Gut microbiota-derived metabolites in CRC progression and causation

Abstract

Background

Based on recent research reports, dysbiosis and improper concentrations of microbial metabolites in the gut may result into the carcinogenesis of colorectal cancer. Recent advancement also highlights the involvement of bacteria and their secreted metabolites in the cancer causation. Gut microbial metabolites are functional output of the host–microbiota interactions and produced by anaerobic fermentation of food components in the diet. They contribute to influence variety of biological mechanisms including inflammation, cell signaling, cell-cycle disruption which are majorly disrupted in carcinogenic activities.

Purpose

In this review, we intend to discuss recent updates and possible molecular mechanisms to provide the role of bacterial metabolites, gut bacteria and diet in the colorectal carcinogenesis.

Summary

Recent evidences have proposed the role of bacteria, such as Fusobacterium nucleaturm, Streptococcus bovis, Helicobacter pylori, Bacteroides fragilis and Clostridium septicum, in the carcinogenesis of CRC. Metagenomic study confirmed that these bacteria are in increased abundance in CRC patient as compared to healthy individuals and can cause inflammation and DNA damage which can lead to development of cancer. These bacteria produce metabolites, such as secondary bile salts from primary bile salts, hydrogen sulfide, trimethylamine-N-oxide (TMAO), which are likely to promote inflammation and subsequently cancer development.

Conclusion

Recent studies suggest that gut microbiota-derived metabolites have a role in CRC progression and causation and hence, could be implicated in CRC diagnosis, prognosis and therapy.

Introduction

Colorectal cancer (CRC) is the third-most common cancer in terms of frequency and second-most common in terms of mortality worldwide (Lichtenstern et al. 2020). Among 1.80 million CRC cases, WHO reported 862,000 deaths in 2018 indicating less than 50% survival. The disease progresses through adenoma-to-carcinoma sequence and takes years to develop. CRC initiates in the colon or rectal epithelial layer triggered by different factors, such as genetics, diet, and environment (Armaghany et al. 2012). Alongside, a number of studies have found that gut microbiota has a profound effect on human health and diseases and may also contribute to CRC causation or progression (Jahani-Sherafat et al. 2018). It is strengthened by the fact that the human gut does not have a uniform distribution of microbiota as the distal part of the large intestine dominates the prior part in terms of bacterial load. Studies reported more cases of cancer in the distal part, providing a hint that microbiota has a role in the development or progression of CRC (Dieterich et al. 2018). As colon and rectum regions have the greatest exposure to microbiota, the implication of gut microbiota in the development of CRC is an active area of research.

Bacteria live in a symbiotic relationship in the gut where the body provides them food and shelter, in turn bacteria produce important nutrients such as vitamin B12, which the human body cannot synthesize, and short-chain fatty acids (SCFA) that are important for gut homeostasis and serve as an energy source for intestinal cells (Fang et al. 2017; Parada Venegas et al. 2019). Any change in diet could imbalance microbial composition, known as dysbiosis. Dysbiosis leads to disturbed host–microbiota relationship which could drive the development of CRC (Vipperla and O’Keefe 2016). Recent evidences have proposed the role of some bacteria, such as Fusobacterium nucleatum, Streptococcus bovis, Helicobacter pylori, Bacteroides fragilis, and Clostridium septicum, in carcinogenesis of CRC (Chew and Lubowski 2001a; Shmuely et al. 2001; Abdulamir et al. 2011; Ahn et al. 2013; Boleij et al. 2015). These bacteria are in increased abundance in CRC patients as compared to healthy individuals and can cause inflammation and DNA damage which can lead to the development of cancer (Dahmus et al. 2018). These bacteria produce toxic metabolites, such as secondary bile salts from primary bile salts, hydrogen sulfide, trimethylamine-N-oxide (TMAO) from choline, indoxyl sulfate from amino acid tryptophan, and many more which are likely to promote inflammation and prolonged inflammation can develop into cancer.

The potential role of several gut bacterial metabolites through pro-inflammatory pathways has been reported recently. For instance, Escherichia coli-derived metabolite colibactin has been reported to cause DNA alkylation and induce double-strand breaks in DNA. Inability to repair double-strand breaks can lead to genomic instability and the development of CRC (Research 2020). P-cresol sulfate (PCS) is another genotoxic metabolite secreted by bacteria that influence cell-cycle kinetics and induce DNA damage in colonic epithelial cells (Andriamihaja et al. 2015; Al Hinai et al. 2018). Besides genetic factors and environmental factors, the contribution of bacterial metabolites is gaining more attention. As bacteria convert dietary intake into metabolic by-products thereby, diet has a significant contribution to metabolites secreted by bacteria. In this review, we will discuss the recent studies that focus on the role of microbiota and microbial metabolites in the carcinogenesis of CRC. We illustrate the intricate relationship between microbial metabolic products and the development of CRC.

Gut microbiota, inflammation and CRC

Gut microbiome is a complex territory of microorganisms. The number of microorganisms present in human gut is one to threefold higher than the number of human cells. The gut microbiota consists of bacteria, fungi, archaea and protozoa. Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria are most abundantly present phyla of human intestine. Any change in diet or environment can trigger the shifting of the protective microflora to pathogenic, known as dysbiosis, plays an important role in progression of colorectal cancer (Gao et al. 2015). Dysbiosis in gut microbiota may cause inflammation, dysplasia and finally, colorectal cancer (Solé et al. 2021). Pathogenic symbiont bacteria also cause localized inflammation in normal tissue of colon and promote the genotoxicity of intestinal epithelial cells and enhance the progression of CRC (Chen et al. 2017). There are studies which show that there is a difference in mucosa-associated bacterial microbiota of CRC patients and normal healthy person. Researchers found out that cancerous tissue has lower microbial diversity than normal non-cancerous tissue (Chen et al. 2012; Ai et al. 2019). A study shows that dominating phylum in healthy subjects is Proteobacteria, while in CRC patients, it is Firmicutes which is present more abundantly (Gao et al. 2015). This study also shows that Lactococcus and Fusobacterium were present in higher abundance and Pseudomonas and Escherichia–Shigella in lower abundance in cancerous tissue samples as compared to adjacent non-cancerous normal tissue samples. There are two strategies to study the colorectal cancer-associated microbiome. One is testing the effect of these specific bacteria by inducing them in animal model such as mice and another is studying the difference of gut microbiota between the healthy control and colorectal cancer patients in humans. There is more research which focuses on studying the difference between gut microbiota. Possible roles of gut microbiota in CRC causation are shown in Fig. 1.

Fig. 1.

Possible roles of gut microbiota in CRC causation. Gut microbiota can promote colorectal cancer progression by involvement of different mechanisms, such as loss of epithelial barrier function, increased pathogenic abundance, microbial dysbiosis and altered metabolism. The resulted dysregulated immune function may release cytokines and lead to cell proliferation. The genotoxic effects of pathogenic bacteria produce metabolites that leads to genetic mutations and chronic inflammation which causes colorectal cancer. ROS reactive oxygen species, IL interleukin, TNF tumor necrosis factor, H2S hydrogen sulfide

Intestinal epithelial cells (IEC) form a physical barrier which separates the gut microbiota from internal intestinal tissue. The main function of intestinal epithelial cells is to form an effective surface barrier which protects the gut from external environment and from invasion of symbiotic bacteria, maintain the intake of nutrients from lumen and it also maintains homeostasis between the gut and environment. Intestinal layer is made up from mucus layer and tight junctions. Epithelial cells are connected together through tight junctions (Capaldo et al. 2017). Mucus layers contain a huge mass of glycoprotein mucus. The function of intestinal barrier is also depending upon gut microbiota and it also contributes to gut homeostasis (Jakobsson et al. 2015). Although the mechanism has not been absolutely elucidated but lipopolysaccharides concentration was found significantly increased in case of colorectal cancer tissues which could signify the CRC chronic inflammation, progression and metastasis. Due to inflammation, colonic epithelial cells become incapable of constructing an efficient surface barrier contrary to bacteria and their metabolites. Resultantly, bacteria invade easily due to loss of barrier function and induce tumorigenesis by promoting inflammation (Gil-Cardoso et al. 2016).

Gut microbiota-derived metabolites in inflammation and CRC

The gut microbiota synthesize a large variety of metabolites or bioactive compounds that contribute to prompting of diseases or normal physiology (Fan et al. 2015). These metabolites are produced from the endogenous compounds that are manufactured by host and microorganisms in addition to the anaerobic fermentation of exogenous undigested dietary components that finally go to large intestine (Rooks and Garrett 2016). The composition of metabolites of the gut microbiota contains volatile small molecules, lipids, proteins and peptides, sugars, secondary bile products or terpenoids, biogenic amines, oligosaccharides, glycolipids, organic acids and amino acids (Wang and Zhao 2018), that could maintain the inflammatory conditions of host (Feng et al. 2018). Several studies have shown the association of metabolites of gut microbiota with inflammatory diseases. Moreover, many reports have emphasized the role of gut metabolites in inflammatory diseases’ pathogenesis for instance obesity, type 2 and type 1 diabetes mellitus (DM), and asthma (Knip and Siljander 2016; Meijnikman et al. 2018). Moreover, gut microbial metabolites have been shown to have a connection to colorectal tumorigenesis. There are many metabolic biomarkers which are reported in CRC (Dalal et al. 2020). Gut microbiota extract energy and generate a number of metabolites that affect the host physiology by decomposing different dietary residues in the intestinal tract. In animal models, early studies showed that gut microbiota can play a role in CRC development by producing microbial metabolites that interfere with the host immune system and cause the release of genotoxic virulence factors (Kostic et al. 2013; Rubinstein et al. 2013; Zackular et al. 2013; Cipe et al. 2015). Gut microbiota-produced metabolites are more easily translocated through the mucosal membrane, modulating cancer tolerance and progression. Increased levels of gut microbiota-derived secondary bile acids, especially deoxycholic acid (DCA), have been linked to the production of CRC (Cross et al. 2014; Zeng et al. 2019). But on the other hand, tumorigenesis is aided by the reduction of certain helpful microbial metabolites, such as butyrate (Wang et al. 2019). SCFAs, secondary bile acids, polyamines, indoles, methylamines, polyphenolics, vitamins, and others have all been identified as gut microbiota's unique metabolites in recent decades (Yan et al. 2016).

Short-chain fatty acids (SCFAs)

The most important metabolites majorly found in the gut microbiota are short-chain fatty acids (SCFAs). The resistant starches and non-absorbable dietary fibers on fermentation by the members of the intestinal microbial community generate propionate, butyrate, and acetate that are not possible to break via host metabolism (Koh et al. 2016). Moreover, SCFAs show anti-inflammatory effects for providing energy to the host by attaching to G protein-coupled receptor 43 (GPR43) (Maslowski et al. 2009), which is expressed in cells of immune system along with the macrophages (Sivaprakasam et al. 2016). Various studies have revealed that SCFAs aid in maintaining a good balance between inflammation and intestinal immunity (Koh et al. 2016; Schulthess et al. 2019). Among SCFA, butyrate plays an important role in antagonizing colonic inflammation and its chief producers are identified as Firmicutes, Clostridia, Eubacterium and Ruminococcaceae (Ohira et al. 2017). Furthermore, the butyrate can display an anti-inflammatory effect partly via the suppression of NF-kB (Lührs et al. 2002), which is a transcription factor that maintains the innate immune and inflammatory responses. Also, it strongly prevents the signaling of interferon-gamma (IFN-g) for enhanced inflammation (Klampfer et al. 2003) and inhibits colon inflammation by targeting peroxisome proliferator-activated receptor-g (PPARg) (Lührs et al. 2002).

Butyrate has been the most researched SCFA, and it has been proposed that it can protect against CRC (McNabney and Henagan 2017). Decrease of butyrate-producing bacterial species and lower fecal butyrate levels have been linked to colon tumorigenesis in several clinical trials, indicating that SCFAs could have anti-carcinogenic properties (Chen et al. 2013). Butyrate prevents tumorigenesis in CRC by modulating the translation of tumor-suppressor genes by specifically inhibiting the function of histone deacetylases (HDACs). It also has an impact by alternate mechanisms, such as cancer cell metabolic rewiring and stimulation of G protein-coupled receptors (GPCRs) signaling mechanisms, which leads to tumor cells’ apoptosis and anti-inflammatory response (O’Keefe 2016). Furthermore, butyrate was shown to induce expression of cell-cycle regulators like p21 and p27, as well as pro-apoptotic genes like FAS, in CRC cell lines by inducing histone acetylation, inhibiting proliferation and promoting apoptosis (Wilson et al. 2010). Butyrate suppresses CRC production by modulating tumor metabolism in addition to epigenetic modifications. SCFAs are thought to have a preventative impact on CRC based on recent data. More research is needed to fully comprehend its role in mediating interactions between the colon epithelium and the gut microbiota during carcinogenesis.

Long-chain fatty acids (LCFAs)

Long-chain fatty acids (LCFAs) have extensively been utilized in dietary supplements that contain fish oils and linoleic acid comprising omega-6 and omega-3 fatty acids (FA). They are considered as vital components which are obtained from diet as they cannot be synthesized by mammals and metabolized to form mediators of bioactive lipid which appears to regulate inflammation via reduction of inflammatory cytokines (Prasad and Bondy 2019). Numerous hydroxy FA, oxy FA, and conjugated linoleic acids (CLAs) are produced in the intestine by commensal bacteria, such as Bacteroides thetaiotaomicron, Enterococcus faecalis, and Lactobacillus plantarum (Kishino et al. 2013). Moreover, LCFAs include palmitic (C16:0), margaric (C17:0), stearic (C18:0), arachidic (C20:0), and behemic (C22:0) acids that are mainly found in vegetable oils, and the major ones are palmitate and stearate which have vital role in CRC. Various studies have been shown that LCFAs have anti-neoplastic properties, but proof for relation between LCFAs and CRC remains inconsistent (Kantor et al. 2014). Many case–control studies (Neoptolemos et al. 1988; Slattery et al. 1997) on palmitic acid and stearic acid have been done to investigate the effect of LCFAs concentration on CRC, but the results seem consistent with no association between LCFAs and CRC.

Tryptophan

The metabolism of amino acid provides large amounts of bioactive metabolites to the host by the microbiota of intestine. Tryptophan is a vital amino acid which is catalyzed through tryptophanase (specially encoded in genome of microbia) and converts it into indole (Devlin et al. 2016). The role of tryptophan which is obtained from diet in maintenance of inflammatory response was found by Rothhammer et al. (2016). Tryptophan is a common constituent of protein-based foods, such as eggs, fish, meat and cheese. Following the consumption of such foods, bacteria, such as Clostridium sporogenes, Lactobacillus etc., in the gut catabolize tryptophan into a range of indole derivatives (such as indole-3-acetic acid, indoxyl-3-sulfate, indole-3-propionic acid and indole-3-aldehyde) that are ligands for the aryl hydrocarbon receptor (AHR). Activating AHR in innate lymphoid cells (ILC) and gut-resident T-cells increases the generation of IL-22 that helps in protection against inflammation in the colon (colitis). Also, the indole metabolites can reduce the detrimental LPS-mediated liver inflammation via altering the NLRP3 route (Beaumont et al. 2018). In an experiment on autoimmune encephalomyelitis on a preclinical model of multiple sclerosis, the metabolites of indole lessened central nervous system inflammation by decreasing pathogenic activity of astrocytes (Rothhammer et al. 2018). Moreover, in in vivo, tryptophan metabolites not only suppress inflammation, but also fix the abdominal wall structure and associate with beneficial bacteria in the intestine, potentially slowing CRC progression. Tryptophan metabolism plays an important role in stopping the growth of CRC, so elucidating the connection between tryptophan metabolism and CRC would aid future research (Zhang et al. 2019).

Trimethylamine N-oxide (TMAO)

A significant microbe-dependent metabolite of gut produced from l-carnitine, betaine and dietary choline is Trimethylamine N-oxide (TMAO) that is metabolized to trimethylamine (TMA) by metabolism of gut microbiota, further converting into TMAO via hepatic flavin monooxygenases (FMOs) (Koeth et al. 2013). Recently, studies have revealed the roles of TMAO in activating inflammatory routes that lead to atherosclerosis (AS) (Chen et al. 2017) which is a chronic inflammatory disease, and its inflammation is continuously induced during the progression of the disease (Tuttolomondo et al. 2012). Moreover, studies have also displayed that enhanced level of TMAO prompted the initiation of NF-kappa B (NF-κB) route and improved the pro-inflammatory genes’ expression involving chemokines, adhesion molecules and inflammatory cytokines (Chen et al. 2017). However, the mechanism describing the role of TMAO in AS participation is still in the exploratory stage. Earlier observational researches have found a connection between CRC and TMAO (Guertin et al. 2017). Microbial genes involved in trimethylamine synthesis were also shown to be over-represented in CRC cohorts (Thomas et al. 2019). Bae et al. (2014) for the first time showed the association of plasma TMAO level with CRC risk among women in the US. Further, various clinical studies positively demonstrated an increased serum/plasma TMAO level among CRC patients, compared to healthy controls, rendering TMAO a potential prognostic marker for CRC (Bae et al. 2014; Liu et al. 2017). Also, urine TMAO can be used as a predictor of CRC which has been shown by research studies (Kim et al. 2010). On the other hand, a few reports have shown that TMAO has a protective effect in the process of CRC (Lunn et al. 2007; Georgescauld et al. 2009). Therefore, the effect of TMAO on cancer needs further study.

Colibactin

Colibactin is a secondary metabolic compound encoded by polyketide synthase (pks) genomic island which is harbored by phylogenetic group B2 of gut bacteria E. coli (Nougayrède et al. 2006). It is a genotoxic compound which induces double-strand DNA breaks by activating DNA-damage signaling and cell-cycle arrest (Nougayrède et al. 2006). Recently in 2020, Dziubańska-Kusibab et al. induced DNA damage by colibactin on colorectal adenocarcinoma cell line, Caco-2 by infecting Caco-2 cells with pks⁺ E. coli and pks⁻ E. coli (Dziubańska-Kusibab et al. 2020). This study finds enriched DNA mutations in pks⁺ E. coli-infected Caco-2 cells which show a strong mutational role of colibactin in colorectal cancer.

Taurine

Taurine is an amino sulfonic acid whose amounts are changed by means of commensal bacteria de-conjugation of majorly bile acids (Yao et al. 2018), generating enhanced levels of luminal taurine. It also appears to prompt the signaling of NLRP6 inflammasome, thereby reducing inflammatory bowel diseases (Levy et al. 2015). It is also considered as a protective metabolite in diabetes and colorectal cancer. In multiple cohorts, metabolomics research indicated a connection between bile acid dysfunction and CRC. A multi-omics analysis found that CRC high-risk patients had higher fecal concentrations of tumor-promoting DCA (Deoxycholic acid) and higher levels of 7-dehydroxylating bacteria (Ocvirk et al. 2020). Another research found that bile acids like DCA were elevated in patients with numerous polypoid adenomas and intra-mucosal carcinomas (Yachida et al. 2019). DCAs, which are thought to play a role in the growth of CRC, were first discovered in mice in 1940 (Cook et al. 1940). DCA penetration caused single-strand DNA breaks in CRC cells, according to in vitro studies (Powolny et al. 2001). In CRC cells, DCA-induced mitochondrial oxidative stress will trigger NF-Κβ signaling, inhibiting apoptosis and promoting tumor progression (Payne et al. 2007). UDCA and tauroursodeoxycholic acid (TUDCA), in contrast to DCA, have been linked to the prevention of colon tumor growth. UDCA blocked cell-cycle progression in colon cancer cells and decreased the development of colon cancer stem-like cells by regulating intracellular reactive oxygen species (ROS) production (Kim 2017). TUDCA, on the other hand, inhibited NF-Κβ signaling in CRC cells and reduced colitis-associated tumorigenesis in mice treated with AOM/DSS (Kim et al. 2019).

Flavonoids

Flavonoids are polyphenolic metabolites which are fermented and hydrolyzed via colonic microbiota like Bacteroides uniformis and Bacteroides distasonis (Cassidy and Minihane 2017). Various research has described that luteolins (a flavonoid) are strong antioxidants that prevent the mast cells which are engaged in inflammatory responses. Also, a study revealed that an analog of luteolin, i.e. tetramethoxyluteolin prevented the discharge of inflammatory mediators, i.e. tumor necrosis factor (TNF), histamine secretion, and beta-hexosaminidase from mast cells (Weng et al. 2015). Moreover, various evidences have shown that dietary flavonoids are associated with a decreased risk of CRC, therefore flavonoid intake might be an important dietary determinant of CRC risk (Theodoratou et al. 2007). Flavonoids, such as flavonols, flavones and anthocyanidins, may potentially decrease the risk of CRC. It has been shown that high intake of flavonols (such as quercetin) may reduce the risk of colon cancer, and high intake of flavones (such as apigenin) may reduce the risk of rectal cancer (Chang et al. 2018). To further confirm these relations, well-designed prospective cohort studies are needed.

Polyamines

Polyamines have been involved in maintaining the induction of inflammatory bowel diseases such as colitis (Levy et al. 2015). Spermidine is a metabolite which is produced either by host or microbiota or consumed by means of diet that acted on the NLRP6 inflammasome route, consequently impacting microbial composition. It is generated via decarboxylation of amino acids that seems to suppress NLRP6 inflammasome assembly and decreases the levels of colonic IL-18. Dysfunction of polyamine synthesis by the host or gut microbiota could be a factor in the development of CRC. A metabolomics study showed that the host and microbiota are involved in a positive feedback mechanism in which polyamines generated by CRC cells stimulate the growth of bacterial biofilms, and bacteria in biofilms produce polyamines to trigger cancer growth. Polyamines were shown to induce intracellular oxidative stress, which led to DNA damage and accelerated carcinogenesis in a mouse model of CRC (Goodwin et al. 2011). Polyamines also cause oncogenic signaling in CRC cells, resulting in increased spermidine and spermine levels, as well as pAKT and catenin expression, both of which facilitate cell proliferation and tumor metastasis (Wang et al. 2017).

N-Acyl amides

N-Acyl amides are bioactive lipids which play an important role in physiology of mammals. Commendamide/N-acyl-3-hydroxypalmitoylglycine is a metabolite found in human gut microbiome that was recognized through functional metagenomic selection of NF-kB activators. It helps in activating a G protein-coupled receptor G2A/GPR132, connected with atherosclerosis and autoimmunity and bears a resemblance to long-chain N-acyl-amides (Cohen et al. 2015).

Involvement of microbial metabolites and their producers in inflammation and CRC is shown in Table 1.

Table 1.

Involvement of microbial metabolites and their producers in inflammation and CRC

S. no	Class of metabolites	Metabolite	Producers	References
1	Short-chain fatty acids (SCFAs)	Acetate	Blautia hydrogenotrophica, Methanobrevibacter smithii, Ruminococcus gnavus	Georgescauld et al. (2009), Gil-Cardoso et al. (2016), Giovannucci (2004)
		Propionate	Ruminococcus obeum, Coprococcus catus, Roseburia inulinivorans	Gil-Cardoso et al. (2016), Giovannucci (2004), Goodwin et al. (2011)
		Butyrate	Ruminococcaceae, Eubacterium, Clostridia, and Firmicutes Roseburia inulinivorans, Eubacterium rectale, Anaerostipes spp.	Giovannucci (2004), Goodwin et al. (2011), Guertin et al. (2017)
2	Long-chain fatty acids (LCFAs)	Conjugated linoleic acids (CLAs) Oxy Fatty acids Hydroxy Fatty acids	Lactobacillus plantarum, Enterococcus faecalis, and Bacteroides thetaiotaomicron	Dieterich et al. (2018)
3	Tryptophan	Indole	Desulfitobacterium hafniense, Clostridium malenomenatum, Clostridium limosum	Gur et al. (2015), Hartwich et al. (2001), Howarth et al. (2008)
		Indoleacetic acid (IAA)	Bacteroides fragilis, Clostridium difficile, Clostridium perfringens, Clostridium putrefaciens	Hartwich et al. (2001), Howarth et al. (2008), Huycke et al. (2002)
		Indoleacrylic acid (IA)	Clostridium sporogenes, Peptostreptococcus russellii, Peptostreptococcus anaerobius, Peptostreptococcus stomatis	Jahani-Sherafat et al. (2018), Jakobsson et al. (2015)
		Indolelactic acid (ILA)	Bacteroides fragilis, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus murinus	Huycke et al. (2002), Jahani-Sherafat et al. (2018), Johnson et al. (2015), Kantor et al. (2014)
		Indolepropionic acid (IPA)	Clostridium sporogenes, Clostridium botulinum, Clostridium caloritolerans	Hartwich et al. (2001), Jakobsson et al. (2015)
		Skatole	Clostridium drakei, Clostridium scatologenes, Lactobacillus spp.	Kasai et al. (2016), Kasper et al. (2020)
		Indolealdehyde (IAld)	Lactobacillus reuteri, Lactobacillus murinus	Kantor et al. (2014)
		Tryptamine	Clostridium sporogenes, Ruminococcus gnavus	Kato et al. (2013)
4	Dietary choline, betaine and l-carnitine	TMAO	Firmicutes and Proteobacteria phyla	Kennedy et al. (2005)
5	Amino sulfonic acid	Taurine	Bilophila wadsworthia	Kim et al. (2017)
6	Flavonoids	Luteolin	Bacteroides distasonis and Bacteroides uniformis	Kim et al. (2017)
7	Polyamines	Spermidine	several members of the Lactobacillus genus	Fischbach and Sonnenburg (2011)
8	N-Acyl amides	Commendamide	Bacteroides vulgatus, Bacteroides dorei, and Bacteroides massiliensis	Gausachs et al. (2017)

Possible role of bacteria in CRC causation

Fusobacterium nucleatum

Fusobacterium nucleatum is a Gram-negative, oral commensal, obligate anaerobic, opportunistic bacteria in humans. It has a role in periodontal disease, cardiovascular diseases, rheumatoid arthritis, IBD and CRC (Chen et al. 2017). There are so many studies which show higher abundance of Fusobacterium nucleatum in colorectal cancer tissues as compared to normal tissue (Kostic et al. 2012; McCoy et al. 2013; Kostic et al. 2013; Viljoen et al. 2015; Kasai et al. 2016; Ye et al. 2017; King et al. 2020). Shorter survival rate is reported in colorectal cancer patients, due to colonization of Fusobacterium nucleatum (Alhinai et al. 2019). In cell line, Fusobacterium nucleatum increase the proliferation of colorectal cancer cell lines but not in non-cancerous cell lines (Rubinstein et al. 2013). Recently in 2020, Kasper et al., done both targeted and untargeted metabolomics to check the effect of viable Fusobacterium nucleatum on metabolic processes in tumor spheroid microenvironment (Kasper et al. 2020). Study reported the higher concentration of polyamines, i.e. N¹,N¹²-diacetylspermine and N¹-acetylspermidine. Amount of these metabolites increased markedly after 24–48 h of infection with F. nucleatum. As we have discussed the role of polyamines in earlier section, polyamines are responsible for oxidative stress which may further lead to DNA damage and carcinogenesis. A recent study suggests that F. nucleatum is also correlated with MSI (microsatellite instability) and CIMP (CpG Island Methylator Phenotype)-positive tumors (Lee et al. 2018). Administration of F. nucleatum in APC^Min/+ mice caused increase in number and size of tumors, diarrhea, enlarged spleen and gut, ascites and reduced survival of APC^Min/+ mice and it also increases the level of inflammatory cytokines in serum as compared to normal uninfected mice. F. nucleatum infections also generate miRNA expression (Yang et al. 2017). Abundance of F. nucleatum in colorectal cancer shows increased levels of IL-17A and TNF- α which are tumor-promoting cytokines (Ye et al. 2017) by activating NF-κB signaling pathway. FadA adhesion protein from F. nucleatum activates β-catenin signaling pathway in intestinal epithelial cells through binding with E-cadherin (Rubinstein et al. 2013, 2019) and results in upregulation of pro-inflammatory and oncogenic gene expression in colorectal cancer cases. Fap2 protein of F. nucleatum interacts with TIGIT (immune inhibitory receptor) and inhibits T cell activation and NK cell-meditated killing of tumor cells (Gur et al. 2015). So, F. nucleatum plays a crucial role in carcinogenesis of colorectal cancer. So, F. nucleatum is one of the pathogens that could play a crucial role in CRC carcinogenesis.

Escherichia coli

Escherichia coli is a Gram-negative, commensal, facultative anaerobic bacteria in human gut. Normally it is not related to diseases but there are some virulent strains of E. coli which colonize in human gut and promote gut-related diseases due to their pathogenic attributes (Buc et al. 2013). There are so many studies which show higher abundance of E. coli in mucosa of CRC patients as compared to healthy control. E. coli has four phylogenetic groups A, B1, B2 and D. The B2 and D groups contain virulence factors and some strains are correlated with intestinal inflammatory diseases which can promote CRC (Martin et al. 2004; Elliott et al. 2013). CIF (cycle-inhibiting factor), CNF (cytotoxic necrotizing factor), CDT (cytolethal distending toxin) and colibactin are studied more extensively for their effect on CRC. As discussed in earlier section, colibactin is a genotoxic compound produced by polyketide synthase (pks). Colibactin induces double-strand DNA breaks by activating DNA damage signaling and cell-cycle arrest (Dziubańska-Kusibab et al. 2020). CIF (cycle-inhibiting factor) induces cell-cycle arrest by rearranging actin cytoskeleton. CNF induces cell-cycle arrest by activating Rho GTPases and COX-2 (Lucas et al. 2017). CDT possesses DNase activity, so it induces cell-cycle arrest, double-strand DNA breaks and apoptosis. So, E. coli could play a major role in CRC carcinogenesis.

Enterococcus faecalis

Enterococcus faecalis is a facultative anaerobic, Gram-positive, gut commensal bacterium in humans. E. faecalis is closely interrelated with S. bovis. Normally it is not related to diseases but there are some studies which show higher abundance of E. faecalis in colorectal cancer patients stool samples as compared to healthy control (Balamurugan et al. 2008). Studies also show its higher abundance in CRC patient’s tissue samples and adjacent mucosa as compare to normal healthy controls (Zhou et al. 2016), so it confirms the link between CRC and E. faecalis. In 2017, a case of E. faecalis bacteremia was reported, in which patient was found positive for colorectal carcinoma (Amarnani and Rapose 2017). In a study on IL-10 knockout mice, E. faecalis promotes colitis, induces dysplasia and leading to CRC (Lucas et al. 2017). E. faecalis produces metabolites, i.e. extracellular superoxide and hydrogen peroxide which induce DNA damage. When rat is infected with E. faecalis, it induces DNA damage in colonic cells (Huycke et al. 2002). As reactive oxygen species (ROS) can cause chromosomal instability, which can promote CRC. A study was done on mammalian cells to investigate the same which shows that E. faecalis cause chromosomal instability due to ROS which involve COX-2 as its expression was increased just after 2 h of infection (Lucas et al. 2017). So, E. faecalis may have a role in carcinogenesis of colorectal cancer.

Streptococcus bovis/gallolyticus

Streptococcus bovis is a Gram-positive bacterium of humans, mostly associated with urinary tract infection, sepsis, endocarditis and colorectal cancer. There are a lot of studies which show higher abundance of S. bovis in colorectal cancer patients as compared to healthy control, so it confirms the link between CRC and S. bovis (Abdulamir et al. 2010; Boleij et al. 2011). S. bovis antigen activates COX-2 which induces cell angiogenesis and suppresses apoptosis, so it can activate the cancer pathway. Experimentations on AOM-treated rats were done in which rats were infected with S. bovis. S. bovis-infected rats develop polyps while there were not found any polyps in uninfected rates which were also treated with AOM (Biarc et al. 2004). Other studies also support that S. bovis infection in AOM-treated mouse advanced the carcinogenesis of CRC by increasing the expression of pro-inflammatory markers which result in development of hyper-proliferative crypts (Lucas et al. 2017). Abdulamir et al. (2011) studied on mucosa and stool samples of human CRC patients and results show abundance of S. bovis and higher expression Nf-KB and IL-8 mRNA in patient’s samples as compared to healthy control samples. So, all these studies show that S. bovis may have a role in inducing inflammation and promoting carcinogenesis of colorectal cancer but any metabolic pathway is not known.

Bacteroides fragilis

Bacteroides fragilis is a Gram-negative, obligate anaerobic, symbiotic gut bacterium in humans. B. fragilis have 2 subtypes: enterotoxigenic B. fragilis (ETBF) and nontoxigenic B. fragilis (NTBF). ETBF is interconnected with diarrheal disease in humans. ETBF secretes fragilysin or BFT enterotoxin which is encoded by the bft gene (Sears 2001). BFT is responsible for a rapid expression of polyamine—spermine oxidase (SMO), which causes SMO-dependent ROS and DNA damage in HT-29/c1 and T84 colonic epithelial cell lines and B. fragilis-infected mice also show alternation in expression of intestinal SMO (Goodwin et al. 2011). In colonic epithelial cells (CEC), BFT toxin cleaves E-cadherin which is a tumor-suppressor protein. Cleavage of E-cadherin activates Wnt signaling and also some other pro-carcinogenic signaling which results in cell proliferation, barrier disruption, inflammation and subsequently leading to colorectal cancer, as found in murine model study (Dai et al. 2019). B. fragilis colonization has been found more pronounced in pre-cancerous and cancerous lesions as compare to early-stage CRC, suggesting that B. fragilis have a role in early carcinogenesis (Zamani et al. 2019). ETBF induces IL-17-mediated pro-carcinogenic inflammatory response in colonic epithelial cells through STAT3 activation and subsequently induces colitis (Dai et al. 2019). So, B. fragilis may have a role in inducing inflammation and promoting carcinogenesis of colorectal cancer.

Helicobacter pylori

Helicobacter pylori is a Gram-negative bacterium of gastric epithelium in humans. H. pylori is well recognized for inducing chronic inflammation. There are studies which connect H. pylori infection with increasing incidences of CRC (Nam et al. 2017). H. pylori promote production of gastrin which increased the expression of COX-2 and BCL-2. BCL-2 is an anti-apoptotic protein, so it reduced apoptosis (Hartwich et al. 2001). Due to overexpression of gastrin, acid production can be disturbed which can disturb intestinal epithelial barrier. Due to perturbation in intestinal epithelial barrier, CRC-related bacteria, such as E. feacalis and B. fragilis, can get an opportunity to grow and colonize (Tatishchev et al. 2012). There are some theories which state that H. pylori produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) which can cause DNA damage. Effect of H. pylori is strain-dependent; as CagA virulence factor-containing strains are more harmful, patients who contain this strain have more chances of having CRC (Lucas et al. 2017). Further, H. pylori promote the production of many pro-inflammatory cytokines like IL-1, IL-6, IL-8, IL-1, INF- γ and TNF-α (Wessler et al. 2017) which proves its role in inflammation and carcinogenesis of CRC.

Clostridium septicum

Clostridium septicum is a Gram-positive, obligate anaerobic, spore-forming bacterium, normally not found in human gut. C. septicum generate α-toxin which is a harmful and haemolytic virulence factor (Kennedy et al. 2005). There are very rare cases that C. septicum is belonging to bacteraemia. Hypoxic and acidic tumor environment can support the growth of C. septicum (Chew and Lubowski 2001b). The link between C. septicum with carcinogenesis of colorectal cancer is poorly known. A comparative study of patients with C. septicum and Streptococcus gallolyticus bacteraemia shows that both the bacteria are related to colorectal neoplasms (CRN), but mortality rate is higher in case of C. septicum (Corredoira et al. 2017). C. septicum activates mitogen-activated protein kinase (MAPK) signaling pathway, results in production of pro-inflammatory cytokine TNF-α (Chakravorty et al. 2015), so it creates an environment suitable for CRC development. The pathway by which C. septicum activates MAPK signaling is not known, which needs to be studied for further information.

Salmonella enterica

Salmonella enterica is a facultative anaerobic, Gram-negative pathogen in humans and animals. In a study, blood samples from US and Netherlands CRC patients were taken and checked for the presence of antibody against Salmonella flagellin. Results show higher level of antibody against S. enterica in pre-cancerous and cancerous cases than in healthy controls (Kato et al. 2013). So, it supports that there is a possible link between S. enterica and colorectal cancer. S. enterica produces AvrA effector protein, which is a pathogenic product. A study was done for checking the effect of AvrA in which mice were infected with AvrA-sufficient Salmonella and AvrA-deficient Salmonella. Results show 100% tumor occurrence in AvrA-infected group, 56.3% in AvrA-deficient Salmonella, while it was only 51.4% in AOM/DSS group (Lu et al. 2014). In another study, administration of Salmonella AvrA expressing strain activates STAT3 signaling pathway (Lu et al. 2016), thus promote the carcinogenesis of CRC.

Peptostreptococcus anaerobius

Peptostreptococcus anaerobius is a Gram-positive, anaerobic bacterium of gut and oral part of humans. P. anaerobius is normally harmless but it may be harmful in immunocompromised conditions (Sun and Kato 2016). There is a higher abundance of P. anaerobius in colorectal cancer patient’s stool samples as compared to healthy controls. Administration of P. anaerobius in mice caused increase in intestinal dysplasia (Tsoi et al. 2017). P. anaerobius interact with TLR4 and TLR2 on colonic epithelial cells and increase ROS level which enhance cholesterol synthesis and cell proliferation (Tsoi et al. 2017). Tsoi et al., analyzed mechanistic cellular pathways after co-culturing of P. anaerobius with 3 different colonic cell lines NCM460, HT-29, and Caco-2 (Tsoi et al. 2017). Results show enrichment of 8 pathways, named TLR signaling, cholesterol biosynthesis, AMP-activated protein kinase signaling, butanoate metabolism, CRC, cell cycle, peroxisome signaling pathways and fatty acid degradation pathway, in all 3 cell lines after co-culturing with P. anaerobius. Results of this study suggest that P. anaerobius promote carcinogenesis by enhancing the expression of oncogenic genes and their signaling pathways. In a study, Apc^Min/+ mice were infected with P. anaerobius which activates Nf-KB signaling, results in pro-inflammatory response as level of cytokines, such as IL-10 and IFN-γ, were increased in P. anaerobius-infected Apc^Min/+ mice (Long et al. 2019). So, P. anaerobius may have a role in enhancing CRC progression.

Role of microbial metabolism of dietary intake in inflammation and CRC

Diet modulates the interactions between host gut microbiota and metabolites precursors to maintain gut health (Forgie et al. 2019; Vernocchi et al. 2020). Researchers exploited the paired microbiome data and discovered the strong coupling of gut microbiota dysbiosis and cancer epidemiology (Wirbel et al. 2019). Earlier studies reported that native Africans are less susceptible to colorectal cancer than African American for the difference in their staple food (O’Keefe et al. 2015). There are evidences to support the mechanism of gradual dysbiosis in gut microbiota influenced by dietary intake and genetic mutations in cancer critical genes, for example, APC, Kirsten-RAS (KRAS) and P53 genes (Gausachs et al. 2017). There are three major macronutrients from diet are carbohydrates, protein and fat fermented by gut microbiota to generate a variety of gut metabolites through different mechanisms. Although reports proposed that plenty of nutrients from protein and fat are inflammatory and mount the neoplastic changes on mucosal cells. Short-chain fatty acids (SCFAs), i.e. butyrate, propionate and acetate are the key fermented products of microbial metabolism for non-digestible dietary carbohydrates (Prasad and Bondy 2019). Butyrate is the energy source for colonocytes (Donohoe et al. 2011). While Bacteroidetes converts simple sugars into SCFAs and hydrogen, Clostridium converts organic acids into additional SCFAs. Abnormal levels of acetate, butyrate, propionate and succinate were found in CRC patients. Fumarate and glucose showed decreased level and lactate was found at increased level of concentration in tissue samples of CRC patients in comparison of healthy individuals. SCFAs can lower the acidity in the stomach to stimulate the blood circulation and proliferation of epithelial cells. At the molecular level, SCFAs regulate the epigenetic modifications, such as histone hyper-acetylation and apoptosis, to reduce the risk of inflammation in CRC (Fischbach and Sonnenburg 2011; den Besten et al. 2013; Rowland et al. 2018).

Colonic bacteria also utilized the glycoprotein in the mucosal layer and used dietary fiber to produce SCFAs as their source of energy. Low-dietary fiber diet can cause the disruption of mucosal barrier, it exposes the mucosal layer to variety of inflammatory and toxic agents. Moreover, high-dietary fiber diet has shown better response to prebiotics, non-fermented fiber absorbs water content, normalizes bowel movements and discourages the interaction of potential carcinogens, i.e. harmful metabolites with mucosal layer (Howarth et al. 2008; Maino Vieytes et al. 2019). Certain bacterial classes including E. coli, Klebsiella, Enterobacteriaceae and Clostridium are involved in fermentation of sulfur-containing amino acids, whereas Clostridium and Bacteroides ferment aromatic amino acids (Diether and Willing 2019). Bacteria also produce metabolites like amines, ammonia, hydrogen sulfide, indoles and phenols which were all linked with CRC carcinogenesis. These metabolites cause DNA damage and inflammation by modification in purine and pyrimidine metabolism intermediates. One of the good examples is Trimethylamine N-oxide (TMAO) generated from choline, betaine and cartinine via l-Cartinine metabolism in red meat by gut microbiota that contribute to inflammation (Chan et al. 2019). The host and bacterial polyamine metabolites cooperatively enhance biofilm formation and cellular proliferation (Johnson et al. 2015). It will create the conditions for oncogenic transformations in colonic epithelial cells. In the settings of lipid metabolism, microbial-derived metabolism can boost the lipase enzyme activity followed up with the conversion of primary bile acids to secondary bile acids in the colon. For example, Bacteroides intestinalis de-conjugate and dehydrate the primary bile acids to convert into secondary bile acids, it can also produce lithocholic and deoxycholic acids, which are cytotoxic with cancer causing anti-apoptotic properties to the colonic epithelial cells. Dietary intake of saturated fats increases the production of bile acids and risks of CRC. The elevated levels of these metabolites can be pro-inflammatory as they generate reactive oxygen and nitrogen species, it also activates NF-kB in intestinal epithelial cells (Ocvirk and O’Keefe 2017; Liu et al. 2020). Dietary intake of low folate and high alcohol consumption is also included in risk factors for CRC. Studies confirm the major risk ratio to rectum, minor in the distal and then proximal colon. Gut bacteria metabolize alcohol to produce acetaldehyde, which in turn is responsible for the progression of alcohol-associated CRC. Other related metabolites promote carcinogenesis causing biochemical, epigenetic, immunological and genetic anomalies ultimately causing chronic inflammation (Giovannucci 2004).

Undoubtedly, diet is an important environmental factor which affects the overall host cellular metabolism (Fig. 2). The dietary intake has profound effects on the development of CRC. It can be correlated with dynamic behavior of environmental factors and inherent properties of genetic alterations. Gut microbiota acts as a community in which microbial interactions generate a metabolic phenotype which help in colonic health and function (Oliphant and Allen-Vercoe 2019; Seesaha et al. 2020). By enlightening the role of gut microbiota in maintaining the nutrients and further effect on host biology can lead to enhanced dietary recommendations and disease management in colorectal cancer.

Fig. 2.

Microbial metabolism of dietary intake. Human gut colonizes myriad of bacteria based on dietary food intake. The nutrients from dietary food items include dietary fiber, bile acids, lipids, polyamines etc. used by gut microbial precursors to produce bacterial metabolites, such as Colibactin, TMAO, secondary bile acids, etc. Diet may preferably change the gut microbial diversity which consequently alter the gut bacterial-derived metabolites. Perturbation of gut microbiota and metabolites induces CRC inflammation. TMAO Trimethylamine N-oxide, CRC colorectal cancer

Conclusion

Numerous studies have reported the alteration of gut microbiota during CRC progression and the numbers of these bacteria, such as F. nucleatum, E. coli, S. enterica, etc., have been implicated in the causation of the disease. Even after a series of studies on linking gut microbiota with CRC progression, underlying mechanisms are not known and more efforts and investigations will be required to support the hypothesis that gut bacteria may be termed as causative agents for CRC progression. In this review, rather than implicating bacteria with CRC, we put effort to mention those studies which have linked gut-microbiota-derived metabolites in the causation and progression of CRC. Recent studies have shed light on the production of colibactin by gut-dwelling E. coli and based on similar kinds of studies, we reckon that microbial metabolites, such SCFA, TMAO, etc., may have role in the progression of CRC. These metabolites may be produced by different gut-dwelling microbial species; hence, it is recommended that future studies should try to shift their focus from bacteria to their metabolites while studying the link between bacteria and CRC. This approach might provide some evidences to explain the underlying mechanisms which are involved in the causation of CRC by bacterial metabolites (Mager et al. 2020).

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