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Identification of transcriptional signatures overlapping in pancreatic ductal development, acute pancreatitis and KrasG12D-driven carcinogenesis

ABSTRACT: To determine the molecular basis of gene regulation in pancreatic ductal epithelial cells, we developed methods for the isolation of this cell population during mouse development and normal adult homeostasis, as well as in conditions with ductal features (acinar-to-ductal metaplasia (ADM), pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC)). Our technique utilizes the specificity of Dolichos biflorus Agglutinin (DBA) lectin marking the entire normal ductal tree, including terminal intercalated ducts (putative sites of stem or progenitor cells) and ductal structures in ADM and PanIN. We used ferromagnetic-labeled DBA lectin to isolate ductal structures. Ductal cells were isolated under the following conditions: (1) Embryonic Development in wild type mice: E14.5, E15.5, E16.5, and postnatal day 1 (P1); (2) Injury and regeneration (pancreatitis) 0, 1, 3, 5 days following cerulein-induced acute pancreatitis. Cerulein is a cholecystokinin analog which produces a self-limited pancreatitis with injury and subsequent regeneration and repair, completed five days after insult; and (3) Pdx1-Cre;LSL-KrasG12D/+ mice aged 10 and 20 weeks that harbor PanIN lesions and a subset develop PDAC. Ductal/PanIN cells were isolated from these mice and appropriate control mice (Pdx1-Cre;Kras+/+).

ORGANISM(S): Mus musculus

PROVIDER: GSE40895 | GEO | 2015/09/30

SECONDARY ACCESSION(S): PRJNA176437

REPOSITORIES: GEO

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Project description:BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here we determine the contribution of epigenetically silenced genes to the development of PDAC. METHODS: We investigated methylated DNAs from PDACs, chronic pancreatitis and normal pancreatic tissues using Methyl-CpG immunoprecipitation followed by microarray hybridization. Promoter methylation of selected genes was confirmed with the Epityper assay. Expression levels were evaluated by quantitative RT-PCR. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanINs), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. RESULTS: A total of 3.8% of 27.800 interrogated CpG islands were hypermethylated in PDAC versus normal and chronic pancreatitis tissues. Hypermethylation was confirmed in 12 out of 13 selected islands and was associated with gene silencing in 4 of them. The most prominently hypermethylated gene, WNK2, was further investigated. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in pancreatic tumor cells than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expression were lower in PDAC and chronic pancreatitis compared to normal tissues both in patients and mouse models. Overexpression of WNK2 led to a reduced cell growth and WNK2 expression in tissues correlated negatively with the expression of pERK1/2, a downstream target of WNK2 responsible for cell proliferation. CONCLUSIONS: WNK2 is downregulated by promoter hypermethylation early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway. 3 types of pancreatic tissue samples: 5 normals, 2 chronic pancreatitis, 7 tumors (PDAC)

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Identification of transcriptional signatures overlapping in pancreatic ductal development, acute pancreatitis and KrasG12D-driven carcinogenesis

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