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

Cigarette smoke enhances the expression of pro-inflammatory signaling genes.

Dysbiosis of gut bacteria is closely related to inflammation, which links carcinogenic factors and tumorigenesis. Therefore, we analyzed the expression of pro-inflammatory genes using a mouse inflammation response and autoimmune PCR array. Compared to smoke-free control mice, 27 upregulated genes and 5 downregulated genes were observed in mice exposed to cigarette smoke (Figure 5D, Supplementary Table 3). Cigarette smoke induced changes in pathways including pro-inflammatory interleukin 17 (IL-17) signaling and tumor necrosis factor (TNF) signaling pathways (Figure 5E). Quantitative reverse transcription PCR (RT-PCR) confirmed that, compared to smoke-free control mice, the expression of pro-inflammatory Il-17a and Cxcl2 increased, while the expression of anti-inflammatory Il-10 decreased in smoke-exposed mice (Figure 5F). Additionally, the relative mRNA expression of Il-17a was positively correlated with the abundance of E. lenta in the colon (Supplementary Figure 5). These findings suggest that cigarette smoke promotes inflammation in colorectal tumorigenesis.

Supplementary Figure 5

Fecal microbiota transplantation from germ-free mice into the intestines of smoke-exposed conventional mice, changes in microbiota.

To confirm the direct effect of cigarette smoke-altered gut microbiota on colorectal tumorigenesis, we performed fecal microbiota transplantation in germ-free mice. Germ-free mice were transplanted with feces from either smoke-exposed or smoke-free conventional mice. Mice were examined after 20 weeks (Figure 6A). We conducted shotgun metagenomic sequencing analysis to explore the colonization of the altered microbiota due to cigarette smoke. Similar to the traditional AOM mouse model, germ-free mice receiving feces from smoke-free mice (GF-AOM) showed significantly reduced α-diversity compared to those receiving feces from smoke-exposed mice (GF-AOMS) (Figure 6B and Supplementary Figure 2C). β-diversity analysis again showed significant separation of gut microbiota between the two groups of germ-free mice (GF-AOMS vs. GF-AOM, PERMANOVA) (Figure 6C). Further analysis revealed 34 differentially enriched bacteria (Figure 6D). In GF-AOMS mice, the abundance of E. lenta significantly increased, while the abundance of Bacteroides was significantly reduced (p<0.05, FC>1.5). Quantitative PCR confirmed that the abundance of E. lenta in the GF-AOMS group was significantly higher than that in the GF-AOM group (p<0.001, Figure 6E).

Figure 6

Figure 6 Notes: Changes in the gut microbiota of germ-free mice transplanted with fecal microbiota from smoke-exposed conventional mice. (A) Schematic of the germ-free mouse model. Germ-free mice were orally administered feces from the AOM + smoking group and the AOM group (n = 8/group). Mice were euthanized at the end of week 20. (B) Fisher statistics (α-diversity) and (C) PCoA analysis (β-diversity) for GF-AOMS and GF-AOM groups. Significance for α and β diversity was obtained through two-tailed Mann-Whitney U test and PERMANOVA, respectively. (D) Differential bacteria between GF-AOMS and GF-AOM groups. Abundance differences were detected using multivariable statistical models (FDR correction, FC>1.5, MaAsLin2). (E) The abundance of Eggerthella lenta species was validated between GF-AOMS and GF-AOM groups by quantitative PCR. (F) Consistent changes in bacterial abundance (p<0.05, smoke-exposed mice vs. smoke-free mice; GF-AOMS mice vs. GF-AOM mice) were observed in both mouse models (germ-free mice and AOM mice). The FC abundance between smoking and non-smoking was calculated. Red dots represent germ-free mouse models, and yellow dots represent conventional mouse models. (G) After strong feeding in germ-free mice compared to the donor, network modules were conserved. Correlation was determined using the SparCC method, and network modules were extracted based on the first-order neighborhood of bacteria. (H) The concentration of TDCA in feces between GF-AOMS and GF-AOM groups was measured by targeted mass spectrometry, p<0.05, two-tailed Mann-Whitney U test. AOM, azoxymethane; FC, fold change; GF-AOM, germ-free mice, feces from AOM donor mice; GF-AOMS, germ-free mice with feces from AOM + smoking donor mice; PCoA, principal coordinate analysis; PERMANOVA, permutational multivariate analysis of variance; TDCA, taurocholic acid.

Further testing of the consistency of microbiota changes between the two mouse models (germ-free mice and conventional AOM mice) was conducted. We found that, compared to GF-AOM mice, the abundance of bacteria in smoke-exposed AOM mice continued to increase in GF-AOMS mice, while the abundance of bacteria that decreased in smoke-exposed AOM mice continued to decrease in GF-AOMS mice compared to GF-AOM mice (Figure 6F). Importantly, we observed an increase in the abundance of E. lenta, while the abundance of the probiotic P. distasonis was lower in smoke-exposed AOM mice and GF-AOMS mice. Correlation analysis showed that the ecological network modules were conserved after fecal microbiota transplantation in germ-free mice compared to donor mice (Figure 6G). Additionally, the fecal TDCA levels in GF-AOMS were also significantly increased compared to GF-AOM mice (Figure 6H).

Cigarette smoke-altered microbiota increases colonic cell proliferation and tumorigenesis in germ-free mice.

Compared to GF-AOM mice, GF-AOMS mice exhibited increased colonic epithelial cell proliferation, as evidenced by a higher proportion of Ki-67 positive cells (Figure 7A) and higher levels of PCNA expression (Figure 7B). Additionally, we treated germ-free mice with AOM and gavaged them with feces from either smoke-exposed (GFAOM-AOMS) or smoke-free mice (GFAOM-AOM) (Supplementary Figure 6A). Compared to GFAOM-AOM mice, GFAOM-AOMS mice showed an increased number and size of colonic tumors (Supplementary Figure 6B). These results in germ-free mice are consistent with observations in conventional mice, indicating that cigarette smoke-altered microbiota and metabolome can directly promote colonic cell proliferation and tumorigenesis.

Supplementary Figure 6

Figure 7

Figure 7 Note: The altered microbiota from cigarette smoke increases colon cell proliferation, impairs intestinal barrier function, and enhances the expression of carcinogenic MAPK/ERK and pro-inflammatory genes in germ-free mice. (A) Representative images of Ki67 positive cell immunohistochemical staining and the proportion of Ki67 positive cells in the colon of germ-free mice. (B) Protein expression of PCNA in the colon of germ-free mice by Western blotting. (C) Protein expression of claudin-3 and ZO-1 in the colon of germ-free mice by Western blotting. (D) LPS concentrations in the serum of GF-AOM and GF-AOMS group mice. (E) Electron microscopy showing the structure of the intestinal barrier in germ-free mice. Arrows point to cell-cell junctions. (F) Differentially expressed genes in the colon epithelium of GF-AOMS group compared to GF-AOM group identified by mouse cancer pathway finder PCR array analysis. (G) Changes in cancer signaling pathways in the GF-AOMS group compared to the GF-AOM group through enrichment analysis. Including enrichment scores >1. Arrows represent the direction of enrichment, calculated by comparing upregulated and downregulated genes in the pathways. Differentially expressed genes in the MAPK signaling pathway are shown in the network. (H) Protein expression of ERK1/2 in the colon of GF-AOM and GF-AOMS group mice by Western blotting. (I) Differentially expressed genes in the colon epithelium of GF-AOMS group compared to GF-AOM group identified by mouse inflammation response and autoimmune array analysis. (J) Changes in inflammatory signaling pathways in the GF-AOMS group compared to GF-AOM group through enrichment analysis. Including enrichment scores >1. Differentially expressed genes in TNF and IL-17 signaling pathways are shown in the network. (K) Gene expression of Il-17a, Cxcl2, and Cxcr2 by quantitative RT-PCR. (a) Tight junctions; (b) Adhesion liquid network; (c) Desmosomes; (d) Paracellular spaces. Asterisks indicate disruption of cell junctions. Data are presented as mean ± SD. *p<0.05; statistical significance determined by two-tailed unpaired Student's t-test. p-values adjusted by Roosevelt (Online Supplement Tables 4, 5). AOM, azoxymethane; ERK, extracellular signal-regulated kinase; GF-AOM, germ-free mice with feces from AOM donor mice; GF-AOMS, germ-free mice with feces from AOM + smoking donor mice; Il, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; PCNA, proliferating cell nuclear antigen; RT-PCR, reverse transcription PCR; TNF, tumor necrosis factor.

The microbiota altered by cigarette smoke impairs the intestinal barrier function of germ-free mice.

We investigated whether the microbiota altered by cigarette smoke affects the intestinal barrier function of germ-free mice. A decrease in the expression of claudin-3 and ZO-1 and an increase in serum LPS levels were observed in GF-AOMS mice (Figure 7C, D). The impaired intestinal barrier function in GF-AOMS mice was also confirmed by EM, which showed widened intercellular junctions in the apical junction complex and paracellular spaces in the colon (Figure 7E). These results in germ-free mice are consistent with those in conventional mice, suggesting that alterations in the microbiota and metabolome due to cigarette smoke may impair intestinal barrier function, potentially further promoting tumorigenesis.

The microbiota altered by cigarette smoke enhances the carcinogenic MAPK/ERK pathway in the colon epithelium of germ-free mice.

To confirm the molecular mechanisms by which the altered microbiota from cigarette smoke promotes colon cell proliferation, we performed a cancer pathway finder array on the colon epithelium of germ-free mice. Compared to GF-AOM mice, we identified 9 upregulated genes and 3 downregulated genes in GF-AOMS mice (Figure 7F, Supplement Table 4). These differentially expressed genes were primarily enriched in the MAPK/ERK signaling pathway. The increased protein expression of phosphorylated-ERK1/2 in GF-AOMS mice compared to GF-AOM mice confirmed the activation of MAPK/ERK signaling (Figure 7G and H). Additionally, we observed a positive correlation between ERK phosphorylation levels and TDCA levels (Supplement Figure 4B). Similarly, using inflammation response and autoimmune PCR arrays, we found that 16 pro-inflammatory genes were upregulated in GF-AOMS compared to GF-AOM mice (Figure 7I, Supplement Table 5), which were also primarily enriched in the TNF and IL-17 signaling pathways (Figure 7J). Key pro-inflammatory genes in the TNF and IL-17 pathways, including Il-17a and C-X-C motif chemokine ligand 2 (Cxcl2) and C-X-C motif chemokine receptor 2 (Cxcr2), were confirmed to be upregulated in GF-AOMS mice by quantitative RT-PCR (Figure 7K). Consistent findings from both conventional and germ-free mouse models indicate that the microbiota altered by cigarette smoke directly induces pro-inflammatory TNF and IL-17 signaling as well as carcinogenic MAPK/ERK signaling in the colon. Therefore, the altered gut microbiota contributes to the tumor-promoting effects of cigarette smoke in colorectal carcinogenesis.

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