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

Background

Colorectal cancer (CRC) is one of the most common cancers worldwide. Although there are many early screening and prevention strategies for colorectal cancer, its burden is expected to increase further. There is evidence supporting the association of lifestyle factors such as diet, smoking, obesity, and exercise with CRC. Smoking increases the risk of lung cancer, with approximately 80% of primary lung cancers attributable to smoking. Smoking also increases the risk of cancers in other organs not directly exposed to cigarette smoke, such as the colon, rectum, pancreas, and kidneys. Studies have shown a significant correlation between smoking and the incidence and mortality of human CRC, and an increased risk of CRC development has also been observed in animal models. However, the mechanisms by which smoking promotes the initiation and progression of CRC remain unclear. An increase in bacterial diversity has been observed after smoking cessation in humans. Reports also indicate that changes in the microbiome and mucin structure are associated with smoking. Furthermore, gut microbes from CRC patients can promote colonic tumorigenesis in recipient mice. However,Gut microbiotathe changes represent a connection between smoking and CRC remain elusive.

In this study, we aimed to determine the role of smoking in CRC development using conventional and germ-free mouse models. We demonstrated that smoking can promote CRC by inducing dysbiosis of the gut microbiota, which affects metabolites, particularly taurodeoxycholic acid (TDCA). Increased gut TDCA can activate the carcinogenic MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2) pathway in colonic epithelium, thereby promoting colonic cell proliferation. Additionally, smoking may impair gut barrier function, further promoting TDCA activation of colonic carcinogenic MAPK/ERK signaling.

Experimental Methods

Male 10-week-old C57BL/6 mice were purchased. All mice were given food and water ad libitum and were housed in a specific pathogen-free environment. They were intraperitoneally injected with the carcinogen azoxymethane (AOM) (10 mg/kg) once a week for 6 consecutive weeks to induce colorectal cancer. At the start of AOM injections, mice were exposed to cigarette smoke (4% at a flow rate of 2000 mL/min) or clean air for 2 hours daily (15 mice per group, consisting of four cages with 3-4 mice each) for 28 weeks. The cigarette smoke was generated by a peristaltic pump. Cigarettes (containing nicotine) (1.0 mg/cigarette) were lit and continuously inhaled by the pump, mixed with fresh air, and then pumped into the smoking chamber. Only fresh air was pumped into the clean chamber (Figure 1A). At the end of week 28, both the cigarette smoke-exposed and the control mice were euthanized.

Figure 1

Figure 1 Legend: Smoking increases colorectal tumorigenesis in mice. (A) Schematic diagram of the smoking or clean chamber designed for cigarette smoke exposure and the AOM-induced cancer model. Mixed fresh air and smoke air were pumped into the smoking chamber, while fresh air was pumped into the clean chamber. Mice were placed in the chamber for 2 hours daily for 28 weeks. AOM (10 mg/kg) was injected intraperitoneally once a week for 6 consecutive weeks starting from day 0. Mice were euthanized at the end of week 28 (AOM group, n = 15; AOM + smoking group, n = 15). (B) Representative images of the colon at the time of sacrifice. The number and size of tumors in the AOM and AOM + smoking groups. (C) Representative images of H&E staining of adenomas in the AOM group and adenocarcinomas in the AOM + smoking group. (D) Incidence of adenomas and adenocarcinomas in the colon of AOM-treated mice. (E) Representative images of Ki67 positive cell immunohistochemical staining and the proportion of Ki67 positive cells in the colon. (F) Protein expression of PCNA in the colon of AOM-treated mice by Western blotting. AOM, azoxymethane; PCNA, proliferating cell nuclear antigen.

Experimental Results

Cigarette smoke promotes colorectal tumorigenesis in mice

To investigate the effect of smoking on colorectal tumorigenesis, AOM-treated C57BL/6 mice were exposed to clean air or cigarette smoke (Figure 1A). In mice exposed to cigarette smoke, both the number and size of colorectal tumors were significantly greater than in the smoke-free control mice (Figure 1B). The presence of colonic adenomas and adenocarcinomas was confirmed by histological examination by a pathologist (Figure 1C). Exposure to cigarette smoke significantly increased the incidence of colonic tumors (Figure 1D). Compared to smoke-free control mice, an increase in epithelial cell proliferation was observed in the colons of mice exposed to cigarette smoke, indicating a significantly higher number of Ki-67 positive cells (Figure 1E) and higher expression levels of the proliferation marker proliferating cell nuclear antigen (PCNA) (Figure 1F).

Cigarette smoke alters the composition and interactions of gut microbiota in mice

Due to the long time the mice lived together, 16S sequencing was used to assess whether there was a cage effect on the microbial community. It was found that the cage effect had no impact on the microbial community at different time points (Supplementary Figure 1). However, the significant impact of smoking on the microbial community occurred at the end of the experiment (end time point) (Supplementary Figure 1), indicating that long-term smoking is a major factor affecting the microbiome. Then, shotgun metagenomic sequencing analysis of fecal samples was performed to determine the potential changes in the gut microbiome caused by cigarette smoke exposure. At baseline (initial time point), there were no significant differences in α and β diversity between smoke-exposed mice and smoke-free control mice (Supplementary Figures 2A, B). After 28 weeks, a significant reduction in α diversity was observed in smoke-exposed mice compared to smoke-free control mice (Figure 2A and Supplementary Figure 2C). PCoA analysis (β diversity) showed that the clustering of the gut microbiome in smoke-exposed mice was significantly different from that in smoke-free control mice (Figure 2B). Twenty bacteria (FC>1.5) were significantly altered in smoke-exposed mice (Figure 2C). Among them, E. lenta and Staphylococcus were enriched, while beneficial gut bacteria, including Lactobacillus reuteri, Parabacteroides distasonis, and Bacteroides dorei, were depleted due to cigarette smoke exposure. The higher abundance of E. lenta in smoke-exposed mice was confirmed by quantitative PCR (Figure 2D). Our published dataset was further used to assess the association of E. lenta with human CRC. Compared to normal subjects (n = 204), CRC patients (n = 185) had significantly higher levels of E. lenta. Among these CRC patients, smokers (n = 30) had higher E. lenta abundance compared to non-smokers (n = 87) (p<0.05) (Supplementary Figures 3A, B).

Supplementary Figure 1

Supplementary Figure 2

Figure 2

Figure 2 Notes: Cigarette smoke modulates the gut microbiome of mice. (A) Fisher statistics (α diversity) and (B) PCoA analysis (β diversity) for the AOM+smoking and AOM groups. The significance of α and β diversity was obtained using two-tailed Mann-Whitney U test and PERMANOVA, respectively. (C) Bacterial differentiation between the AOM+smoking group and the AOM group. Abundance differences were detected using a multivariable statistical model (FDR correction, FC>1.5, MaAsLin2). (D) The abundance of Eggerthella lenta species between the AOM+smoking group and the AOM group was verified by quantitative PCR. (E) Ecological network of differential bacteria between the AOM+smoking group and the AOM group. Correlations were determined using the SparCC method. Visualization was performed for correlations >0.6 between AOM+smoking and AOM groups with differences in correlation strength. AOM, azoxymethane; FC, fold change; PCoA, principal coordinate analysis; PERMANOVA, permutational multivariate analysis of variance.

Interactions between microorganisms may contribute to disease progression. We studied the ecological network of interactions between bacteria, focusing on the abundance differences between smoke-exposed mice and smoke-free control mice (Figure 2E). It was observed that the symbiotic and co-exclusion interactions between bacteria were significantly different between smoke-exposed mice and smoke-free control mice.

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

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