Experimental conclusion

We have established a new mechanism for cigarette smoke-induced colorectal tumorigenesis mediated by gut microbiota for the first time. Smoking is a risk factor for CRC. However, the understanding of the mechanisms underlying smoking-induced CRC is limited. Although several studies have shown an association between gut microbiota and smoking, it is still unclear whether the microbiota altered by exposure to smoking plays a significant role in the onset and progression of CRC. To fill this critical gap, we conducted experiments using an AOM-induced CRC mouse model and germ-free mice transplanted with cigarette smoke-altered microbiota, involving long-term exposure of mice to cigarette smoke.

This study demonstrated that smoking can promote colon tumorigenesis by modulating the composition of gut microbiota and inducing gut microbiota dysbiosis. In particular, E. lenta, which has been reported to be associated with CRC, was significantly enriched in mice exposed to cigarette smoke. On the other hand, P. distasonis, which was depleted in mice exposed to cigarette smoke, has been shown to increase the expression of tight junction proteins in the colon and attenuate colorectal tumorigenesis. Lactobacilli (L. reuteri, L. jensenii, and L. crispatus), known for their ability to inhibit pathogenic bacterial infection or proliferation, were found to be less abundant in mice exposed to cigarette smoke. Further evidence showed an antagonistic relationship between the enriched E. lenta and the depleted lactobacilli species in mice exposed to cigarette smoke.

It was also found that the bile acid biosynthesis pathway was significantly enriched in the fecal metabolites of mice exposed to smoke. Primary bile acids are synthesized in the liver and secreted into the intestine via bile ducts. The gut symbiotic microbiota converts primary bile acids into secondary bile acids, which are then primarily absorbed by the terminal ileum and colon, with a small amount excreted in feces. Due to their tumor-promoting properties, bile acids are receiving increasing attention. Among the secondary bile acids derived from gut microbiota, we confirmed that TDCA was significantly increased in the feces of mice exposed to smoke. Previous studies have shown that TDCA can promote colorectal and esophageal adenocarcinoma. The administration of TDCA into the colonic lumen increased the frequency of colorectal tumors induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We further found that changes in fecal metabolites were significantly correlated with changes in gut microbiota abundance, with TDCA showing the strongest positive correlation with E. lenta. This result is supported by previous studies indicating that E. lenta has the ability to dehydroxylate primary bile acids and expresses bile acid 3β-HSDH. These characteristics enable it to participate in the synthesis of secondary bile acids, thereby affecting the concentration of TDCA in feces.
 

We further assessed the intestinal barrier function of the mice and determined that it was impaired in mice exposed to cigarette smoke. The expression of two important tight junction proteins, claudin-3 and ZO-1, was reduced in mice exposed to cigarette smoke. Impaired intestinal tight junctions increase colonic permeability, allowing more intestinal toxic metabolites (such as TDCA) to enter the intercellular space of colonic epithelial cells, activating the MAPK/ERK signaling pathway.

Next, we investigated the molecular mechanisms by which cigarette smoke promotes colon tumorigenesis and revealed significant enrichment and activation of the MAPK/ERK pathway induced by cigarette smoke. This result is consistent with the function of TDCA, which has been reported as a ligand that activates the MAPK/ERK pathway. Activation of the MAPK/ERK pathway is important for cell proliferation and has been shown to be involved in the pathogenesis, progression, and carcinogenesis of human CRC. Additionally, we found that the pro-inflammatory IL-17 and TNF pathways were induced by cigarette smoke. IL-17 has been shown to promote the development of colon cancer. Previous studies indicated that E. lenta is enriched in IBD patients and can induce IL-17a through the action of cardiac glycoside reductase 2. We also found that E. lenta may activate the pro-inflammatory cytokine IL-17 pathway to induce tumorigenesis. Activation of the TNF-α pathway induces colitis and colorectal carcinogenesis. These findings collectively suggest that the activation of carcinogenic MAPK signaling and pro-inflammatory IL-17 and TNF signaling pathways triggered by smoke-related gut dysbiosis and metabolite changes contributes to cigarette smoke-related colon tumorigenesis.
 

In germ-free mice with fecal microbiota transplantation, we further studied the direct role of smoke-altered gut microbiota in tumorigenesis. The use of cigarette smoke-altered gut microbiota alone increased the proliferation of colonic epithelial cells in germ-free mice. The gut microbiota composition of recipient germ-free mice was similar to that of the gut microbiota composition in donor conventionally raised mice exposed to smoke. In particular, E. lenta was significantly enriched in both smoke-exposed AOM mice and GF-AOM mice. The cigarette smoke-altered gut microbiota also increased fecal TDCA levels and induced activation of the MAPK/ERK, IL-17, and TNF signaling pathways in the colonic epithelium of GF-AOM mice. These findings collectively suggest that cigarette smoke alters the gut microbiota, promoting the occurrence of colon tumors by increasing TDCA levels in the colon and further activating carcinogenic MAPK/ERK, IL-17, and TNF signaling pathways in the colonic epithelium. Additionally, the cigarette smoke-altered gut microbiota impaired the colonic epithelium, as evidenced by the reduced expression of tight junction proteins claudin-3 and ZO-1. Intestinal barrier dysfunction may facilitate the entry of carcinogenic TDCA into the intercellular space of colonic epithelium and further activate the carcinogenic MAPK/ERK signaling pathway.
 

This study demonstrates the promoting effect of smoking on CRC through the modulation of gut microbiota composition. Compared to continuous smokers, quitting smoking is associated with improved CRC-specific survival rates. Therefore, quitting smoking is one of the practical methods to prevent CRC, at least by reconstructing a healthy gut microbiome.
 

In summary, this study first demonstrates that cigarette smoke promotes colon tumorigenesis by inducing gut microbiota dysbiosis. Smoke-induced gut microbiota dysbiosis can increase TDCA levels in the colon, which then activates carcinogenic MAPK/ERK, IL-17, and TNF signaling pathways in the colonic epithelium. Furthermore, intestinal barrier dysfunction caused by gut microbiota dysbiosis may further promote TDCA activation of the MAPK/ERK signaling pathway.
 

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