Cigarette smoke promotes the occurrence and development of colorectal cancer by regulating the gut microbiota and related metabolites (Part II)

Cigarette smoke alters metabolites associated with the gut microbiome in feces.

Changes in fecal metabolites after smoke exposure were determined by liquid chromatography, and mass spectrometry (MS)/MS analysis of mouse feces. Orthogonal partial least squares discriminant analysis showed that the fecal metabolite profiles of smoke-exposed mice were significantly different from those of smoke-free mice (Figure 3A). Compared to smoke-free control mice, 41 metabolites were altered in the feces of mice exposed to cigarette smoke (Figure 3B). The altered metabolites were enriched or depleted in different metabolomic signaling pathways (Figure 3C). Bile acid biosynthesis was the most enriched pathway in smoke-exposed mice compared to smoke-free mice. In the gut, bacteria convert primary bile acids into secondary bile acids, which are partially absorbed by the terminal ileum and colon. Among these secondary bile acids, TDCA is known to be pro-carcinogenic. The abundance of TDCA was significantly increased in smoke-exposed mice (Figure 3B). Targeted MS further confirmed the elevated fecal TDCA in smoke-exposed mice (p = 0.0094) (Figure 3D).

Figure 3

Figure 3 Notes: Cigarette smoke alters metabolites associated with the gut microbiome in feces. (A) There is a significant difference in fecal metabolite profiles between the AOM+smoking group and the AOM group, with OPLS-DA being a supervised multivariate regression analysis to identify distinguishable patterns between different groups. The x-axis captures variation between groups, while the y-axis captures variation within groups. (B) Differential metabolites between the AOM+smoking group and the AOM group, two-tailed Mann-Whitney U test. (C) Enrichment analysis of differential metabolites between the AOM+smoking group and the AOM group. (D) Concentration of TDCA in feces of the AOM+smoking group and the AOM group determined by targeted mass spectrometry p<0.05, two-tailed Student's t-test. (E) Correlation analysis of bacteria with differential metabolites using partial Spearman correlation. (F) Linear correlation between TDCA and Eggerthella lenta, adjusted and unadjusted by linear model (smoke exposure/smoke-free). AOM, azoxymethane; OPLS-DA, orthogonal partial least squares discriminant analysis; TDCA, taurodeoxycholic acid.

To determine the potential association between the microbiome and metabolites, a correlation analysis between bacteria and metabolites was performed using partial Spearman correlation. E. lenta was observed to have the most positive correlation with TDCA, while the two probiotics, L. jensenii and L. crispatus, which were depleted in smoke-exposed mice, showed a negative correlation with TDCA (Figure 3E). E. lenta has the ability to remove primary bile acids and expresses bile acid 3β-hydroxysteroid dehydrogenase (3β-HSDH); therefore, it is involved in the synthesis of secondary bile acids and affects the levels of TDCA in feces. Consequently, we measured the abundance of the gene encoding the 3β-HSDH enzyme, and a significantly higher gene abundance was observed in smoke-exposed mice compared to smoke-free mice (Supplementary Figure 3C). Consistent with this, we further confirmed a positive correlation between the abundance of E. lenta and the concentration of TDCA in feces (Figure 3F). Therefore, gut microbiome dysbiosis and altered metabolites may jointly promote the occurrence of colon tumors.

Supplementary Figure 3

Cigarette smoke impairs intestinal barrier function.

To study the effect of smoking on intestinal barrier function, the expression levels of colonic tight junction proteins, claudin-3 and Zonula occludens-1 (ZO-1), as well as serum lipopolysaccharide (LPS) levels were analyzed. Cigarette smoke significantly reduced the levels of claudin-3 and ZO-1 determined by Western blotting (Figure 4A) and immunofluorescence staining (Figure 4B). Pathologists further confirmed the impaired tight junctions under electron microscopy (Figure 4C). Meanwhile, we found that serum LPS levels were significantly increased in smoke-exposed mice compared to smoke-free mice (Figure 4D). These results collectively indicate that cigarette smoke leads to impaired intestinal barrier function.

Figure 4

Figure 4 Notes: Cigarette smoke impairs intestinal barrier function. (A) Protein expression of claudin-3 and ZO-1 in the colon of AOM-treated mice by Western blotting. (B) Protein expression of claudin-3 and ZO-1 in the colon of the AOM-treated model by immunofluorescence staining. (C) Representative images of the colonic barrier structure in AOM-treated mice. Arrows point to the cell-cell junctions under electron microscopy. (a) Tight junction; (b) Adherens junction; (c) Desmosome; (d) Gap junction. Asterisks indicate disrupted cell junctions. (D) LPS concentrations in the serum of AOM and AOM+smoking group mice. Data are presented as mean ± SD. Statistical significance was determined by unpaired Student's t-test. AOM, azoxymethane; 4′,6-diamidino-2-phenylindole; LPS, lipopolysaccharide.

Cigarette smoke enhances carcinogenic MAPK/ERK signaling in the colonic epithelium.
To gain molecular insights into the tumor-promoting effects of smoking, the expression of cancer-related genes in the colonic epithelium was analyzed using a mouse cancer pathway finder PCR array. Compared to smoke-free control mice, we observed 19 upregulated genes and 7 downregulated genes in smoke-exposed mice (Figure 5A, Supplementary Table 2). Enrichment analysis indicated that the mitogen-activated protein kinase (MAPK) signaling pathway was the top pathway activated by cigarette smoke (Figure 5B). Previous studies have reported that TDCA can activate the ERK subfamily of the MAPK pathway, thus, the activation of MAPK/ERK in the colonic epithelium of smoke-exposed mice was assessed compared to control mice. Activation of MAPK/ERK signaling by smoking was confirmed by elevated levels of phosphorylated ERK1/2, which is a key mediator protein in the MAPK/ERK pathway (Figure 5C). Additionally, a positive correlation was observed between ERK phosphorylation levels and TDCA levels (Supplementary Figure 4A). These findings suggest that cigarette smoke induces ERK1/2 phosphorylation and activates the MAPK/ERK signaling pathway to promote colon tumorigenesis.

Figure 5

Figure 5 Caption: Cigarette smoke enhances the expression of carcinogenic MAPK/ERK pathways and pro-inflammatory pathways in colonic epithelium. (A) Differentially expressed genes in the colonic epithelium of the AOM+smoking group compared to the AOM group were analyzed using the mouse cancer pathway finder PCR array, with FC=AOM+smoking/AOM positive log2(FC)=high expression in the AOM+smoking group and negative log2(FC)=high expression in the AOM group. This includes FC between AOM+smoking and AOM>2. (B) Enrichment analysis shows that cancer signaling pathways are altered in the AOM+smoking group compared to the AOM group. Arrows indicate the direction of enrichment, calculated by comparing upregulated and downregulated genes in this pathway. Differentially expressed genes in the MAPK signaling pathway are shown in the network. (C) Western blot analysis of protein expression of ρ-ERK1/2 in the colon of AOM-treated mice. (D) Comparison of differentially expressed genes in the colonic epithelium of AOM+smoking mice with AOM mice through mouse inflammation response and autoimmune array analysis. (E) Enrichment analysis shows that inflammatory signaling pathways are altered in the AOM+smoking group compared to the AOM group. Differentially expressed genes in the TNF and IL-17 signaling pathways are shown in the network. (F) Quantitative RT-PCR analysis of gene expression of Cxcl2, Il-17a, and Il-10. Statistical significance was determined by two-tailed unpaired Student's t-test. P-values were adjusted for FDR (Supplementary Tables 2, 3). AOM, azoxymethane; ERK, extracellular signal-regulated kinase; FC, fold change; Il, interleukin; MAPK, mitogen-activated protein kinase; RT-PCR, reverse transcription PCR; TNF, tumor necrosis factor.

Supplementary Figure 4

Citation: Cigarette smoke promotes colorectal cancer through modulation of gut microbiota and related metabolites.