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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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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.
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pone-0052133-g002: 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.

Mentions: The Notch receptor family contains four members, Notch1–4. Given previous reports suggesting functional overlap between the different receptors, we investigated the effect of inhibiting multiple members of the Notch receptor family simultaneously on K-ras induced ADM in vitro. Notch signaling requires the transcriptional co-activator proteins Mastermind-like (MAML1–3) for transcription of downstream target genes. When overexpressed, a truncated form of MAML1 (aa 13–74) acts as a potent dominant-negative mutant, inhibiting Notch-mediated transcriptional activation [24]. We isolated acinar cells from PDX-1-Cre;LSL-KrasG12D mice and infected them with an adenovirus expressing DNMAML1 fused to GFP. Even in the presence of DNMAML1-GFP, PDX-1-Cre;LSL-KrasG12D acinar cells maintained the ability to transdifferentiate to cytokeratin positive ductal cysts at day 2, similar to cells expressing a control adenovirus (Figure 2A–B). In order to verify that Notch signaling is inhibited in the presence of DNMAML1, we analyzed expression of Hes1, a downstream effector of Notch signaling. Acinar cells infected with the control adenovirus express Hes1; however, Hes1 expression is reduced when cells are infected with Ad-DNMAML1-GFP (Figure 2C). To further examine the effect of globally inhibiting Notch signaling, we analyzed the effect of a gamma secretase inhibitor, DAPT, on acinar transdifferentiation. PDX-1-Cre;LSL-KrasG12D acinar cells treated with DAPT maintained the ability to form ctyokeratin positive ductal cysts (Figure S2). Overall, these results indicate that in the presence of oncogenic K-ras, Notch pathway signaling is not required for 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)

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.
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pone-0052133-g002: 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.
Mentions: The Notch receptor family contains four members, Notch1–4. Given previous reports suggesting functional overlap between the different receptors, we investigated the effect of inhibiting multiple members of the Notch receptor family simultaneously on K-ras induced ADM in vitro. Notch signaling requires the transcriptional co-activator proteins Mastermind-like (MAML1–3) for transcription of downstream target genes. When overexpressed, a truncated form of MAML1 (aa 13–74) acts as a potent dominant-negative mutant, inhibiting Notch-mediated transcriptional activation [24]. We isolated acinar cells from PDX-1-Cre;LSL-KrasG12D mice and infected them with an adenovirus expressing DNMAML1 fused to GFP. Even in the presence of DNMAML1-GFP, PDX-1-Cre;LSL-KrasG12D acinar cells maintained the ability to transdifferentiate to cytokeratin positive ductal cysts at day 2, similar to cells expressing a control adenovirus (Figure 2A–B). In order to verify that Notch signaling is inhibited in the presence of DNMAML1, we analyzed expression of Hes1, a downstream effector of Notch signaling. Acinar cells infected with the control adenovirus express Hes1; however, Hes1 expression is reduced when cells are infected with Ad-DNMAML1-GFP (Figure 2C). To further examine the effect of globally inhibiting Notch signaling, we analyzed the effect of a gamma secretase inhibitor, DAPT, on acinar transdifferentiation. PDX-1-Cre;LSL-KrasG12D acinar cells treated with DAPT maintained the ability to form ctyokeratin positive ductal cysts (Figure S2). Overall, these results indicate that in the presence of oncogenic K-ras, Notch pathway signaling is not required for 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