistry and quantification of positive cells per high-power field (HFP) for Ki67 and (D) cleaved caspase 3 in 1-day-old animals.

Figure 6

HIF2α stabilization-induced pancreatitis in young mice. Immunohistochemistry and quantification of positive cells per high-power field for (A) Ki67 and (B) cleaved caspase 3 in 2-week-old animals (n = 2 animals per genotype). Immunohistochemistry and quantification of positive cells per high-power (HFP) field for (C) Ki67 and (D) cleaved caspase 3 in 4-week-old animals (n = 2 animals per genotype).

Figure 7

Immunostaining for lineage markers in 9-week-old HIF2α stabilized pancreata. Immunohistochemistry and quantification of positive cells per high-power field (HFP) for (A) Mist1 and (B) Sox9. (C) Immunohistochemistry for the endocrine marker chromogranin. (D) Gomori trichrome staining and (E) CD45 immunohistochemistry and quantification. All staining was in 9-week-old animals (n = 5).

Figure 8

HIF2α stabilization causes gene expression changes consistent with chronic pancreatitis. qPCR for (A) markers of fibrosis and (B) ER stress in control and HIF2α stabilized animals. (C) qPCR array results, including table of genes analyzed. All data represented as levels in HIF2α stabilized vs control pancreas. Green = higher expression in control; red = higher expression in Hif2α. All changes represented as magnitude of log2(fold-change). (D) qPCR validation of changed targets from qPCR array in control and HIF2α stabilized animals.

Figure 9

HIF2α stabilization causes endocrine pancreas dysfunction. (A) Immunofluorescence for insulin and 4′,6-diamidino-2-phenylindole (DAPI) in 9-week-old animals (n = 3 animals per genotype). (B) Glucose tolerance test (n = 5 animals per genotype) and (C) glucose-stimulated insulin secretion (n = 3 animals per genotype) in 9-week-old animals.

Figure 10

HIF2α stabilization during pancreatic cancer initiation leads to MCNs. (A) Transgenic mouse scheme. (B) Gross morphology of 2 separate KC;HIF2α animals at 9 weeks of age. Arrows indicate cysts, arrowhead indicates spleen. (C) H&E evaluation of human MCN and KC; HIF2α pancreata (n = 7 KC;HIF2α pancreata and 3 human MCN samples).

Figure 11

HIF2α stabilization results in mucinous cystic neoplasm formation in multiple mouse models. Transgenic mouse scheme for (A) Ptf1a-Cre; LSL-Kras^G12D; HIF2a-LSL/+ (KC;HIF2α) and (B) Ptf1a-Cre; LSLKras-^G12D; VHL^fl/fl (KC;VHL^fl/fl) animals. H&E histology analysis at 1, 3, 7, and 9 months in (C) KC (n = 14), (D) KC;HIF2α (n = 10), and (E) KC;VHL^fl/fl animals (n = 9).

Figure 12

KC;HIF2α animals have MCN-like features. (A) Periodic acid–Schiff (PAS) staining, and immunohistochemistry for (B) CK19, (C) ER, (D) vimentin, (E) Muc1, and (F) Muc5ac in 9- to 10-week-old KC and KC;HIF2α animals (n = 2–4 pancreata per genotype for each immunostaining condition). (G) qPCR analysis of gene expression levels in KC and KC;HIF2α pancreata at 9–10 weeks of age (n = 6).

Figure 13

Wnt pathway is activated in KC;HIF2α animals. (A) qPCR analysis for gene expression levels of Wnt pathway targets in KC and KC;HIF2α animals at 9–10 weeks of age (n = 3). (B) Immunohistochemistry for Lef1 in KC and KC;HIF2α animals (n = 2 pancreata per condition). (C) Immunofluorescence for Lef1 in human MCN tissue (n = 2 human MCN samples). DAPI, 4′,6-diamidino-2-phenylindole.