Abstract
Colorectal cancer (CRC) is a highly heterogeneous disease, shaped by genetic and epigenetic factors, and influenced by its microenvironment. Epigenetic alterations, particularly aberrant DNA methylation, strongly influence CRC development and prognosis. CRC progression occurs through three major pathways: Chromosomal Instability (CIN), characterised by mutations in tumour suppressor genes and oncogenes; Microsatellite Instability (MSI), arising from defects in DNA mismatch repair pathways, commonly due to promoter hypermethylation and reduced expression of DNA mismatch repair genes; and the CpG Island Methylator Phenotype (CIMP), marked by widespread gene promoter hypermethylation, particularly in CpG dinucleotide dense regions – termed CpG islands. Emerging evidence also implicates the gut microbiome in CRC pathogenesis. Notably, microbial species such as Fusobacterium spp. have been implicated in influencing host methylation patterns and promoting tumourigenesis. Previous research conducted in this group revealed relatively high Fusobacterium sp. levels in CRC tumour samples characterised by MSI, and found that treating cell lines with Fusobacterium reduces expression of DNA mis-match repair genes.
We investigated the association between Fusobacterium sp. abundance and DNA methylation profiles of CRC tumour cells. Oxford Nanopore Technologies (ONT)’s reduced representation methylation sequencing (RRMS) protocol was used on ten CRC tumour resection samples with known Fusobacterium levels. We then identified regions of differential methylation between CRC samples abundant in Fusobacterium sp., and CRC samples with no detected Fusobacterium sp. Finally, existing transcriptomic data was utilised to draw connections between differential methylation profiles, and gene expression.
We identified seven genes (SOX1, TFAP2C, NKX2-2, HIC1, OTX2, ADAMTS17 and ZBTB7A) with differentially methylated promoter regions, six of which (SOX1, TFAP2C, NKX2-2, HIC1, OTX2, and ZBTB7A) are transcription factors characterised as tumour suppressors or oncogenes in CRC. Using existing transcriptomic data, we found that expression of TFAP2C, HIC1 and ZBTB7A was inversely associated with Fusobacterium-linked methylation profiles. Pathway analysis showed enrichment for transcriptional regulation, the dysregulation of which is heavily implicated in disease development, prognosis and response to treatment. These findings suggest that aberrant Fusobacterium abundance may impact transcription-regulation via methylation-mediated regulation of tumour-associated genes, providing new insight into the interplay between the microbiome and epigenetics in CRC.