Walnut Substantially Alters the DNA Methylation Profile in Colon Cancer Stem Cells

Abstract

The generation of cancer stem cells (CSCs) may involve epigenetic reprogramming which leads to the loss of expression of genes specific to the differentiated state and regains stem cell‐specific characteristics. DNA methylation plays a critical role in the loss of pluripotency and developmental plasticity. We recently reported that walnut phenolic extracts (WPEs) can reduce the stemness of colon CSCs by suppressing their self‐renewing capacity and enhancing the differentiation ability. To explore the underlying epigenetic mechanism, we conducted a targeted bisulfite sequencing from colon CSCs, which were isolated from HCT116 colon cancer cells by the fluorescence‐activated cell sorting and treated with or without 40ug/ml WPE (n=3). Among 3 million targeted CpG sites, 127,607 sites were hypomethylated and 237,302 sites were hypermethylated by WPE treatment. Methylated CpG site and methylation levels were analyzed by the Bismark program (version 0.9.0). From differentially methylated sites (DMS) data we selected differentially methylated regions (DMR) that include at least 3 CpGs with the same direction of DNA methylation change (fold change>1.5, P<0.01). Among total 145 DMRs 71 DMRs were hypomethylated and 74 were hypermethyated due to WPE treatment. The average size of DMR is 330 bp. The DAVID program revealed that genes associated with fertilization (CD9, FUT10, ATP8B3) and energy derivation by oxidation of organic compound (NDUFB10, EPM2A, NDUFS3) include hypermethylated DMRs, while hypomethylated DMRs are found in genes associated with mitochondrial organization (FXN, SH3GLB1, TMEM70), embryonic development ending in birth or egg hatching (SMO, HOXB7, FGF9), and regulation of apoptosis (MCL1, SH3GLB1PDCD6). Using DMS data (hypermethylated or hypomethylated, 1.5 fold change) along with RNA‐seq data (transcriptionally induced or reduced, 1.5 fold change) we classified into 4 groups: 1) hypomethylated and induced, 2) hypermethylated and reduced, 3) hypomethylated and reduced, and 4) hypermethyated and induced. The hypomethylated and induced group includes genes associated with oxidative phosphorylation, regulation of cellular protein metabolic process, and mitochondrial ATP synthesis coupled electron transport. The hypermethylated and reduced group includes genes associated with DNA repair, regulation of cell cycle process, and transcription. The hypomethylated and reduced group includes genes associated with regulation of mitotic cell cycle, phospholipid metabolic process, and cell division. The hypermethyated and induced group includes genes associated with translation, cholesterol biosynthesis process, and glucose catabolic process. Collectively, walnut treatment of colon CSC induces substantial DNA methylation changes in genes associated with development, metabolism, cellular process and mitochondrial function along with their expression changes.Support or Funding InformationThis work has been supported by the California Walnut Commission and the Korean Food Research Institute.