Alcohol is a consistently identified risk factor for colon cancer. However, the molecular mechanism underlying its effect on normal colon crypt cells remains poorly understood. We employed RNA-sequencing to asses transcriptomic response to ethanol exposure (0.2% vol:vol) in 3D organoid lines derived from healthy colon (n = 34). Paired regression analysis identified 2,162 differentially expressed genes in response to ethanol. When stratified by colon location, a far greater number of differentially expressed genes were identified in organoids derived from the left versus right colon, many of which corresponded to cell-type specific markers. To test the hypothesis that the effects of ethanol treatment on colon organoid populations were in part due to differential cell composition, we incorporated external single cell RNA-sequencing data from normal colon biopsies to estimate cellular proportions following single cell deconvolution. We inferred cell-type-specific changes, and observed an increase in transit amplifying cells following ethanol exposure that was greater in organoids from the left than right colon, with a concomitant decrease in more differentiated cells. If this occurs in the colon following alcohol consumption, this would lead to an increased zone of cells in the lower crypt where conditions are optimal for cell division and the potential to develop mutations.

Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited because of the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles, we performed in vitro aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50 μmol/L aspirin or vehicle control for 72 hours and subjected to bulk RNA sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant (q < 0.10) DEGs prior to deconvolution. We provide replication of these findings in an independent population-based RNA-sequencing dataset of mucosal biopsies (BarcUVa-Seq), where a significant enrichment for overlap of DEGs was observed (P < 2.2E−16). Single-cell deconvolution revealed changes in cell composition, including a decrease in transit-amplifying cells following aspirin treatment (P = 0.01). Following deconvolution, DEGs included novel putative targets for aspirin such as TRABD2A (q = 0.055), a negative regulator of Wnt signaling. Weighted gene co-expression network analysis identified 12 significant modules, including two that contained hubs for EGFR and PTGES2, the latter being previously implicated in aspirin chemoprevention. In summary, aspirin treatment of patient-derived colon organoids using physiologically relevant doses resulted in transcriptome-wide changes that reveal altered cell composition and improved understanding of transcriptional pathways, providing novel insight into its chemopreventive properties. Prevention Relevance: Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transit-amplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis.

Tobacco smoke and red/processed meats are well-known risk factors for colorectal cancer (CRC). Most research has focused on studies of normal colon biopsies in epidemiologic studies or treatment of CRC cell lines in vitro . These studies are often constrained by challenges with accuracy of self-report data or, in the case of CRC cell lines, small sample sizes and lack of relationship to normal tissue at risk. In an attempt to address some of these limitations, we performed a 24-hour treatment of a representative carcinogens cocktail in 37 independent organoid lines derived from normal colon biopsies. Machine learning algorithms were applied to bulk RNA-sequencing and revealed cellular composition changes in colon organoids. We identified 738 differentially expressed genes in response to carcinogens exposure. Network analysis identified significantly different modules of co-expression, that included genes related to MSI-H tumor biology, and genes previously implicated in CRC through genome-wide association studies. Our study helps to better define the molecular effects of representative carcinogens from smoking and red/processed meat in normal colon epithelial cells and in the etiology of the MSI-H subtype of CRC, and suggests an overlap between molecular mechanisms involved in inherited and environmental CRC risk.

Background Familial adenomatous polyposis (FAP) is an inherited colorectal cancer (CRC) syndrome resulting from germ line mutations in the adenomatous polyposis coli (APC) gene. While FAP accounts for less than 1% of all CRC cases, loss of APC expression is seen in > 80% of non-hereditary CRCs. To better understand molecular mechanisms underlying APC-driven CRC, we performed an epigenome-wide analysis of colon organoids derived from normal-appearing colons of FAP patients versus healthy subjects to identify differentially methylated regions (DMRs) that may precede the onset of CRC. Results We identified 358 DMRs when comparing colon organoids of FAP patients to those of healthy subjects (FDR < 0.05, |mean beta difference| = 5%). Of these, nearly 50% of DMRs were also differentially methylated in at least one of three CRC tumor and normal adjacent tissue (NAT) cohorts (TCGA-COAD, GSE193535 and ColoCare). Moreover, 27 of the DMRs mapped to CRC genome-wide association study (GWAS) loci. We provide evidence suggesting that some of these DMRs led to significant differences in gene expression of adjacent genes using quantitative PCR. For example, we identified significantly greater expression of five genes: Kazal-type serine peptidase inhibitor domain 1 (KAZALD1, P = 0.032), F-Box and leucine-rich repeat protein 8 (FBXL8, P = 0.036), TRIM31 antisense RNA 1 (TRIM31-AS1, P = 0.036), Fas apoptotic inhibitory molecule 2 (FAIM2, P = 0.049) and (Collagen beta (1–0)galactosyltransferase 2 (COLGALT2, P = 0.049). Importantly, both FBXL8 and TRIM31-AS1 were also significantly differentially expressed in TCGA-COAD tumor versus matched NAT, supporting a role for these genes in CRC tumor development. Conclusions We performed the first DNA methylome-wide analysis of normal colon organoids derived from FAP patients compared to those of healthy subjects. Our results reveal that normal colon organoids from FAP patients exhibit extensive epigenetic differences compared to those of healthy subjects that appear similar to those exhibited in CRC tumor. Our analyses therefore identify DMRs and candidate target genes that are potentially important in CRC tumor development in FAP, with potential implications for non-hereditary CRC.