Selective epigenetic alterations in RNF43 in pancreatic exocrine cells from high-fat-diet-induced obese mice; implications for pancreatic cancer

Objective Pancreatic cancer (PC) originates and progresses with genetic mutations in various oncogenes and suppressor genes, notably KRAS, CDKN2A, TP53, and SMAD4, prevalent across diverse PC cells. In addition to genetic mutations/deletions, persistent exposure to high-risk factors, including obesity, induces whole-genome scale epigenetic alterations contributing to malignancy. However, the impact of obesity on DNA methylation in the presymptomatic stage, particularly in genes prone to PC mutation, remains uncharacterized. Results We analyzed the methylation levels of 197 loci in six genes (KRAS, CDKN2A, TP53, SMAD4, GNAS and RNF43) using Illumina Mouse Methylation BeadChip array (280 K) data from pancreatic exocrine cells obtained from high-fat-diet (HFD) induced obese mice. Results revealed no significant differences in methylation levels in loci between HFD- and normal-fat-diet (NFD)-fed mice, except for RNF43, a negative regulator of Wnt signaling, which showed hypermethylation in three loci. These findings indicate that, in mouse pancreatic exocrine cells, high-fat dietary obesity induced aberrant DNA methylation in RNF43 but not in other frequently mutated PC-related genes. Supplementary Information The online version contains supplementary material available at 10.1186/s13104-024-06757-0.


Introduction
Pancreatic cancer (PC), ranking seventh as the leading cause of cancer-related deaths worldwide in 2018 [1], poses challenges owing to late diagnosis and poor outcomes resulting from the absence of early symptoms.This nature of PC underscores the need for therapeutic strategies as well as cancer prevention, which has attracted significant social attention, particularly given the lack of reliable screening tests for asymptomatic individuals during the early stages.Therefore, characterizing early stage PC to elucidate the mechanisms by which high-risk factor exposure transduces presymptomatic tissue into cancerous tissues is crucial.
Recognized risk factors for PC include obesity, chronic pancreatitis, diabetes, aging, male sex, and smoking [2,3].A possible contributor to the transition from presymptomatic pancreatic tissue to cancerous tissue is epigenetic modification: reversible but heritable changes in gene expression without amino acid mutation and known to undergo modification through lifestyle-and environmental-factors.DNA methylation, a well-known epigenetic event, regulates gene expression (i.e., level of normal protein) and microRNA stability, influencing various biological processes, including development, genetic imprinting, immune response, and aging.
In our recent study, we performed an epigenome-wide analysis of isolated pancreatic exocrine cells obtained from high fat-diet (HFD)-induced obesity (DIO) mice.Obesity induces whole-genome scale abnormalities in DNA methylation in the presymptomatic stage, with enrichment in cellular processes, such as DNA repair, transcription regulation, and cell proliferation [25].Comparing differentially methylated regions (DMRs) with those in stage IB PC, we identified three potential pathway candidates for PC development.However, the specific impact of obesity on the methylation levels of individual PC-driver genes, including KRAS, CDKN2A, TP53 and SMAD4, in DIO mice remains unknown.Therefore, we aimed to investigate alterations in methylation levels across all CpG islands within these genes in DIO mice.

Discussions
In DIO mice, abnormal methylation was observed in three loci in mouse RNF43 of pancreatic exocrine cells, contrasting with unaltered CpG islands in KRAS, CDKN2A, TP53, SMAD4 and GNAS.Obesity alone was unlikely to induce aberrant methylation in these genes; therefore, the prominent contribution of these genes in PC arose from genetic alterations: mutations/deletions.
Despite reports of decreased CDKN2A levels owing to the hypermethylation of its promoter in approximately 15% of PC cases [23], our results revealed no influence of HFD on CDKN2A methylation.Obesity may not have induced CDKN2A hypermethylation at the presymptomatic stage but induced aberrant methylation in later PC development.
We examined the methylation status of six genes in pancreatic exocrine cells of DIO mice, assuming their significant roles in PC development.Notably, the methylation levels of KRAS, CDKN2A, TP53, SMAD4 and GNAS remained unaffected by HFD-induced obesity, suggesting that the epigenetic effects of obesity may not converge onto identical genes undergoing mutations/deletions during PC development.In a previous study, we identified over 300 DMRs in DIO mice, proposing three pathways for PC development: (i) cell hypertrophy (involving PLC, PKC, SMAD2/3 and TRKA); (ii) metabolic control (involving CREB and AMPK); and (iii) potassium regulation (involving K + -channel).The epigenetic effects of obesity on PC may preferentially involve genes in these pathways rather than those well-known for mutations/ deletions.These findings provide key molecular insights into PC pathogenesis and potential biomarkers development through obesity-induced epigenetics.

Limitations
The current study has several limitations.Notably, metabolic differences between mouse and human pancreatic tissues could lead to variations in obesity-induced epigenetic effects.Furthermore, there is a need to increase the sample size to strengthen our findings.The expression level of RNF43 in obese mice was not evaluated, indicating a gap that necessitates future research to explore the correlation between DNA methylation levels at three loci, RNF43 expression levels, and subsequent abnormal augmentation of Wnt signaling.Besides DNA methylation, epigenetic modifications involve histone modifications (e.g., acetylation and deacetylation of histones) and chromatin accessibility [29,30].These modifications result from three biochemical reactions, including writers (adding chemical groups to DNA or histones), erasers (removing epigenetic modifications), and readers (recognizing specific epigenetic marks).Investigating how obesity-induced epigenetic changes may influence histone modifications and chromatin accessibility, and the mechanisms of these epigenetic modifications through biochemical reactions, remains an area for future exploration.

Mice and diets
To minimize confounding risk factors associated with human PC, particularly sex, we exclusively examined female C57BL/6J mice (Charles River Japan, Tokyo).Mice were housed separately and given ad libitum access to HFD or NFD from 5 to 60 weeks of age (tissue collection).The HFD group (n = 7) received feed containing fat equivalent to 60% of the total calories (D12492, Research Diets Inc., New Brunswick, NJ).The NFD group (n = 5) received feed containing fat equivalent to 10% of the total calories (D12450J Research Diets Inc).

Mouse pancreatic exocrine cells cultures
All procedures for the experiments using mice were approved by the Animal Committee of Saitama Medical University (protocol:2499).All methods were performed following experimental procedures mirroring those described in our previous study [25].Briefly, mice were euthanized at approximately 60-week old with CO 2 gas, and pancreatic tissues were excised.The tissues were minced, digested, and triturated in Hanks' balanced salt solution (HBSS) containing 5% fetal bovine serum (FBS), 0.25 mg/ml trypsin inhibitor and 25 ng/ml epidermal growth factor (EGF) (Corning Life Sciences, Tewksbury, MA).Isolated pancreatic cells were passed through the mesh, rinsed with the medium, and plated.

DNA methylation analysis
Methylation analyses followed the protocols outlined in our previous study (Araki 2023).Briefly, cultured pancreatic exocrine cells were recovered at ∼ 4 days in vitro and genomic DNA was prepared; genomic DNA (∼ 1 µg) was bisulfite-treated using a DNA methylation kit (Takara Bio).We analyzed methylation using an Infinium Mouse Methylation BeadChip array, validated to contain 280,754 CpG sites.Statistical analyses were conducted using Python (v3.6) and GenomeStudio Methylation Module (v1.8) available in GenomeStudio (v2011.1),with individual probes filtered based on mean q < 0.05 (FDR = 0.1, Abs(delta) > 0.2 [i.e., > 20% change in β-value]).CpG site gene information for mouse KRAS, CDKN2A, TP53 and SMAD4 is shown in Supplementary Tables 1 and Fig. 2. Human PC stage IB methylation data (n = 3) were extracted from the National Cancer Institute portal (GDC portal, https://portal.gdc.cancer.gov/).Stage IB represents PC with tumor sizes ranging between 2and 4-cm (https://www.cancer.gov/).The database lacks data on earlier PC stages, rendering Stage IB the earliest available.Probe information and the signal data were converted and imported into GenomeStudio.