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. 2012;7(12):e52133.
doi: 10.1371/journal.pone.0052133. Epub 2012 Dec 19.

Notch1 is not required for acinar-to-ductal metaplasia in a model of Kras-induced pancreatic ductal adenocarcinoma

Jacqueline L Avila  1 , Scott Troutman, Amy Durham, Joseph L Kissil
Affiliations

Notch1 is not required for acinar-to-ductal metaplasia in a model of Kras-induced pancreatic ductal adenocarcinoma

Jacqueline L Avila et al. PLoS One. 2012.

Abstract

Pancreatic ductal adenocarcinoma is believed to arise from precursor lesions termed pancreatic intraepithelial neoplasia (PanIN). Mouse models have demonstrated that targeted expression of activated K-ras to mature acinar cells in the pancreas induces the spontaneous development of PanIN lesions; implying acinar-to-ductal metaplasia (ADM) is a key event in this process. Recent studies suggest Notch signaling is a key regulator of ADM. To assess if Notch1 is required for K-ras driven ADM we employed both an in vivo mouse model and in vitro explant culture system, in which an oncogenic allele of K-ras is activated and Notch1 is deleted simultaneously in acinar cells. Our results demonstrate that oncogenic K-ras is sufficient to drive ADM both in vitro and in vivo but that loss of Notch1 has a minimal effect on this process. Interestingly, while loss of Notch1 in vivo does not affect the severity of PanIN lesions observed, the overall numbers of lesions were greater in mice with deleted Notch1. This suggests Notch1 deletion renders acinar cells more susceptible to formation of K-ras-induced PanINs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Notch1 is not required for oncogenic K-ras mediated ADM in vitro.
(A) Pancreatic explants from wildtype and PDX-1-Cre;Notch1lox/lox mice embedded in collagen either untreated (control) or treated with EGF (20 µg/mL). Cells are immunostained for expression of pan-cytokeratin (red) at day 5. Nuclei are stained with Hoechst dye (blue). Scale bar, 20 µm. Arrows indicate cytokeratin-positive ductal cells. Representative brightfield images are shown at day 5 in the presence of EGF. Scale bar, 100 µm. (B) Quantitative analysis of percent ductal cyst conversion on day 5 in explants isolated from wildtype and PDX-1-Cre;Notch1lox/lox mice. n = 3 for each group. (C) Pancreatic explants from PDX-1-Cre;LSL-KrasG12D and PDX-1-Cre;LSL-KrasG12D;Notch1lox/lox mice were isolated at day 2 in the absence of EGF. Cells are immunostained for pan-cytokeratin (red) and Hoechst dye (blue). Scale bar, 20 µm. Arrows indicate cytokeratin-positive ductal cells. Representative brightfield images of cyst formation are shown. Scale bar, 100 µm. (D) Quantitative analysis of percent ductal cyst conversion at day 2 in explants isolated from PDX-1-Cre;LSL-KrasG12D (n = 5) and PDX-1-Cre;LSL-KrasG12D;Notch1lox/lox mice (n = 6). (E) Western blot analysis of Notch1 expression in acinar cells isolated from PDX-1-Cre;LSL-KrasG12D and PDX-1-Cre;LSL-KrasG12D;Notch1lox/lox mice; tubulin as loading control. Three samples are shown for each genotype.
Figure 2
Figure 2. DNMAML expression does not inhibit oncogenic K-ras mediated ADM in vitro.
(A) Pancreatic explants from Pdx1-Cre;LSL-KrasG12D mice at Day 2, infected with control adenovirus (Ad-empty) or Adenovirus expressing DNMAML (Ad-dnMAML/GFP). Representative brightfield images of ductal cysts shown. Scale bar, 100 µm. (B) Pdx1-Cre;LSL-KrasG12D acinar explants expressing DNMAML-GFP form cytokeratin positive cysts. Explants were stained with antibodies against GFP (green) and pan-cytokeratin (red). Nuclei are counterstained with Hoechst (blue). Images are representative of 3 separate experiments. Scale bar, 20 µm. (C) DNMAML expression inhibits Notch signaling in Pdx1-Cre;LSL-KrasG12D acinar explants. Explants were stained with antibodies against GFP (green) and Hes1 (red). Nuclei are counterstained with Hoechst (blue). Scale bar, 20 µm.
Figure 3
Figure 3. Notch1 deletion in mature acinar cells does not accelerate spontaneous PanIN development.
(A) Schematic of experimental design. Mice were treated with tamoxifen at 4 weeks of age to activate K-rasG12D and delete Notch1 expression. Pancreatic tissue was collected 3 months later. (B) Histological analysis of pancreas tissue from Elastase1-CreERT2;Notch1lox/lox, Elastase1-CreERT2;KrasG12D, and Elastase1-CreERT2;KrasG12D;Notch1lox/lox mice. Higher magnifications of the boxed areas are seen below the images. Scale bar for top images, 100 µm. Scale bar for higher magnification, 50 µm. (C) PCR analysis of Cre-mediated recombination of the LSL-KrasG12D and Notch1lox/lox loci. Genomic DNA was isolated from the tail (T) and pancreas (P) of each mouse. For the Notch1 PCR, the presence of a band indicates deletion of the floxed Notch1 gene. For the Kras PCR, the larger band represents deletion of the STOP cassette.
Figure 4
Figure 4. Notch1 deletion does not alter PanIN development following acute pancreatitis.
(A) Schematic of experimental design. Mice were treated with tamoxifen at 8 weeks of age to activate K-rasG12D and delete Notch1 expression. Two weeks later, mice were treated with caerulein for 2 consecutive days to induce acute pancreatitis. Pancreatic tissue was collected 3 weeks following the final caerulein treatment. (B) Histological analysis of pancreas tissue from Elastase1-CreERT2;Notch1lox/lox, Elastase1-CreERT2;KrasG12D, and Elastase1-CreERT2;KrasG12D;Notch1lox/lox mice following acute pancreatitis. PanIN-3 lesions are shown in Elastase1-CreERT2;KrasG12D and Elastase1-CreERT2;KrasG12D;Notch1lox/lox pancreas tissue. Scale bar, 30 µm. (C) Fibroplasia and inflammation observed in Elastase1-CreERT2;KrasG12D and Elastase1-CreERT2;KrasG12D;Notch1lox/lox pancreas tissue. Scale bar, 100 µm. (D) Atypical flat lesions (AFLs) were observed in Elastase1-CreERT2;KrasG12D and Elastase1-CreERT2;KrasG12D;Notch1lox/lox pancreas tissue. Scale bar, 100 µm. (E) Analysis of the number of PanIN lesions found per pancreas in Elastase1-CreERT2;KrasG12D (n = 5) and Elastase1-CreERT2;KrasG12D;Notch1lox/lox (n = 6) following acute pancreatitis.
Figure 5
Figure 5. Notch1 functions in a cell autonomous manner.
(A) Representative images of orthotopic tumors in Pdx1-cre;Notchlox/+ and Pdx1-Cre;Notch1lox/lox mice. Tumor cells (T) are seen adjacent to normal pancreatic tissue. Scale bar, 300 µm (B) Quantitative analysis of pancreas weight and percentage of tumor volume in Pdx1-Cre;Notchlox/+(n = 5) and Pdx1-Cre;Notch1lox/lox (n = 6) mice 2 weeks after orthotopic injections. Box plots depict median values, lower and upper quartiles, and maximum and minimum observed values.
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