GUT: Yu Jun's team at the University of Chinese in Hong Kong solves the mystery of bowel cancer caused by smoking

Colorectal cancer (CRC) is one of the most common cancers worldwide, and there is plenty of evidence that lifestyle habits such as diet, smoking, obesity and exercise are all strongly associated with the onset of CRC.

Smoking not only increases the risk of lung cancer, but also promotes cancerous changes in organs such as the colon, kidneys and pancreas. Studies have shown that smoking significantly increases the incidence and mortality of colorectal cancer in humans, and smoking has also been observed in animal models to increase the risk of colorectal cancer.

So a very direct question, how exactly does smoking promote the occurrence and development of CRC? This is not yet clear.

Recently, a research team led by Yu Jun of the University of Chinese in Hong Kong published important research results at Gut [1].

They found that smoking led to changes in the composition of the intestinal flora and its metabolites, leading to intestinal barrier dysfunction and activation of tumorigenic and pro-inflammatory signaling pathways, thereby promoting the development of colorectal tumors.

Their study suggests that controlling enterobacteria may be a potential strategy to prevent colorectal cancer in smokers.

Screenshot of the first page of the paper

Smoking is closely related to the morbidity and mortality of CRC, and it is necessary to explore the mechanism of occurrence.

On the one hand, studies have shown that smoking increases the diversity of bacteria in the human gut [2, 3] and alters the structure of the gut microbiota and mucins[4]; on the other hand, existing studies have confirmed a correlation between changes in the gut microbiota and the occurrence of colorectal cancer [5], and intestinal flora derived from patients with CRC promotes colon cancer in recipient mice.

But whether changes in the gut microbiota represent a link between smoking and colorectal cancer are unclear.

To clarify this relationship, Professor Yu Jun's team exposed Azomethane (AOM)-treated C57BL/6 mice to clean air or cigarette smoke for 2 hours a day, and after 28 weeks, the changes in microbiomes and metabolites in mouse feces were analyzed by shotgun metage sequencing and liquid chromatography-mass spectrometry.

Experimental molding flowchart

We know that intraperitoneal injection of AOM induces CRC in mice, so will there be a difference in the occurrence of CRC in mice exposed to two different air environments?

The results showed that the number and size of colorectal tumors in mice exposed to cigarette smoke increased significantly compared with the mice in the control group, and both immunohistochemistry and Western blotting showed a significant increase in the proportion of Ki67+ cells in the intestinal epithelium, which meant that the exposure of cigarette smoke promoted the proliferation of colon epithelial cells in mice.

Cigarette smoke promotes the development of colon cancer in mice

The researchers then used shotgun metagenomic sequencing to analyze the fecal microbiomes of the two groups, and they found that after a 28-week treatment, the alpha diversity of the intestinal flora of the mice in the cigarette exposure group was significantly reduced, and there were significant differences in beta diversity clustering.

Cigarette smoke regulates the intestinal flora of mice

Compared to the control group of mice, 20 species of bacteria exposed to cigarette smoke underwent significant changes (p1.5), of which Eggerthella. Lenta and Staphylococcus capitis were significantly enriched, and the intestinal probiotics Lactobacillus reuteri, Parabacteroides distasonis and Bacteroides dorei decreased. In addition, they also found significant changes in the correlation of the gut microbiota of the two groups of mice, and in the cigarette smoke exposure group, E. lenta showed a negative correlation with the abundance of several probiotics, that is, accompanied by E. The enrichment of lenta, the abundance of several probiotics is significantly reduced.

In addition to sequencing, the researchers also analyzed the fecal metabolites of mice by liquid chromatography-mass spectrometry, and found that the fecal metabolites of the two groups of mice had significant differences, with 41 metabolites altered (the content of p taurine deoxycholic acid (TDCA) was significantly increased in cigarette-exposed mice, TDCA is a recognized carcinogen in secondary bile acids).

So what is the association between altered gut flora and fecal metabolites in the cigarette exposure group? Through correlation analysis, the researchers found that E. Lenta showed the strongest positive correlation with TDCA, while two reduced probiotics, Lactobacillus jensenii and Lactobacillus crispatus, showed an inverse correlation with TDCA. Therefore, the dysregulated intestinal flora and its metabolites are likely to be an important driver of CRC in mice.

Partial Spearman correlation analysis of gut bacteria with differential metabolites

The team then studied the effect of cigarette smoke on the intestinal barrier function of mice, and they measured the expression level of colonic tight junction protein and found that cigarette smoke significantly reduced the expression level of claudin-3 and latched small band-1 (ZO-1), and the serum level of lipopolysaccharides (LPS) was significantly improved. These results suggest that cigarette smoke leads to impaired intestinal barrier function.

Cigarette smoke reduces the expression of claudin-3 and ZO-1

In order to understand the effect of smoking on the formation of proto-cancer from a molecular perspective, Yu Jun's research team conducted genetic testing on mouse intestinal epithelial cells, and compared with the non-smoking group, the intestinal epithelial cells in the cigarette exposure group had 19 genes upregulated and 7 genes downregulated. The enrichment results showed that the altered genes were mainly enriched in the mito-activated protein kinase (MAPK) signaling pathway.

Previous literature has reported that TDCA can activate the ERK subfamily of the MAPK pathway, so the team evaluated the activation of the MAPK/ERK pathway in the intestinal epithelium of two groups of mice. ERK1/2 is a key regulatory protein in the MAPK/ERK pathway, and researchers found that cigarette smoke increases the phosphorylation level of ERK1/2, and phosphorylated ERK1/2 is positively correlated with TDCA. The above results suggest that cigarette smoke induces phosphorylation of ERK1/2 and activates the MAPK/ERK pathway, thereby promoting the development of colon cancer.

Subsequently, in order to identify the changes in the expression of inflammation-related genes, Yu Jun's team detected the expression of inflammation and self-immunity-related genes in the intestinal epithelial cells of two groups of mice. The results showed that the differential genes of the two groups were mainly enriched in the interleukin 17 (IL-17) and tumor necrosis factor (TNF) signaling pathways. The qPCR results validated an increase in the transcriptional levels of the pro-inflammatory factors Il17a and Cxcl2 in the cigarette exposure group and a decrease in the anti-inflammatory factor Il10. This suggests that cigarette smoke promotes the occurrence of inflammation in the colon.

Two chips of mouse intestinal epithelial cells detect gene expression results

To confirm the direct effect of intestinal flora alterations caused by cigarette smoke on colorectal tumors, they performed fecal flora transplantation on germ-free mice, transplanting feces from mice in the non-smoking group (GF-AOM) and cigarette exposure group (GF-AOMS) to the colon site of mice, respectively.

Similar to the previous results, the alpha diversity of the GF-AOMS intestinal flora was significantly reduced, and the beta diversity analysis showed significant separation from GF-AOM mice. Lenta remains the most abundant bacterium in GF-AOMS, and TDCA levels have also increased significantly. In addition, in GF-AOMS, the microbiota of cigarette smoke group sources also increased colon cell proliferation, impaired intestinal barrier function, and enhanced the expression of the oncogenic signaling pathway MAPK/ERK and pro-inflammatory genes. Therefore, changes in the intestinal flora contribute to the pro-tumor effect of cigarette smoke in the development of colorectal cancer.

Schematic diagram of the mechanism

In general, this study found that smoking can induce intestinal dysbiosis and promote the occurrence of colon tumors by regulating the composition of the intestinal flora; metabolic smoking significantly promotes the biosynthesis of TDCA. Lenta bacteria are significantly positively correlated; smoking impairs the intestinal barrier and activates the tumorigenic MAPK/ERK signaling pathway.

This study is the first to establish a novel gut microbiome-mediated mechanism of colorectal tumorigenesis caused by cigarette smoke, suggesting that smoking cessation is at least one of the viable ways to prevent colorectal cancer by rebuilding a healthy gut microbiota.

Bibliography:

[1]. Bai X, Wei H, Liu W, et al. Cigarette smoke promotes colorectal cancer through modulation of gut microbiota and related metabolites [published online ahead of print, 2022 Apr 6]. Gut. 2022;gutjnl-2021-325021. doi:10.1136/gutjnl-2021-325021

[2]. Biedermann L, Zeitz J, Mwinyi J, et al. Smoking cessation induces profound changes in the composition of the intestinal microbiota in humans. PLoS One. 2013;8( 3):e59260. doi:10.1371/journal.pone.0059260

[3]. Biedermann L, Brülisauer K, Zeitz J, et al. Smoking cessation alters intestinal microbiota: insights from quantitative investigations on human fecal samples using FISH. Inflamm Bowel Dis. 2014;20(9):1496-1501. doi:10.1097/MIB.0000000000000129

[4]. Allais L, Kerckhof FM, Verschuere S, et al. Chronic cigarette smoke exposure induces microbial and inflammatory shifts and mucin changes in the murine gut. Environ Microbiol. 2016;18(5):1352-1363. doi:10.1111/1462-2920.12934

[5]. Yu J, Feng Q, Wong SH, et al. Metagenomic analysis of faecal microbiome as a tool towards targeted non-invasive biomarkers for colorectal cancer. Gut. 2017;66(1):70-78. doi:10.1136/gutjnl-2015-309800

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