Assessment of Colorectal Cancer Risk Factors through the Application of Network-Based Approaches in a Racially Diverse Cohort of Colon Organoid Stem Cells

Abstract

Numerous demographic factors have been associated with colorectal cancer (CRC) risk. To better define biological mechanisms underlying these associations, we performed RNA sequencing of stem-cell-enriched organoids derived from the healthy colons of seven European Americans and eight African Americans. A weighted gene co-expression network analysis was performed following RNA sequencing. Module-trait relationships were determined through the association testing of each module and five CRC risk factors (age, body mass index, sex, smoking history, and race). Only modules that displayed a significantly positive correlation for gene significance and module membership were considered for further investigation. In total, 16 modules were associated with known CRC risk factors (p < 0.05). To contextualize the role of risk modules in CRC, publicly available RNA-sequencing data from TCGA-COAD were downloaded and re-analyzed. Differentially expressed genes identified between tumors and matched normal-adjacent tissue were overlaid across each module. Loci derived from CRC genome-wide association studies were additionally overlaid across modules to identify robust putative targets of risk. Among them, MYBL2 and RXRA represented strong plausible drivers through which cigarette smoking and BMI potentially modulated CRC risk, respectively. In summary, our findings highlight the potential of the colon organoid system in identifying novel CRC risk mechanisms in an ancestrally diverse and cellularly relevant population.

Keywords: WGCNA; aging; body mass index; colon organoid; colorectal cancer risk; network analysis; racial disparities; smoking; stem cell.

Figure 1

Overview of the palevioletred2 module. (A) Palevioletred2 module members are represented as nodes connected to other members through edges. The number of edges connected to each node provides a visual representation of the MM for each gene (node), where nodes with greater MMs display more connections. ME expressions of palevioletred2 were significantly greater in AA than EA colon stem cells. (B) Violin plots display significant differences in gene expression in TCGA-COAD of palevioletred2 module members that were mapped to CRC GWAS loci.

Figure 2

Overview of the lightpink3 module. (A) Lightpink3 module members are represented as nodes connected to other members through edges. Given the size of the module, a networking threshold of 0.1 was set to reduce poorly weighted connections prior to viewing in Cytoscape. The number of edges connected to each node provides a visual representation of the MM for each gene (node), where nodes with greater MMs display more connections. (B) Violin plots display significant differences in gene expression in TCGA-COAD of lightpink3 module members that were mapped to CRC GWAS loci.

Figure 3

Overview of the lightsteelblue module. (A) Lightsteelblue module members are represented as nodes connected to other members through edges. ME expression was increased with increasing age. Given the size of the module, a networking threshold of 0.19 was set to reduce poorly weighted connections prior to viewing in Cytoscape. The number of edges connected to each node provides a visual representation of the MM for each gene (node), where nodes with greater MMs display more connections. (B) Violin plots display significant increases in gene expression in TCGA-COAD tumors of lightsteelblue module members that were mapped to CRC GWAS loci.

Figure 4

Overview of the purple module. (A) Purple module members are represented as nodes connected to other members through edges. ME expression was increased in former and current smokers. Given the size of the module, a networking threshold of 0.22 was set to reduce poorly weighted connections prior to viewing in Cytoscape. The number of edges connected to each node provides a visual representation of the MM for each gene (node), where nodes with greater MMs display more connections. (B) Violin plots display significant increases in gene expression in TCGA-COAD tumors of purple module members that were mapped to CRC GWAS loci.

Figure 5

Overview of the darkred module. (A) Darkred module members are represented as nodes connected to other members through edges. ME expression was increased in former and current smokers. Given the size of the module, a networking threshold of 0.22 was set to reduce poorly weighted connections prior to viewing in Cytoscape. The number of edges connected to each node provides a visual representation of the MM for each gene (node), where nodes with greater MMs display more connections. (B) Violin plots display significant decreases in gene expression in TCGA-COAD tumors of darkred module members that were mapped to CRC GWAS loci.