Abstract
BackgroundAltered microbiome composition and aberrant promoter hypermethylation of tumor suppressor genes (TSGs) are two important hallmarks of colorectal cancer (CRC). Here we performed concurrent 16S rRNA gene sequencing and methyl-CpG binding domain-based capture sequencing in 33 tissue biopsies (5 normal colonic mucosa tissues, 4 pairs of adenoma and adenoma-adjacent tissues, and 10 pairs of CRC and CRC-adjacent tissues) to identify significant associations between TSG promoter hypermethylation and CRC-associated bacteria, followed by functional validation of the methylation-associated bacteria.ResultsFusobacterium nucleatum and Hungatella hathewayi were identified as the top two methylation-regulating bacteria. Targeted analysis on bona fide TSGs revealed that H. hathewayi and Streptococcus spp. significantly correlated with CDX2 and MLH1 promoter hypermethylation, respectively. Mechanistic validation with cell-line and animal models revealed that F. nucleatum and H. hathewayi upregulated DNA methyltransferase. H. hathewayi inoculation also promoted colonic epithelial cell proliferation in germ-free and conventional mice.ConclusionOur integrative analysis revealed previously unknown epigenetic regulation of TSGs in host cells through inducing DNA methyltransferase by F. nucleatum and H. hathewayi, and established the latter as CRC-promoting bacteria.8Z6GNsVPEC4pSjR8q4gW5YVideo abstract.
Highlights
Colorectal cancer (CRC), the fourth most common cancer and the fifth leading cause of cancer death globally [1], occurs as a result of intricate interactions between host and environmental factors, among which gut dysbiosis is strongly associated with disease occurrence [2, 3]
colorectal cancer (CRC), and their adjacent tissues showed that F. nucleatum, Hungatella hathewayi, and Parvimonas spp. were the top three bacteria that showed significant enrichment in tumor tissues compared to corresponding adjacent controls
We found that incubation with F. nucleatum and H. hathewayi led to significant increases in global DNA methylation (5-mC) levels in NCM460 (p < 0.05 for H. hathewayi and p < 0.05 for F. nucleatum), HCT116 (p < 0.05 for H. hathewayi and p < 0.01 for F. nucleatum) and HT29 (p < 0.01 for H. hathewayi and p < 0.05 for F. nucleatum) compared to phosphate-buffered saline (PBS) treatment or incubation with non-tumorigenic
Summary
Colorectal cancer (CRC), the fourth most common cancer and the fifth leading cause of cancer death globally [1], occurs as a result of intricate interactions between host and environmental factors, among which gut dysbiosis is strongly associated with disease occurrence [2, 3]. Transplantation of stool from CRC patients into germ-free and conventional mice confirmed the causal link of gut microbes to CRC development [11] These evidences collectively support a key functional role of bacteria in CRC pathogenesis. Both genetic and epigenetic pathways are involved in CRC development, in which the latter aberrantly silences tumor suppressor genes (TSGs) via multiple mechanisms, including promoter hypermethylation and histone modifications [12]. DNA methylation involves the covalent addition of a methyl group to the 5-carbon of the cytosine ring within CG dinucleotides of gene promoters by DNA methyltransferases (DNMTs) to form 5-methylcytosine (5-mC) In this regard, gene-specific promoter hypermethylation has been widely reported in CRC with many methylated genes functionally verified as bona fide TSGs (e.g., APC, MLH1, PTEN, RUNX3, CDX1/2) [13]. We performed concurrent 16S rRNA gene sequencing and methyl-CpG binding domain-based capture sequencing in 33 tissue biopsies (5 normal colonic mucosa tissues, 4 pairs of adenoma and adenoma-adjacent tissues, and 10 pairs of CRC and CRC-adjacent tissues) to identify significant associations between TSG promoter hypermethylation and CRC-associated bacteria, followed by functional validation of the methylation-associated bacteria
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