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)

Project description:Background: A better understanding of the pancreatic adenocarcinoma (PDAC) immune microenvironment is critical to improving outcomes. Myeloid cells are of particular importance for PDAC progression. Circulating monocytes are recruited and hijacked by the tumor to become immune suppressive tumor-associated macrophages. Meanwhile, tissue resident macrophages are innately anti-inflammatory and pro-fibrotic. Open chromatin and transcription factor activity provide source and function data for myeloid cells. Thus, we explore single nuclear assay for transposase accessible chromatin (ATAC) sequencing (snATAC-Seq), a method to analyze open gene promoters and transcription factor binding, as an important means for discerning the myeloid composition in human PDAC tumors. Methods: Frozen pancreatic tissues (benign or PDAC) were digested into single nuclei suspensions. snATAC-Seq and sn-Multiome (ATAC + RNA) libraries were prepared using 10X Chromium technology and sequenced by an Illumina sequencer. Signac was used for preliminary analysis, clustering, and differentially accessible chromatin region identification. snMultiome data was used to validate cell annotations for the snATAC-Seq data. Results: Unique populations comprised the benign and tumor samples. Myeloid cell transcription factor activities were higher in tumor samples than benign pancreatic samples, enabling us to further stratify myeloid populations in PDAC tumors. GO enrichment analysis showed myeloid cells can activate immune response in tumors. Subcluster analysis revealed eight distinct myeloid populations. GO enrichment demonstrated unique functions of different myeloid populations, including IL-1b signaling (recruited monocytes) and intracellular protein transport (dendritic cells). Conclusions: These data suggest snATAC-Seq as a method for retrospective analysis of frozen human pancreatic tissues to identify myeloid populations. Understanding the source specific potential function of myeloid cells is important for patient prognosis in PDAC.

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Gene expression of inflammatory monocytes/macrophages after ablative radiotherapy in pancreatic adenocarcinoma

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