Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids

Abstract

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.

Novel insights into the molecular mechanisms underlying risk of colorectal cancer from smoking and red/processed meat carcinogens by modeling exposure in normal colon organoids

“…Network analyses have been used in research into other complex genetic traits to identify candidate genes involved in GWAS [43]. We have also previously made use of WGCNA to unravel the relationship between aspirin and CRC risk loci in colon organoids [15]. From our WGCNA, we identified candidate GWAS-related genes LARP4 and PIP4K2C as respective hub genes for the plum4 and lightsteelblue modules.…”

“…Genes rarely act in isolation, and expression of genes within related pathways are usually coordinated in such a way that they can be identified using systems level approaches. We have previously found that WGCNA led to the identification of modules driven both by aspirin-related genes and CRC loci in a similarly sized cohort of aspirin treated colon organoids [15]. Here, we generated a network of gene co-expression in our colon organoid model using WGCNA [31] and determined whether modules comprised within this network were differentially associated with carcinogens exposure (Supplementary Figure 2).…”

“…To contextualize the carcinogens-related DEGs we identified, we analyzed RNA-seq data from a large population-based cohort of normal colon mucosal biopsies (BarcUVa-Seq) cohort (Supplementary File 3). We have previously performed deconvolution analyses of this dataset [15] and used cell scores those cell scores to adjust for cell composition within our subsequent regression model. Given that our interest only lay within the 738 DEGs identified in colon organoids, we set a validation threshold at P < 0.05 for these genes.…”

“…Analysis parameters are reported in Supplementary Table 5. BarcUVa-Seq data was deconvoluted for use as validation in a previous study [15], and the same cell scores were used here.…”

“…Three-dimensional (3D) normal human colon organoids are an important model for the study of the stem cell niche of the colon crypt [12], benefitting from an increased cellular heterogeneity relative to CRC cell lines [12,13] and the ability to control dose administration relative to most data collected from biopsies. We have previously shown that exposure of colon organoids to ethanol [14] or aspirin [15] can reveal candidate genes implicated in CRC risk through the analysis of bulk RNA-sequencing (RNA-seq) followed by single cell deconvolution to adjust for cellular content.…”