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Proteome-wide Investigation of Proline Hydroxylation in Pancreatic Ductal Adenocarcinoma Using DiLeu Isobaric Labeling Strategy

ABSTRACT: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer marked by a dense, collagen-rich extracellular matrix that promotes tumor progression. In this study, we developed a high-throughput method combining enhanced hydrophilic interaction chromatography (HILIC) and 12-plex N,N-dimethyl leucine (DiLeu) isobaric tags for efficient quantitative analysis of hydroxyproline. Applied to human pancreatic tissues, this approach identified the largest hydroxyproline dataset reported for the pancreas. Our analysis reveals significant changes in proline hydroxylation in benign tumors, PDAC, and adjacent normal tissues, highlighting key proteins like collagen and GAPDH as potential targets for further investigation into PDAC progression.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Lingjun Li

PROVIDER: MSV000095824 | MassIVE |

REPOSITORIES: MassIVE

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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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Proteome-wide Investigation of Proline Hydroxylation in Pancreatic Ductal Adenocarcinoma Using DiLeu Isobaric Labeling Strategy

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