Relationship between intestinal microbiota and colorectal cancer

Abstract

The human gastrointestinal tract hosts a complex and vast microbial community with up to 10¹¹-10¹² microorganisms colonizing the colon. The gut microbiota has a serious effect on homeostasis and pathogenesis through a number of mechanisms. In recent years, the relationship between the intestinal microbiota and sporadic colorectal cancer has attracted much scientific interest. Mechanisms underlying colonic carcinogenesis include the conversion of procarcinogenic diet-related factors to carcinogens and the stimulation of procarcinogenic signaling pathways in luminal epithelial cells. Understanding each of these mechanisms will facilitate future studies, leading to the development of novel strategies for the diagnosis, treatment, and prevention of colorectal cancer. In this review, we discuss the relationship between colorectal cancer and the intestinal microbiota.

Key Words: Sporadic, Colorectal, Cancer, Intestinal, Microbiota

Core tip: Microbiota’s role in providing intestinal homeostasis is not as an audience, but it is active. Both the composition of microbiota and its metabolic activity impact the sensitivity of the host and can cause many pathologies including colorectal cancer.

INTRODUCTION

Colorectal cancer is the third commonest cancer type worldwide and causes 600000 deaths every year[1]. Because colorectal cancer patients are frequently asymptomatic in the early phase of the disease, diagnosis at this stage presents a significant clinical challenge. Detection of early stage cancers (stages 1-2) allows curative surgery with a 5-year survival rate of 80%. However, survival rates decrease to approximately 10% for metastatic and late stage tumors[2]. Although there are currently methods for the early diagnosis methods, including computed tomography, colonoscopy, and blood tests, it is expected that evaluation of the intestinal microbiota will prove to be a valuable method allowing earlier diagnosis of colorectal cancer.

In humans, a relationship between cancer and microorganisms has been demonstrated in a number of organs, with the most well-known example being the relationship between Helicobacter pylori and gastric cancer and mucosa-associated lymphoid tissue lymphoma[3].

In adults, while the bacterial population in the stomach and small intestine is smaller (10³-10⁴ CFU/g contents), increased concentrations of microorganisms are found in the colon (10¹¹-10¹² CFU/g contents) compared with the upper gastrointestinal tract. The majority of these microorganisms exist in a favorable symbiotic relationship with humans[3,4]. The intestinal microbiota develops specific to individual variation and environmental conditions beginning at birth[5].

Recently, etiology of colorectal cancer has been shown to be related to genetic mutations, diet, inflammatory processes, lifestyle, and the gut microbiota, with up to 95% of colorectal cancer thought to sporadically develop in individuals with no genetic predisposition[6].

The colonic microbiota is thought to contribute to the development of colorectal cancer by controlling the epithelial cell proliferation and differentiation, synthesizing essential nutrients and bioactive products, preventing the reproduction of pathogenic organisms, and stimulating the immune system[7]. In this review, studies investigating the role of the intestinal microbiota in the development of colorectal cancer development are discussed.

MICROBIOTA OF THE HUMAN INTESTINE

There are 100 billion bacteria in the human intestine with an approximate weight equivalent to 1.5-2 kg. Bacteroidetes and Firmicutes are the major species of the adult intestinal microbiota with the next most frequent species being Actinobacteria, Proteobacteria, and Verrucomicrobia[8].

Normally, colonic bacteria exist in a mutually beneficial symbiotic relationship with humans without adverse effects on the host cells. In situations where this balance is deregulated because of a number of possible causes, the numbers and species of harmful bacteria increase, providing a basis for the development of inflammatory and chronic disease. Changes in the intestinal microbiota have been shown to be associated with obesity, fatty liver, type 1 and 2 diabetes, kidney disease, arthritis, inflammatory bowel disease, and colorectal cancer[9-13]. However, the precise relationship between changes in the microbiota and colorectal cancer has yet to be fully elucidated.

FACTORS INFLUENCING GASTROINTESTINAL MICROBIOTA

The intestinal microbiota is affected by a number of factors, such as antibiotics, diet, and inflammation[4-18]. A number of studies have reported a high degree of similarity in the intestinal microbiota between members of the same family but a low degree of similarity between heterozygous mice despite being housed in the same cage[9,14,19].

The intestinal microbiota of mice fed standard low-in-fat nutrients has been shown to change within a few weeks with particularly great changes in the composition of Bacteroidetes and Firmicutes species. After mice returned to a low-fat diet, a particularly significant reduction in Mollicutes, a species of Firmicutes, was observed[9,20]. Similar changes have observed with diets high in fat, particularly in obese people, genetically obese mice, and obesity-resistant mice[9,14,21]. Transfer of colon microbiota from mice fed a high-fat diet to mice fed a low-in-fat diet has been shown to accelerate tumor growth suggesting diet-induced changes in the colon microbiota may have a synergistic effect with genetic factors on tumor development[22]. Diet-related changes in intestinal microbiota have also been shown to be associated with colorectal cancer[23].

MICROBIAL INFLUENCE ON COLORECTAL CANCER

The relationship between the intestinal microbiota and disease has drawn increased attention in recent years. In particular, recent studies have demonstrated strong associations between the development of colorectal cancer and intestinal bacteria. In these studies, DNA damage caused by superoxide radicals, genotoxin formation, increased T-cell proliferation, and activation of procarcinogenic pathways through a number of receptors have all been shown to contribute to cancer development[24-27].

The enzymatic activation or detoxification of carcinogens, and therefore modulation of their tumorigenic activity, has been shown to be influenced by the intestinal microbiota[24,28-35]. In the 1960s, it was observed that germ-free rats exposed to the glycoside, cyasin, did not develop intestinal tumors. Conversely, germ-free rats directly exposed to methylazoximethanol, a sub-active metabolite of cyasin, did develop intestinal tumors[36]. As the formation of methylazoximethanol depends on bacterial β-glucosidase enzyme activity[36], this study was a potent demonstration of the effect of the intestinal microbiota on bioactive carcinogenic compounds. Subsequent research has revealed that the intestinal microbiota converts latent carcinogens to bioactive forms through a number of enzymes, including β-glucuronidase, β-glucosidase, azoreductase, and nitroreductase[37]. Azoxymethane (AOM) is the most frequently used experimental colon carcinogen. AOM is first hydrolyzed in the liver to methylazoximethanol and conjugated to glucuronic acid before bilious excretion into the intestine where it is converted into a highly reactive methyl carbon ion by bacterial β-glucuronidase[34,37,38]. Interestingly, it has been reported that inhibition of β-glucuronidase activity significantly decreases the tumor-inducing potential of AOM in rats[39]. Furthermore, probiotic bacteria, such as Lactobacillus and Bifidobacterium species, have been shown to have anti-carcinogenic effects through the inactivation of microbial enzymes involved in procarcinogenic activation[40]. For example, Lactobacillales, such as L. Casei and L. Acidophilus suppress β-glucuronidase, azoreductase, and nitroreductase activity[41,42]. This balance between the activation and detoxification of potential carcinogens underlies the activation of host oncogenes and tumor suppressors (Figure 1).

Figure 1 The factors releated to intestinal microbiota promotes neoplasia in the gastrointestinal tract.

In the study by Boleij et al[43] investigating the expression of the Bacteroides fragilis gene (BFT) in colonoscopic samples from 49 healthy individuals and 49 colorectal cancer patients, BFT gene expression was detected more frequently in samples from colorectal cancer patients. When comparing early and late stage cancer patients, BFT gene expression was more frequently detected in late stage cancer patients.

DNA damage and chromosomal instability are early genetic events in the development of colorectal cancer. As with aneuploidy, chromosomal instability is associated with long-term inflammatory bowel disease (IBD) and frequently a precedent event in the subsequent development of colorectal cancer[44-46]. Enterococcus faecalis (E. faecalis), an intestinal bacteria, has been repeatedly found to induce aneuploidy in colonic epithelial cells in monoassociated interleukin (IL)-10 -/- rats and cause aggressive colitis[47,48]. Inhibitors of reactive oxygen and nitrogen species can prevent aneuploidy induced by E. faecalis[49]. These findings demonstrate that intestinal microbiota (particularly specific species) can induce RONS and lead to carcinogenesis.

In intestinal hemostasis, the protective role of the microbiota is thought to be through an effect on epithelial cell proliferation and apoptosis. The main mechanism underlying this effect has been proposed as the conversion of dietary fiber into short chain fatty acids (SCFA), such as acetate, propionate, and butyrate, through microbial fermentation. These SCFAs, particularly butyrate, are readily absorbed easily by the colon and are used as a primary energy source. In addition to significant anti-inflammatory effects[50,51], SCFAs stimulate cell proliferation and differentiation in non-neoplastic normal colon, promote intestinal hemostasis, and the resolution of intestinal injury[51,52]. In addition, SCFAs demonstrate a trans-effect on cancer cells. In particular, butyrate induces apoptosis in colorectal cancer cell lines through a number of mechanisms but predominantly via inhibition of histone deacetylase and activation of intrinsic/mitochondrial apoptosis[53-57].

However, SLC5A and GPR109A, the two major receptors of butyrate, provide protection in the early phases of tumorigenesis as they are frequently inactivated in human cancers[58-60]. It is believed that regulation of microbiota species responsible for the production of butyrate will have efficacy in the treatment of gastrointestinal diseases[61,62]. Therefore, probiotics and in-absorbable food are thought to alter the intestinal microbiota leading to a beneficial increase in the production of short chain fatty acids[63].

Although the development of colorectal cancer has not been attributed to any specific microorganism, a number of cancer-promoting bacteria have been identified (Table 1).

Table 1 The relationship between bacterial types and colorectal cancer.

Bacteria	Subject of study	Evidence	Ref.
Helicobacterhepaticus	Animal	Augments azoxymethane induced, and spontaneous colorectal cancer in mice	[64-69]
H. hepaticus + H.bilis	Animal	Dual infection induces colorectal cancer in mice	[70,71]
H. typhlonius + H. rodentium	Animal	Dual infection in neonates induces colorectal cancer in mice	[72,73]
Streptococcus bovis	Human	S.bovis bacteremia and endocarditis associated with human colorectal cancer	[74-77]
	Animal	Augments azoxymethane induced colorectal cancer in rats	[78]
	Human	Increased humoral immune response to S.bovis antigenRpL7/L12, sassociated with increased risk for colorectal cancer	[79]
Bacteroides fragilis	Animal	Enterotoxigenic B.fragilis augments spontaneous colorectal cancer in mice	[26]
	Human	Increased prevalence of enterotoxigenic B.fragilis in human colorectal cancer	[80]
	Human	Increased prevalence in tumor vs normal colonic tissue by quantative PCR analysis	[81]
	Human	Increased prevalence in tumor vs normal colonic tissue by quantative PCR analysis	[43]
B. vulgatus	Animal	Induces azoxymethane induced, colorectal cancer in mice	[82]
Escherichia coli	Human	Increased mucosa-associated Escherichia coli in human colorectal cancer	[83]
Citrobacter rodentium and C. freundii	Animal	Etiologic agent of transmissible murine colonic hyperplasia	[84]
	Animal	Augments spontaneous and 1,2 dimethylhydrazine induced colorectal cancer in mice	[85,86]
Fusobacterium nucleatum	Human	Increased prevalence in tumor vs normal colonic tissue by quantative PCR analysis	[87]
	Human	Increased prevalence in tumor vs normal colonic tissue by quantative PCR analysis and 16S ribosomal RNA Gene V3 pyrosequencing analysis	[88]
	Human	Increased prevalence in tumor vs normal colonic tissue by quantative PCR analysis	[89]
	Animal	16S ribosomal RNA Gene V3 pyrosequencing analysis	[90]
Enterococcus faecalis	Human	Increased in the feces of colorectal cancer patients by quantative PCR analysis	[91]
Furmicutes	Animal	16S ribosomal RNA Gene V3 pyrosequencing analysis	[90]
Akkermansia muciniphila	Human	16S ribosomal RNA Gene V4 pyrosequencing analysis and Gas Chromatography-Mass Spectrometry	[92]
Methanobrevibacterium	Human	Increased prevalence in tumor vs normal colonic tissue by quantative PCR analysis and 16S ribosomal RNA Gene V3 pyrosequencing analysis in fecal samples	[89]

PCR: Polymerase chain reaction; RNA: Ribonucleic acid; H. Hepaticıus: Helicobacter hepaticus; H. bilis: Helicobacter bilis; H. typhlonius: Helicobacter typhlonius; H. Rodentium: Helicobacter rodentium; B. vulgatus: Bacteroides vulgatus; C. freundii: Citrobacter freundii.

In rats, Helicobacter hepaticus increases the development of colorectal cancer related to experimental colitis and spontaneous colorectal cancer[65,67]. Bacteroides fragilis is a widespread intestinal bacteria and a potential cause of spontaneous colon tumorigenesis in rats as an enterotoxigenic variant[26].

Exclusion of opportunist pathogens by colonic bacteria may represent a natural defense against colorectal cancer. Similarly, food containing species of Lactobacillus and Bifidobacteria, used as probiotics, provide a number of protective benefits against inflammatory bowel diseases[93-95]. Upon colonizing the host and on the condition of the formation of an additional biofilm, probiotic bacteria have been shown to prevent the adhesion and invasion of pathogen types, maintain host tight junction protein structure, decrease host cytokine production, modulate inflammation and immunity, and neutralize carcinogens and toxins[96-100].

Intestinal microbiota have been shown to cause the release of host antibacterial lectins, stimulate antimicrobial host epithelial responses, and deplete subsets of potentially pathogenic bacteria providing a protective role against abnormal immune responses.

In a study by Sobhani et al[81] of 179 individuals undergoing colonoscopy (60 colorectal cancer, 119 normal), significantly greater levels of Bacteroides/Prevotella bacterial DNA were found in patients with colorectal cancer. Further, it was shown that a greater proportion of IL-17 immunomodulatory cells were isolated from patients with colorectal cancer.

In a study by Gao et al[88] in 2015 examining colon samples from 30 healthy and 31 cancer patients, distal and proximal colon microbiota from both healthy individuals and cancer patients were evaluated using the 16S RNA V3 sequence. No significant difference was observed between proximal and distal colon microbiota; however, in patients with colorectal cancer, Firmicutes and Fusobacteria were over-represented and Proteobacteria were under-represented. Further, Lactococcus and Fusobacterium were identified more often, and Pseudomonas and Escherichia–Shigella less often, in tissues from patients with colorectal cancer compared to those without cancer[88].

In a study by Zhu et al[90] using the 1,2-dimethylhydrazine cancer model, V3 sequences of 16S ribosomal RNA isolated from intestinal microbiota samples from rats with cancer and healthy rats were determined. While Firmucutesin was more frequently observed in rats with colorectal cancer, Bacteroidetes and Spirochetes were less commonly observed. There was no significant difference in the Proteobacteria types between the two groups; however, Prevotella, Lactobacillus, and Treponema were more frequently detected in healthy rats. Furthermore, while Fusobacterium was not observed in healthy rats, it could be identified specifically in cancer rats[90]. In a study of feces samples from healthy individuals and colorectal cancer patients, Akkermansia muciniphila was identified 4 times as often in colorectal cancer patients than healthy individuals[92].

As emphasized in many studies discussed above, intestinal microbiota have a substantial impact on intestinal health through controlling the immune and inflammatory response to individual species of intestinal microbiota, the activation or detoxification of carcinogens, the stimulation of DNA damage and chromosomal instability, dysregulation of the balance between proliferation and apoptosis, and prevention of invasion by pathogens.

CONCLUSION

Although colorectal cancer development is a complex process, recent studies have shown that the microbiota is actively involved.

Recently, we have developed a greater understanding of the effect of the microbiota on bowel health and diseases, including esophagitis/Barrett’s esophagus, stomach cancer, IBD, and colorectal cancer. However, while a strong relationship between gastrointestinal diseases and the microbiota content is evident, many questions remain unanswered. One of the most clinically challenging issues is to understand how a change in intestinal microbiota will likely impact on the course of disease. Knowledge obtained from dysbiotic microbiota research in germ-free animals and clinical studies involving a variety of intestinal diseases will help provide answers to these important questions. Further, there is currently a lack of data regarding which microorganisms in the microbiota cause disease and are protective.

Continuous improvements in the development of increasingly cost-effective research methods, gene sequencing technology, and high productivity techniques are expected to provide substantial information regarding the healthy and dysbiotic microbiota composition. This information will facilitate functional experiments utilizing cause and effect animal models.

Understanding the relationship between pathology and the microbiota is important; however, the role of microbiota in pathogenesis has yet to be fully elucidated. Therapeutic microbial transplantation has been trialed in metabolic syndrome and also has utility in the treatment of colorectal cancer; however, this technique has many limitations including infection and the promotion of autoimmune disease. Despite this, there is hope that treatments targeting the human microbiota may provide therapies for the prevention and treatment of colorectal cancer in the future.

In summary, the microbiota plays an active role in intestinal homeostasis. Both the composition of microbiota and its metabolic activity have an impact on the host susceptibility to disease and can directly contribute to a number of varied pathologies, including colorectal cancer.