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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 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.
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pone-0052133-g003: 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.

Mentions: In order to investigate the role of Notch1 in acinar-to-ductal metaplasia in vivo, we utilized an Elastase1-CreERT2-driven mouse model. This transgene permits tamoxifen-inducible Cre activation specifically in adult acinar tissue [25]. Activation of oncogenic Kras in the mature acinar compartment results in the spontaneous development of PanIN lesions [4]. Extensive ADM was observed preceding the onset of PanIN lesions in this model, implying acinar cells might represent the cell of origin for PanIN development. Elastase1-CreERT2;LSL-KrasG12D and Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox mice were generated, and treated with tamoxifen at 4 weeks of age (Figure 3A). Additionally, Elastase1-CreERT2;Notch1lox/lox mice were generated to establish the effect of Notch1 deletion on adult acinar tissue. At 3 months following tamoxifen treatment, pancreas tissue from Elastase1-CreERT2;Notch1lox/lox mice appeared grossly normal (Figure 3B). In contrast, pancreas tissue from both Elastase1-CreERT2;LSL-KrasG12D and Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox demonstrated mild to moderate fibroplasia and fibrosis. Additionally, low-grade PanIN1A lesions were observed in 1 of 6 Elastase1-CreERT2;LSL-KrasG12D mice and 1 of 7 Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox mice (Figure 3B, Figure S3A). Analysis by PCR demonstrated recombination at both the Kras and Notch1 gene loci in the pancerata of these mice (Figure 3C). These results demonstrate that in the context of activated Kras, deletion of Notch1 in adult acinar tissue does not accelerate spontaneous PanIN development. Additionally, the results suggest that Notch1 is not required for K-ras induced PanIN development, as an Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox mouse developed a PanIN1A lesion. However further studies, with larger animal cohorts, are required to conclusively establish this point and to determine whether Notch1 deletion renders acinar cells more susceptible to K-ras induced transformation, in line with previous studies [20].


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526595&req=5

pone-0052133-g003: 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.
Mentions: In order to investigate the role of Notch1 in acinar-to-ductal metaplasia in vivo, we utilized an Elastase1-CreERT2-driven mouse model. This transgene permits tamoxifen-inducible Cre activation specifically in adult acinar tissue [25]. Activation of oncogenic Kras in the mature acinar compartment results in the spontaneous development of PanIN lesions [4]. Extensive ADM was observed preceding the onset of PanIN lesions in this model, implying acinar cells might represent the cell of origin for PanIN development. Elastase1-CreERT2;LSL-KrasG12D and Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox mice were generated, and treated with tamoxifen at 4 weeks of age (Figure 3A). Additionally, Elastase1-CreERT2;Notch1lox/lox mice were generated to establish the effect of Notch1 deletion on adult acinar tissue. At 3 months following tamoxifen treatment, pancreas tissue from Elastase1-CreERT2;Notch1lox/lox mice appeared grossly normal (Figure 3B). In contrast, pancreas tissue from both Elastase1-CreERT2;LSL-KrasG12D and Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox demonstrated mild to moderate fibroplasia and fibrosis. Additionally, low-grade PanIN1A lesions were observed in 1 of 6 Elastase1-CreERT2;LSL-KrasG12D mice and 1 of 7 Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox mice (Figure 3B, Figure S3A). Analysis by PCR demonstrated recombination at both the Kras and Notch1 gene loci in the pancerata of these mice (Figure 3C). These results demonstrate that in the context of activated Kras, deletion of Notch1 in adult acinar tissue does not accelerate spontaneous PanIN development. Additionally, the results suggest that Notch1 is not required for K-ras induced PanIN development, as an Elastase1-CreERT2;LSL-KrasG12D;Notch1lox/lox mouse developed a PanIN1A lesion. However further studies, with larger animal cohorts, are required to conclusively establish this point and to determine whether Notch1 deletion renders acinar cells more susceptible to K-ras induced transformation, in line with previous studies [20].

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