(T1430-10-67) Butyrate Drives Metabolic Rewiring and Epigenetic Reprogramming in Human Colon Cancer Cells

Purpose: Butyrate (B) is a short-chain fatty acid produced by dietary fiber, known to inhibit histone deacetylases (HDACs) and possess cancer-preventive/anticancer effects. However, the role of B in metabolic rewiring, epigenomic reprogramming, transcriptomic network, NRF2 signaling, and eliciting cancer-preventive effects in colorectal cancer (CRC) HCT116 cell remains unclear.

Methods: Cell Viability Test
HCT116 cells were seeded in a 96-well plate at a density of 4 × 103 cells per well for overnight and then treated with either 0.1% DMSO as vehicle control or various concentrations of NaB for 1-, 3-, 5-day in DMEM medium supplemented with 1% FBS.
Luciferase Reporter Activity Assay
HepG2-C8 cells were plated in 12-well plates at a density of 1 × 105 cells per well for 24 h, and then treated with 0.1% DMSO or various concentrations of NaB in DMEM supplemented with 1% FBS for 24 h. ARE-luciferase activity was determined using the luciferase activity assay kit (Promega, Madison, WI, USA). The reporter lysis buffer was used to lysate the cells and 10 µL of the cell lysate supernatant was measured for NRF2-ARE activity using a Sirius luminometer (Berthold Detection System GmbH, Pforzheim, Germany).
Isolation of Nucleic Acids and Next-Generation Sequencing (NGS)
HCT116 cells were plated in 10 cm cell culture plates at a density of 2 × 105 cells per dish for overnight and then treated with the vehicle control, 1 mM NaB for 5 days. The cell culture medium dissolved with 1 mM NaB was changed every other day and cells were harvest at day 5. RNA and DNA were extracted using an AllPrep DNA/RNA Mini Kit (Qiagen, Valencia, CA, USA). Total four RNA samples (n = 2) and two DNA samples (n = 1) were subject to RNA-seq and SureSelect Methyl-seq respectively. Each RNA or DNA sample was pooled by three independent biological replicates.

Results: Sodium butyrate (NaB) dose-dependently inhibits the growth of CRC HCT116 cells. NaB inhibits NRF2/NRF2-target genes and blocks NRF2-ARE signaling. NaB increases NRF2 negative regulator KEAP1 expression through inhibiting its promoter methylation. Associative analysis of DEGs (differentially expressed genes) from RNA-seq and DMRs (differentially methylated regions) from CpG methyl-seq identified the tumor suppressor gene ABCA1 and tumor promote gene EGR3 are correlated with their promoters’ CpG methylation indicating NaB regulates cancer markers through modulating their promoter methylation. NaB activated the mitochondrial tricarboxylic acid (TCA) cycle while inhibited the methionine metabolism which are both tightly coupled to the epigenetic machinery. NaB regulates the epigenetic enzymes/genes including DNMT1, HAT1, KDM1A, KDM1B, and TET1. Altogether, B's regulation of metabolites coupled to the epigenetic enzymes illustrates the potential underlying biological connectivity between metabolomics and epigenomics

Conclusion: B regulates KEAP1/NRF2 signaling, drives metabolic rewiring, CpG methylomic, and transcriptomic reprogramming contributing to the overall cancer-prevention/anticancer effect in the CRC cell model.

NaB-mediated cell viability and regulation of NRF2/NRF2-target genes in HCT116 cells

DNA Methyl-seq and RAN-seq profiles in HCT116 cells regulated by NaB. A) Distribution of annotated DMRs by genomic features including Distal Intergenic, Promoter (≤1kb, 1–2kb, and 2–3kb), 1st Intron and other regions as shown in the legend.

Metabolomic profiles of HCT116 cells treated with NaB. A) PCA plot of metabolomic profiles in HCT116 cells after control and 1, 5, and 10 mM NaB treatment; B) top 30 regulated metabolites after NaB treatment; C) metabolism pathway analysis, and D) pathway enrichment analysis of HCT116 cells after 10 mM NaB treatment.