Early epigenetic downregulation of WNK2 kinase during pancreatic ductal adenocarcinoma development

Abstract

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. We analyzed enriched, highly methylated DNAs from PDACs, chronic pancreatitis (CP) and normal tissues using CpG island microarrays and identified WNK2 as a prominent candidate tumor suppressor gene being downregulated early in PDAC development. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanIN), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in tumor than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expressions were lower in PDAC and CP compared with normal tissues both in patients and mouse models. Overexpression of WNK2 led to reduced cell growth, and WNK2 expression in tissues correlated negatively with pERK1/2 expression, a downstream target of WNK2 responsible for cell proliferation. Downregulation of WNK2 by promoter hypermethylation occurs early in PDAC pathogenesis and may support tumor cell growth via the ERK-MAPK pathway.

Genomic sequencing identifies WNK2 as a driver in hepatocellular carcinoma and a risk factor for early recurrence

“…WNK kinases play an important role in cell cycle progression, metabolic adaptation, antiapoptosis, invasion, and metastasis. 29 Epigenetic silencing of WNK2 has been found in gliomas, meningiomas, and pancreatic ductal adenocarcinomas; [30][31][32] however, the functional impact of genetic mechanisms affecting WNK2 is still unknown, especially in cancer. 29 We showed that in addition to somatic mutations in WNK2, copy number loss occurred in nearly 30% of patients, and WNK2 expression in the tumor samples was also downregulated.…”

WNK4 is an Adipogenic Factor and Its Deletion Reduces Diet-Induced Obesity in Mice

“…The total RNA was reverse-transcribed using Omniscript reverse transcriptase (Qiagen, Hilden, Germany). Sequences of RT-PCR primers employed were as follows: mouse C/EBPβ sense, 5′-GTTTCGGGAGTTGATGCAATC-3′, C/EBPβ antisense, 5′-AACAACCCCGCAGGAACAT-3′; mouse CUL3 sense, 5′-GGATGAGTTCAGGCAACATC-3′, mouse CUL3 antisense, 5′-GCATGTCTTGGTGCTGGTGG-3′; human perilipin sense, 5′- ACCCCCCTGAAAAGATTGCTT-3′, antisense, 5′- GATGGGAACGCTGATGCTGTT-3′; human PPARγ2 sense, 5′-GCAGGAGATCTACAAGGACTTG-3′, antisense, 5′- CCCTCAGAATAGTGCAACTGG-3′; human β-actin sense, 5′-CCGTCTTCCCCTCCATCG-3′; antisense, 5′- GTCCCAGTTGGTGACGATGC-3′; the mouse C/EBPα, mouse PPARγ, mouse β-actin, mouse GAPDH, mouse FAS, mouse LPL, mouse FABP4, mouse WNK2, mouse WNK3, mouse WNK4, mouse KLHL2, mouse KLHL3, mouse F4/80, and mouse adiponectin primers were described previously (Zeniya et al, 2013, Madsen et al, 2003, O'reilly et al, 2006, Dutruel et al, 2014, Suganami et al, 2005). Relative mRNA levels were normalized by endogenous reference genes (β-actin and GAPDH).…”

Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

“…This is particularly significantly considering that cytoplasmic pERK has been detected in a large number of patient PDAC samples (Pham et al. , 2008; Dutruel et al. , 2014).…”