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

Avila JL, Troutman S, Durham A, Kissil JL - PLoS ONE (2012)

Bottom Line: Pancreatic ductal adenocarcinoma is believed to arise from precursor lesions termed pancreatic intraepithelial neoplasia (PanIN).Recent studies suggest Notch signaling is a key regulator of ADM.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.

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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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.
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pone-0052133-g001: 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.

Mentions: We have recently demonstrated that loss of Notch1 in a mouse model of K-ras-induced PDAC leads to increased PanIN incidence and progression [20]. To further investigate the mechanism of Notch1 mediated PanIN suppression, we examined the role of Notch1 in ADM using an in vitro explant culture model. Acinar cell clusters isolated from an adult mouse pancreas transdifferentiate to form cytokeratin-19 positive ductal cysts when embedded in a collagen matrix and treated with growth factors such as EGF or TGF-α [21]. In order to examine the effect of Notch1 deletion on cyst formation, we utilized PDX-1-Cre;Notch1lox/lox mice, which allow for conditional deletion of Notch1lox/lox alleles specifically in pancreatic epithelial cells at day 8.5 of embryonic development [22]. Acinar cells isolated from PDX-1-Cre;Notch1lox/lox mice formed ductal cysts in the presence of EGF at comparable rates to wildtype acinar cells (Figure 1A and 1B). The acinar origin of the isolated cells was verified by immunostaining for the acinar marker, amylase (Figure S1). These results indicate loss of Notch1 does not accelerate ADM and that Notch1 is not required for EGF-induced ADM in vitro.


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

Avila JL, Troutman S, Durham A, Kissil JL - PLoS ONE (2012)

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.
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Related In: Results  -  Collection

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pone-0052133-g001: 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.
Mentions: We have recently demonstrated that loss of Notch1 in a mouse model of K-ras-induced PDAC leads to increased PanIN incidence and progression [20]. To further investigate the mechanism of Notch1 mediated PanIN suppression, we examined the role of Notch1 in ADM using an in vitro explant culture model. Acinar cell clusters isolated from an adult mouse pancreas transdifferentiate to form cytokeratin-19 positive ductal cysts when embedded in a collagen matrix and treated with growth factors such as EGF or TGF-α [21]. In order to examine the effect of Notch1 deletion on cyst formation, we utilized PDX-1-Cre;Notch1lox/lox mice, which allow for conditional deletion of Notch1lox/lox alleles specifically in pancreatic epithelial cells at day 8.5 of embryonic development [22]. Acinar cells isolated from PDX-1-Cre;Notch1lox/lox mice formed ductal cysts in the presence of EGF at comparable rates to wildtype acinar cells (Figure 1A and 1B). The acinar origin of the isolated cells was verified by immunostaining for the acinar marker, amylase (Figure S1). These results indicate loss of Notch1 does not accelerate ADM and that Notch1 is not required for EGF-induced ADM in vitro.

Bottom Line: Pancreatic ductal adenocarcinoma is believed to arise from precursor lesions termed pancreatic intraepithelial neoplasia (PanIN).Recent studies suggest Notch signaling is a key regulator of ADM.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Oncogenesis Program, The Wistar Institute, Philadelphia, PA, USA.

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.

Show MeSH
Related in: MedlinePlus