Gut Microbiota in Colorectal Cancer: Biological Role and Therapeutic Opportunities

Simple Summary Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths worldwide. In this review article, we examine the role of gut microbiota in the development and progression of CRC. We also examine the use of gut microbiota as a biomarker to predict CRC and its possible therapeutic outcome. Abstract Colorectal cancer (CRC) is the second-leading cause of cancer-related deaths worldwide. While CRC is thought to be an interplay between genetic and environmental factors, several lines of evidence suggest the involvement of gut microbiota in promoting inflammation and tumor progression. Gut microbiota refer to the ~40 trillion microorganisms that inhabit the human gut. Advances in next-generation sequencing technologies and metagenomics have provided new insights into the gut microbial ecology and have helped in linking gut microbiota to CRC. Many studies carried out in humans and animal models have emphasized the role of certain gut bacteria, such as Fusobacterium nucleatum, enterotoxigenic Bacteroides fragilis, and colibactin-producing Escherichia coli, in the onset and progression of CRC. Metagenomic studies have opened up new avenues for the application of gut microbiota in the diagnosis, prevention, and treatment of CRC. This review article summarizes the role of gut microbiota in CRC development and its use as a biomarker to predict the disease and its potential therapeutic applications.


Introduction
Colorectal cancer (CRC) is one of the major causes of cancer mortality in humans, accounting for 9.4% of deaths worldwide in 2020 [1]. It is the third most common cancer in males and the second most common cancer in females. The incidence of CRC has increased in recent years. In 2020, there were 1.9 million new cases and 0.9 million deaths worldwide [1,2]. It has been estimated that by 2030, there would be about 2.2 million cases and 1.1 million CRC deaths per year [3]. The incidence of CRC has been rising rapidly in developing countries, whereas stabilizing or decreasing trends have been observed in developed countries where rates remain high [4]. Mutations in many genes have been identified that are associated with CRC, and some with high frequencies of occurence are listed in Table 1. The first gene mutation identified was the adenomatous polyposis coli (APC) gene [5]. The initial formation of polyps occurs in response to mutations in tumorsuppressor genes, such as APC, which is a component of the Wnt/β-catenin pathway and controls cell proliferation. Additionally, mutations may also take place in genes involved in DNA repair, such as hMSH2, contributing to colorectal tumorigenesis. Some of these genetic alterations are also inherited. The majority of CRC cases are sporadic [6], and heritable CRC accounts for only 12-35% of cases [7]. CRC development is multifactorial and influenced by host genetic and environmental factors. CRC development usually takes decades, during which mutations are accumulated in oncogenes and tumor suppressor genes [8]. The sequence of genetic changes leading to CRC is commonly referred to as the adenoma-carcinoma sequence [9]. Tumors are more frequent in the distal region than in the proximal region of the large intestine [10] reflecting differences in the luminal environments of these two regions [11].
In this review, we discuss the relationship between gut microbiota and CRC development. We restrict our discussion mainly to bacteria, which have gained much attention in recent years due to their involvement in human health and diseases. We also discuss the potential role of gut microbiota in the diagnosis, treatment, and prevention of CRC.

Human Gut Microbiota
The human gut contains about 40 trillion microorganisms that constitute the gastrointestinal microbiota [12]. The number of microorganisms in the human gut is about three times greater than the total number of cells in the human body [12]. The human gut microbiome is sometimes referred to as the "forgotten organ" [13,14]. The human colon is considered to be one of the most densely populated microbial ecosystems [12,15]. The colorectum harbors about 30 trillion bacteria that constantly crosstalk with the intestinal epithelium, immunological cells, and mucosal barrier [16,17]. The human gut microbiome represents a complex microecosystem, the composition of which varies between individuals. The human gut microbial genome (microbiome) is at least two orders larger than the human genome [16]. The gut microbiota is acquired during the initial stages of life from the mother. The colonization of the gut by microorganisms is affected by the type of delivery [18] and the type of diet [19]. Babies born through vaginal delivery are exposed to vaginal microbes, such as Lactobacillus, while babies born through C-section are exposed to skin microbes, such as Staphylococcus and Corynebacterium. Breast milk and formula milk also affect the types of microorganisms that colonize the gut. According to a study published in 2018, the composition and structure of gut microbiota are predominantly shaped by environmental factors and only 1.9% of the gut microbiome is heritable [20].
The gut microbiota is critical for normal gut physiology, including digestion, biosynthesis of vitamins, generation of heat, gut immunity, and maintenance of gut homeostasis [15,[29][30][31][32][33][34]. It is estimated that microbial metabolism results in the generation of about 70% of body heat at rest [35,36], which is important for maintaining a stable gut and body

Enterococcus faecalis
Enterococcus faecalis is a commensal Gram-positive, facultative anaerobic bacterium. Studies have confirmed an increased abundance of E. faecalis in the feces of patients with CRC [123,124]. However, the role of E. faecalis in CRC development remains controversial [194]. Many studies have suggested its ability to generate reactive oxygen species (ROS) and extracellular superoxide that can damage colonic epithelial cell DNA, leading to mutations and CRC [124,[195][196][197]. E. faecalis also produces metalloprotease, which can affect the intestinal epithelial barrier and induce inflammation [198]. Contrary to this, other studies indicate that E. faecalis is an important probiotic microorganism [199] and may have a role in CRC prevention [200][201][202].

Streptococcus bovis/gallolyticus
Streptococcus bovis/gallolyticus is a Gram-positive bacterium associated with endocarditis [203]. The first case of endocarditis-associated CRC was reported in 1951 [204]. Studies have confirmed the association of S. bovis/gallolyticus infection with CRC [205][206][207][208][209] and the prevalence of S. bovis/gallolyticus in CRC tissues [210]. The exact mechanism by which S. bovis/gallolyticus induces carcinogenesis is still to be characterized, but studies have indicated its involvement during the early stages of colorectal carcinogenesis [205,206,208,211], and therefore, it may serve as an early marker for CRC screening. S. gallolyticus uses Pil3 pilus for adhesion to and translocation across colonic epithelial cells [212,213]. Additionally, bacterial type VII secretion systems (T7SS) may mediate the interactions of S. gallolyticus with host cells and are important for its virulence [214]. S. gallolyticus probably caused CRC development by upregulating β-catenin levels and inducing inflammation via IL-1, IL-8, and COX-2 [210]. Oral gavage with S. bovis/gallolyticus has been found to increase the tumor burden in azoxymethane (AOM) and dextran sulfate sodium (DSS) mouse models of tumorigenesis [215,216].

Helicobacter pylori
Helicobacter pylori is a Gram-negative, microaerophilic bacterium that is found in the stomach and is responsible for peptic ulcers, chronic gastritis, mucosa-associated lymphoid  [237], upregulates MAP kinase and ERK1/2 expression [238] and the Wnt/β-catenin signaling pathway [239], and activates vascular endothelial growth factor [240], thereby inducing epithelial cell proliferation. H. pylori infections are also associated with methylations on CpG islands [241], but the direct role of H. pylori in hypermethylation remains controversial.
H. pylori infections are associated with an increase in Proteobacteria, Acidobacteria, and Spirochaetes and a decrease in Firmicutes, Bacteriodetes, and Actinobacteria [242], suggesting that H. pylori infection is associated with dysbiosis [243]. Studies have also found an inverse relationship between H. pylori abundance and bacterial diversity [244,245].

Peptostreptococcus anaerobius
Peptostreptococcus anaerobius is a Gram-negative anaerobic bacterium commonly found in the oral cavity and the gut. It promotes carcinogenesis by modulating immune cells and interacting with toll-like receptors, TLR-2 and TLR-4, on colon cells to induce ROS formation [125,126]. The binding of P. anaerobius to colon cancer cells is mediated by its surface protein, putative cell wall binding repeat 2 (PCWBR2), which interacts with α2/β1 integrins on colon cells. The binding of P. anaerobius was found to activate the PI3K-Akt pathway, stimulating inflammation and cell proliferation [126].

Parvimonas micra
Several oral diseases, including endodontic abscesses, odontogenic infections, periodontitis lesions, and other infections, are frequently associated with Parvimonas micra [246]. A recent study using an isolated strain from a CRC patient showed that it could promote coloncyte proliferation via the enhancement of Th17 cell infiltration and the oncogenic Wnt signalling pathway [247].

Biomarkers for CRC Screening
For diagnosis of CRC, the identification of novel biomarkers, which are reliable and non-invasive, is desirable. The identification of microbial biomarkers is helpful for designing non-invasive tools for CRC diagnosis. It has been estimated that the use of accurate tests for screening average-risk individuals can reduce the incidence and mortality associated with CRC [248]. Current clinical diagnostic procedures, such as the fecal occult blood test (FOBT), have limited sensitivity for detecting CRC [249].
Though fecal immunochemical testing (FIT) has high sensitivity for the detection of CRC [250], it can detect CRC with a sensitivity of 79% [249] and colorectal adenomas with a sensitivity of 25-27% [251].
CRC-enriched bacteria may serve as potential diagnostic bacterial markers [102,109]. Large-scale studies on fecal metagenomes have identified microbial signatures that can

Microbial Mechanisms Involved in Colorectal Carcinogenesis
The key mechanisms by which gut microbiota induce colorectal carcinogenesis include genotoxins and virulence factors, gut microbial metabolites, inflammation pathways, oxidative stress, and anti-oxidative defense modulation.

Bacterial Genotoxins and Virulence Factors
During biological evolution, gut bacteria developed pathogenicity by acquiring various virulence factors that enabled them to penetrate the gut mucosal barrier and intestinal epithelial cells [172,273,274], which form a barrier between human tissues and microbiota.
A breach in this barrier results in inflammation [275]. Virulence factors are responsible for pro-carcinogenic and disease-promoting effects [276]. CRC-associated E. coli strains have Afa and Eae adhesins, which allow them to adhere to and invade intestinal epithelial cells [277], and activate inflammation pathways [278]. F. nucleatum binds to E-cadherin via its FadA virulence factor, activating the β-catenin signaling pathway and promoting colorectal carcinogenesis [115,279,280].
Many pathogenic bacteria produce toxins that are associated with the development of CRC. ETBF produces B. fragilis toxin (BFT), which activates the NF-κB and Wnt/βcatenin pathways [186,188], leading to increased cell proliferation, DNA damage, and release of pro-inflammatory mediators [281][282][283]. BFT can also hydrolyze the extracellular domain of E-cadherin [117,284]. Many gut microbes produce genotoxins that damage DNA. Cyclomodulins, such as colibactin, cytolethal distending toxins (CDT), cycle inhibiting factors, and cytotoxic necrotizing factors (CNFs), are genotoxins that induce DNA damage and interfere with the cell cycle [127,128,[285][286][287][288]. CDTs and colibactin are considered true genotoxins as they directly mediate DNA damage by inducing double-stranded DNA breaks [128,285,289]. CDTs are well-characterized toxins produced by most Gram-negative bacteria associated with CRC, such as Escherichia and Campylobacter [290]. The CdtA and CdtC subunits allow interactions with host cells, and the CdtB subunit can translocate to the nucleus and damage host cell DNA [128,285,291,292]. CDTs also induce the production of IL-6, TNF-α, NF-κB, and cyclooxygenase 2 [67]. Colibactin induces DNA damage, ROS formation, and cell cycle arrest [68,128]. Targeting colibactin production has been shown to reduce tumors in a mouse model [293]. Although toxin-producing bacteria represent a small proportion of the gut microbiota, an analysis of CRC tissue samples suggests a high expression of these toxins [294]. Therefore, targeting these toxins may have therapeutic implications in CRC.

Gut Microbial Metabolites and Products
Some metabolites produced by gut microbes are linked to CRC [295,296]. The microbial products that affect CRC development are secondary bile acids, acetaldehyde, trimethylamine-N-oxide (TMAO), and glucuronidase.

Secondary Bile Acids
Bile acids are a type of steroid acid found in bile. They are involved in the emulsification and absorption of fats and the elimination of cholesterol. Cholic acid (CA) and chenodeoxycholic acid (CDCA) are primary bile acids synthesized in the liver. Secondary bile acids, such as deoxycholic acid (DCA) and lithocholic acid (LCA), are produced from primary bile acids by the action of anaerobic microorganisms in the colon [297], which use bile acids as a source of energy [298]. It has been found that people with high-fat diets are susceptible to CRC [299,300], probably because a high-fat diet increases the secretion of primary bile acids, which are converted by gut microbes to secondary bile acids [299,301,302]. Feeding mice with secondary bile acids increases inflammation and induces CRC [301].
DCA can modulate intracellular signaling and gene expression [303]. It can also induce the expression of orphan nuclear receptor Nur77 [304] and downregulate the expression of miR-199a-5p [305]. Nur77 promotes tumorigenesis by upregulating anti-apoptotic BRE (brain and reproductive-organ-expressed protein) and angiogenic VEGF (vascular endothelial growth factor) [304]. miR-199a-5p can target CAC1 (CDK2-associated cullin domain 1), a novel cell cycle regulator widely expressed in CRC, for degradation and, therefore, functions as a tumor suppressor. DCA can directly induce oxidative DNA damage and tumor formation [301,306]. DCA induces epithelial-mesenchymal transition and activates vascular endothelial growth factor receptor 2, leading to intestinal carcinogenesis [307]. LCA is known to induce the expression of urokinase-type plasminogen activator receptor (uPAR), which may activate the MAPK signaling pathway and contribute to cancer progression and metastasis [308,309].
Secondary bile acids can activate G-protein-coupled bile acid receptor 1 (GPBAR1), inducing epithelial cell proliferation [310]. Secondary bile acids can promote DNA damage (by producing ROS and RNS) [311,312], regulate gene expression and membrane permeability [313], and activate epidermal growth factor receptor (EGFR) pathway signaling [303,314,315] and the protein kinase C pathway [316]. In addition, bile acids have strong antimicrobial properties and cause changes in the gut microbiome by selectively killing microbes. This leads to an increase in the population of CRC-associated Bacteroidetes and Gamma-proteobacteria [93].

Acetaldehyde
Acetaldehyde is produced from ethanol by the activity of aerobic and facultative anaerobic bacteria in the gut. Excessive consumption of ethanol is considered a risk factor for various cancers, including CRC [321]. Acetaldehyde is highly toxic and procarcinogenic. It can damage DNA and impair DNA excision repair, promoting colorectal carcinogenesis [322].

Trimethylamine-N-oxide (TMAO)
Trimethylamine-N-oxide (TMAO) is produced by a reaction between flavin monooxygenase and trimethylamine (TMA), which is a microbial metabolite produced from red meat and fats. A diet rich in fats and red meat leads to the production of more TMAO because L-carnitine (a TMA) is processed by gut microbes to form TMAO [323]. TMAO has been linked to increased risk of cardiovascular diseases [323][324][325] and CRC development [326,327]. TMAO causes CRC probably by inducing DNA damage, inflammation, oxidative stress, and protein misfolding [328,329].

Glucuronidase
High fecal glucuronidase activity is found in patients with CRC [330]. The liver inactivates some carcinogens by glucuronic-acid-mediated conjugation, which are excreted through the digestive tract. This process may be reversed in the colon by bacterial glucuronidase, reactivating carcinogens. Inhibiting bacterial glucuronidase in a mouse model can reduce the number of tumors [331], indicating that bacterial glucuronidase is responsible for CRC progression. Moreover, bacterial glucuronidase also affects the activity of some anti-tumor drugs [332], influencing the treatment outcome.

Inflammation and Host Immunity
Inflammation is an adaptive response of the host immune system. Significant inflammation is not caused by healthy microbiota as the host immune system is programmed to recognize normal gut microorganisms. There is a constant cross-talk between host immune cells and gut microbes, selecting and tolerating commensal microbes and eliminating pathogenic ones [333]. Alterations in the gut microbiota play an important role in inflammation [334], which promotes CRC development [335]. The inflammatory events associated with CRC development include DNA damage by ROS and RNS produced by macrophages, neutrophils, and dendritic cells (DCs) and the production of cyclooxygenase-2 [336]. Moreover, the invasion of the intestinal mucosa by pathogens triggers the activation of immune cells and the release of cytokines and growth factors [337], which drive the inflammation process. Persistent inflammation results in epithelial cell proliferation, angiogenesis, and inhibition of apoptosis, leading to cancer [338,339]. The pro-inflammatory cytokines secreted by macrophages and T cells, such as IL-6 and TNF, trigger the differentiation of pro-inflammatory Th17 cells. The prolonged presence of Th17 cells and elevated levels of associated cytokines, such as IL-17 and IL-22, are associated with poor survival in CRC [337]. Studies have found that limiting Th17 cells reduces the risk of carcinogenesis [127,340]. IL-6 is an important cytokine required for angiogenesis and the activation of STAT3 [341]. Increased serum IL-6 and TNF levels have been used as prognostic markers for poor survival of CRC patients [342,343]. Inflammation-associated factors can also activate oncogenes [344] or inactivate tumor suppressor genes [345].
Inflammation is associated with the development of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease. IBDs are associated with increased risk of developing CRC, called colitis-associated cancer [346][347][348], as 20% of patients with ulcerative colitis develop CRC [349]. Individuals with pancolitis are at a higher risk of developing CRC than patients with limited colitis [350]. Meta-analyses have indicated a risk of 18.4% for patients with ulcerative colitis [351] and 8.3% for patients with Crohn's disease [352] to develop CRC. Colitis stimulates carcinogenesis by inducing the expansion of genotoxic bacteria [68]. The gut microbiota of patients with IBDs shows increased abundance of Proteobacteria, particularly Enterobacteriaceae, such as E. coli [353,354]. The role of colibactin-producing E. coli in inducing inflammation and intestinal tumors was elucidated in a study carried out in IL10-deficient mice treated with the genotoxic agent azoxymethane. In such a mouse model lacking pks + E. coli, fewer intestinal tumors developed than in similarly treated mice with pks + E. coli [68]. ETBF promotes inflammation by activating STAT3 and NF-κB signaling in colonic epithelial cells [33,355]. The NF-κB pathway is an important regulator of the genes encoding TNF and COX-2, which are usually overexpressed in IBDs and CRC [356]. B. fragilis also induces the expression of spermine oxidase in colonocytes, which induces ROS production and DNA damage [283].
Other bacteria, such as Citrobacter rodentium and Mycobacterium, can also promote inflammation, inducing IBDs [357][358][359]. Patients with IBDs are more likely to be affected by CRC due to changes in the gut microflora homeostasis [360]. IBDs are also associated with dysbiosis with lower Firmicutes and Bacteriodetes as compared to healthy subjects [361]. Long-term use of NSAIDs is known to reduce the risk of CRC, suggesting a link between inflammation and CRC development [362].
Pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs) and nucleotidebinding oligomerization (NOD)-like receptors (NLRs), play an important role in recognizing the specific molecular patterns of pathogenic microorganisms called microorganismassociated molecular patterns (MAMPs) [363]. PRRs recognize microbial surface molecules, such as peptidoglycan, flagellin, lipoproteins, lipoteichoic acid, and lipopolysaccharides. Lipoteichoic acid specifically binds to CD14 or TLR-2, inducing the secretion of proinflammatory factors [364,365]. TLRs are a major class of PPR expressed in macrophages and dendritic cells. They recognize microbes and activate an immune response if the mucosal barrier is disrupted. TLR signaling initiates immune defense by producing pro-inflammatory cytokines and also enhances barrier function, preventing microbial invasion [366]. There are two important TLR pathways: myeloid differentiation factor 88 (MyD88) adaptor-protein-dependent and TRIF-dependent [367,368]. In the MyD88dependent pathway, the downstream activators are NF-κB and MAPK [368]. Studies have suggested the role of MyD88 in CRC induction [369]. In AOM-treated APC min/+ mice, the inactivation of MyD88 resulted in a decrease in tumor number [370].
TLR-2 expression is important in maintaining the gut microbiota composition and suppressing inflammation [371,372]. It has been found that the number of tumors increases in TLR2-deficient mice compared to control mice [373], suggesting that TLR2 is important for maintaining gut homeostasis [374]. An increase in TLR4 activates NF-κB, which induces COX-2 expression and an increased risk of CRC [375]. High levels of TLR4 and MyD88 in CRC patients increase the risk of liver metastasis and also affect survival [376,377]. Inhibition of TLR4 expression protects against CRC [378]. A study also found that chronic activation of TLR9 may induce hyperproliferation and CRC development [379].
NLRs are located in the cytoplasm of immune and non-immune cells. NLR activation triggers the production of pro-inflammatory cytokines and autophagy [380]. A study found a significant difference in NLR signaling between the tumor and non-tumor tissues of patients with CRC [381]. NOD1or NOD2-deficient APC min/+ and AOM/DSS-treated mice show asignificant increase in CRC numbers [382]. NOD2 mutations are also associated with Crohn's disease and increased risk of CRC [383][384][385][386].

Oxidative Stress
Oxidative stress is caused by imbalance between oxidative molecules, such as ROS and RNS, and anti-oxidative defenses [387]. Oxidative stress affects biomolecules, damages cell membranes, and induces DNA breaks and mutations [387,388]. Oxidative stress induces NF-κB and up-regulates the expression of pro-inflammatory cytokines and anti-apoptotic signaling [338]. The production of ROS has been directly linked with CRC induction. ROS are produced by the gut microbiota or host immune cells, such as macrophages and neutrophils, in response to inflammation induced by pathogenic bacteria or other external environmental factors [389][390][391]. RNS are produced by some bacterial species, such as Lactobacillus and Bifidobacterium [392][393][394]. Species, such as E. faecalis, can produce hydroxyl radicals [195,395] that contribute to CRC development by inducing point mutations and chromosomal breaks [388,396]. H. pylori also induces oxidative stress, resulting in gastric carcinogenesis [397].
Various anti-oxidative defense mechanisms, such as DNA repair, balance oxidative stress [398,399]. Only the base-excision repair system accounts for more than 10,000 repairs in the colon cells per day [400]). Anti-oxidative defense mechanisms are found to be altered in CRC [401,402]. Studies have found downregulation of the MMR system by some enteropathogenic E. coli strains [277,403] as well as in a colitis-induced CRC mouse model [404]. Using APC min/+ MMR-deficient mice, Belcheva et al. [405] found that gut microbes could induce CRC in MMR-deficient epithelial cells.

Diet and the Risk of CRC
Diet plays an important role in the development and progression of many cancers, including CRC [406] (Table 3 and Figure 2). Diet is the major determinant of gut microbiota [407], and changes in diet are accompanied by changes in the fecal microbiota within a few days of diet change [28,408]. High intake of red meat, processed meat, and fats increases the risk of CRC [326,327,409,410], while a high intake of dietary fiber can decrease this risk [411]. Processed meat and red meat are classified as Class 1 and Class 2A carcinogens, respectively, by the WHO. The risk of CRC increases linearly with the intake of processed and red meat [412]. The heme iron in red meat is converted into carcinogenic N-nitroso compounds, which contribute to CRC development [413]. In addition, heme iron also increases mucin-degrading bacteria, such as Akkermansia muciniphila, leading to gut barrier function impairment and CRC [414]. Heme causes persistent intestinal dysbiosis, with an increase in Proteobacteria, resulting in inflammation and hyperproliferation of the intestinal epithelium [415]. A diet rich in meat was found to increase Bacteroidetes and decrease Firmicutes [408]. Zimmer et al. [416] reported that fecal samples from vegans showed a significant reduction in Enterobacteriaceae compared to omnivorous control subjects. Vegetarians, therefore, have a 20% lower risk of developing CRC than non-vegetarians [417].   [416] reported that fecal samples from vegans showed a significant reduction in Enterobacteriaceae compared to omnivorous control subjects. Vegetarians, therefore, have a 20% lower risk of developing CRC than non-vegetarians [417].
A diet rich in complex carbohydrates increases the abundance of probiotic bacteria, such as Bifidobacterium longum, Bifidobacterium breve, and Bifidobacterium thetaiotaomicron [418] and reduces the growth of opportunistic bacteria, such as Enterobacteriaceae [419]. On the other hand, excessive consumption of refined sugars results in the proliferation of pathogenic bacteria, such as Clostridioides difficile and Clostridium perfringens [420]. A diet rich in resistant starch increases the abundance of Firmicutes, such as Ruminococcus bromii, while a diet rich in wheat increases the abundance of butyrate-producing Lachnospiraceae [419]. A low carbohydrate diet is associated with a decrease in butyrateproducing Firmicutes and Actinobacteria [419,421].
Dietary fibers are important factors that affect gut microbial composition and diversity [422]. It has been found that patients with colorectal adenomas have a low intake of dietary fibers [423]. Dietary rice bran intake has been shown to modify gut microbiota and increases the anti-cancer metabolites, myristoylcarnitine and palmitoylcarnitine, in a mouse model of CRC [424]. Cellulose intake has been found to decrease inflammation and tumor formation and increases survival rate in an AOM/DSS mouse model of CRC [425].
Butyrate and propionate can alter chromatin state (by inhibiting histone deacetylases) [439], downregulate pro-inflammatory cytokines, such as IL-6 and IL-12 [440,441], and induce apoptosis [442]. Butyrate and propionate can activate the AP-1 signaling pathway, which controls cell proliferation and apoptosis [443]. Faecalibacterium prausnitzii is an important butyrate-producing bacterium found in the intestine [444], which has drawn much attention in recent years. A decrease in F. prausnitzii has been reported in patients with IBDs [445] and CRC [81,153]. Propionate suppresses CRC by promoting the degradation of euchromatic histone-lysine N-methyltransferase 2 [446]. SCFAs show anti-inflammatory effects and regulate colonic regulatory T cells [435]. Although there is much supporting evidence that dietary fiber decreases the risk of CRC, several cohort studies have failed to find a link between high fibre intake and lower risk of CRC [447,448].
High-protein and low-carbohydrate diets increase the production of toxic metabolites, such as amines, ammonia, phenolic compounds, indoles, and N-nitroso compounds, due to bacterial fermentation [449][450][451]. Many of these toxic metabolites may cause mutations, increasing the risk of carcinogenesis [35,452]. N-nitroso compounds promote DNA alkylation [11]. Ammonia is a potent carcinogen that promotes mucosal damage and adenocarcinoma in rat models [453]. Protein-rich diets are also associated with high fecal glucuronidase activity [454]. The fermentation of sulfur-containing amino acids by sulfate-reducing bacteria, such as Desulfovibrio, Desulfobacter, and Desulfobulbus, results in the generation of sulfides. Desulfovibrio is a gut bacterium that uses lactate to generate hydrogen sulfide [455], which is genotoxic [456] and damages DNA, probably by generating ROS [452]. It also inhibits butyric acid oxidation [457] and increases cell proliferation in vitro [458]. High-sulfur microbial diets are associated with increased risk of distal colon and rectal cancers [459].
The association between fat intake and CRC was first established by Drasar and Irving in 1973 [460]. Several studies have confirmed that high fat intake is a risk factor for CRC. A high-fat diet increases secondary bile acid formation, affecting the composition of the gut microbiota. Increased levels of deoxycholic acid increase resistance to apoptosis [306], induce ROS formation, damage DNA, and activate NF-κB [313]. Therefore, a high-fat diet increases the risk of CRC [461]. Contrary to these findings, studies on low-fat or highfibre diets have failed to demonstrate a decreased risk for CRC [462][463][464][465][466]. In a landmark epidemiological study involving 61,463 Swedish women, Terry et al. [463] concluded that fat intake is not associated with CRC. A study by Taira et al. [467] in rats found that switching from a low-fat diet to a high-fat diet resulted in an increase in Firmicutes and a decrease in Bacteriodetes. In a similar study, Higashimura et al. [468] found that a high-fat diet decreased Lactobacillales and increased Clostridium. A recent study found that a high fat diet induced gut microbial dysbiosis and gut barrier dysfunction in mice, driving colorectal tumorigenesis [469]. Another recent study found that a high-fat diet promoted gut barrier dysfunction and inflammation in the colorectum and liver, contributing to CRC tumorigenesis and metastasis [470].
Urolithins are microbial metabolites of fruits and nuts rich in ellagic acid that inhibit Wnt signaling and have anti-carcinogenic effects [471,472]. Glucosinolates are plant secondary metabolites that have a protective role against CRC [473]. Kaempferol, a polyphenol found in fruits and vegetables, reduces tumor burden and restores damaged intestinal barrier in Apc min/+ mice [474]. Many dietary phytochemicals are modified by gut microbiota to produce phenolic substances that are known to inhibit pro-inflammatory mediators, such as NF-κB and TNF. Studies on mouse models have shown that ketogenic diets inhibit glycolysis and cancer cell proliferation [475,476]. The effect of ketogenic diet on gut microbiota wasstudied in a mouse model of autism, where ketogenic diet significantly increased the Firmicutes/Bacteroidetes ratio [477]. A Mediterranean diet modulates the gut microbiota by enriching anti-inflammatory-environment-promoting bacteria, thus preventing CRC [478]. Since diet affects the composition of gut microbiota, it may influence susceptibility to CRC.

Gut Microbiota in CRC Treatment
Studies have shown that the gut microbiota can influence the therapeutic effects of cancer therapies by modulating the response, efficacy, and toxicity of chemotherapy, radiotherapy, and immunotherapy [479,480]. Dietary interventions through probiotics and prebiotics have been shown to influence the outcome of most cancer therapies. Many clinical trials are being conducted to know the efficacy of probiotics on CRC treatment (Table 4).

Probiotics
Probiotics are live microorganisms that provide multiple health benefits when administered in adequate amounts [481]. Probiotics modify the gut microflora composition by replacing pathogenic microbes with beneficial microbes [482]. Probiotics provide many health benefits, including regulation of the immune system, reduction in colitis and blood cholesterol, inhibition of pathogenic bacteria, and prevention of CRC [483][484][485][486]. Studies have found that probiotics prevent the colonization of pathogenic microbes by competing for nutrients [487] or adhering to epithelial cells or mucus [488] and, thus, help prevent intestinal infections [489,490]. Additionally, probiotics can produce certain metabolites that inhibit pathogen growth [491,492]. By lowering the risks of intestinal infections and inflammation, probiotics may prevent CRC development.
Since CRC is linked to gut microflora dysbiosis, restoring normal gut microflora through probiotics is one of the new approaches for IBD and CRC treatment. Probiotics inhibit CRC by reducing inflammation and carcinogenic microbial metabolites [484,493], downregulating inflammation pathways [494], producing short-chain fatty acids, antioxidants, and anti-cancer compounds [495,496], reducing the expression of cyclooxygenase-2 [497] and cell proliferation [62,498], inducing cancer cell apoptosis [499], and stimulating the expression of tumor suppressor genes [500]. Many studies have confirmed the positive effects of probiotics in the treatment of IBDs, CRC, and other cancers [486,501,502]. The key probiotics are Bifidobacterium and Lactobacillus (Figure 3). The administration of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bl-04 affects the gut microbial profile. Probiotics have been shown to increase butyrate-producing bacteria, such as Faecalibacterium, and decrease CRC-associated bacteria, such as Fusobacterium [503]. Kuugbee et al. [504] reported that a probiotic cocktail containing Lactobacillus acidophilus, Bifidobacterium bifidum, and Bifidobacterium infantum, with oligofructose and maltodextrin, modulates the gut microbiota and reduces colon cancer development by suppressing apoptosis and inflammation. A combination of Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12 with inulin reduces cell proliferation and improves epithelial barrier function [505]. Benito et al. [506] reported that a combination of Bifidobacterium bifidum and Lactobacillus gasseri along with quercetin inhibited CRC development in Apc min/+ mice. Clostridium butyricum can decrease the incidence of tumors in mice by decreasing the number of Th2 and Th17 cells and reducing the secretion of factors associated with inflammation, such as IL-22 and NF-κB [507]. C. butyricum is also known to inhibit high-fat diet-induced CRC development in Apc min/+ mice [508]as well as change gut microbial composition, and decrease the incidence and size of CRC [509]. Apart from these genera, Streptococcus thermophilus has been found to reduce tumor formation in mice, through β-galactosidase-dependent production of galactose that activates oxidative phosphorylation and downregulates the Hippo pathway kinase [510]. ministration of Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bl-04 aff gut microbial profile. Probiotics have been shown to increase butyrate-producin ria, such as Faecalibacterium, and decrease CRC-associated bacteria, such as Fusob [503]. Kuugbee et al. [504] reported that a probiotic cocktail containing Lactobac dophilus, Bifidobacterium bifidum, and Bifidobacterium infantum, with oligofruct maltodextrin, modulates the gut microbiota and reduces colon cancer developm suppressing apoptosis and inflammation. A combination of Lactobacillus rhamn and Bifidobacterium lactis Bb12 with inulin reduces cell proliferation and impro thelial barrier function [505]. Benito et al. [506] reported that a combination of Bif rium bifidum and Lactobacillus gasseri along with quercetin inhibited CRC develop Apc min/+ mice. Clostridium butyricum can decrease the incidence of tumors in mic creasing the number of Th2 and Th17 cells and reducing the secretion of factors a ed with inflammation, such as IL-22 and NF-κB [507]. C. butyricum is also know hibit high-fat diet-induced CRC development in Apc min/+ mice [508]as well as cha microbial composition, and decrease the incidence and size of CRC [509]. Apa these genera, Streptococcus thermophilus has been found to reduce tumor form mice, through β-galactosidase-dependent production of galactose that activate tive phosphorylation and downregulates the Hippo pathway kinase [510].

Bifidobacterium
Bifidobacterium is a Gram-positive, non-motile, anaerobic bacterium found in the human gut. The ratio of Bifidobacterium to E. coli has been used as an indicator of gut microflora. A decrease in Bifidobacterium and an increase in E. coli have been observed in CRC [511]. It has been found that an oral administration of Bifidobacterium alone can influence the immune response against CRC [512]. Bifidobacterium may also enhance chemotherapeutic efficacy by reducing glucuronidase activity [513]. An oral administration of B. breve significantly improves ulcerative colitis [514]. B. breve reduces tumor growth in MC38 colon carcinoma-bearing mice and boosted the efficacy of cancer therapeutics [515]. The strains of B. infantis and B. breve interact with toll-like receptors (TLRs) and can activate intestinal dendritic cells, Foxp3 + regulatory T cells, and IL-10-producing Tr1 cells [516,517].

Lactobacillus
Lactobacillus is a Gram-positive, facultative anaerobic bacterium found in most probiotics. Lactobacillus can reduce the incidence of CRC by inducing apoptosis, reducing the expression of β-catenin and NF-κB [519], and modulating cytokine-producing dendritic cells [520]. It also regulates the expression of toll-like receptors and enhances intestinal epithelial barrier function [504,521,522]. L. rhamnosus GG and L. acidophilus inhibit STAT3 and NF-κB signaling, downregulating the expression of Th17 cells [523,524]. L. rhamnosus GG decreases tumor burden in a mouse model of CRC by increasing colonic CD8 T-cell responses [525].
Studies have confirmed that oral administration of L. casei strain significantly improves ulcerative colitis [526] and decreases the incidence of CRC in high-risk patients [527]. L. casei produces a metabolite, ferrichrome, which can trigger apoptosis in tumor cells through the JNK pathway [499]. Administration of L. salivarius Ren [528] and L. paracasei [529] can suppress CRC development in 1, 2-dimethylhydrazine-induced rat models. L. paracasei subsp. paracasei NTU 101 in combination with 5-fluorouracil (5-FU) was effective in reducing CRC cell viability [530]. It has been found that L. acidophilus NCFM suppresses tumor growth in mouse model by reducing the expression of CXCR4 and downregulating MHC class I in tumor cells [62]. L. rhamnosus and L. plantarum have been shown to stimulate mucin production [531,532]. It has been found that L. acidophilus and L. bulgaricus inhibit H. pylori adherence to GES-1 cells. L. bulgaricus, in particular, was found to inhibit IL-8 production by GES-1 cells by modulating the TLR4/IκBα/NF-κB pathway [533]. L. bulgaricus decreases intestinal inflammation by decreasing the levels of IL-6, TNF-α, IL-17, IL-23, and IL-1β and, thus, has a potential chemopreventive effect on colitis associated colon cancer [534]. L. reuteri strain ATCC PTA 6475 and ATCC 53608 were found to inhibit enteropathogenic E. coli [535]. L. reuteri restricts colon tumor growth and increases tumor reactive oxygen species. L. reuteri and its metabolite, reuterin, are downregulated in mouse and human CRC [536]. A study found that L. plantarum and L. salivarius could augment IL-18 production in a rat model of CRC [537]. A recent study found that administration of L. gallinarum could inhibit colorectal tumorigenesis in Apc min/+ mice and in AOM/DSStreated mice [538]. Another recent study found that L. coryniformis MXJ32 enhanced the expression of tight junction proteins and alleviated intestinal inflammation by downregulating the expression of inflammatory cytokines, decreasing the number of tumors and the average tumor diameter [539].

Prebiotics
Prebiotics are food components that provide health benefits by maintaining a healthy gut microbiota [540]. Many dietary components act as prebiotics. Clinical trials have found that prebiotic administration increases the abundance of probiotic strains, such as Ruminococcus, Faecalibacterium, Rosebura, and Akkermansia [541][542][543]. Prebiotic oligosaccharides can inhibit pathogen colonization by interacting with bacterial receptors and preventing pathogens from attaching to epithelial cells [544]. A study on polydextrose found its beneficial effects on maintaining healthy gut microbiota [545]. Fructans and galactooligosaccharides increase the abundance of beneficial bacteria, such as Bifidobacterium and Lactobacillus, and increase fecal butyrate concentration [546]. Inulin, a polysaccharide found in artichokes, bananas, asparagus, and wheat, decreases the formation of precancerous lesions by inhibiting the activity of glucuronidase and decreasing pH and concentration of indole, phenol, and p-cresol in the colon [547]. Inulin intake has also been shown to increase the abundance of Bifidobacterium [548]. Agro-oligosaccharides alter the production of SCFAs and secondary bile acids and, thus, control CRC development [468]. Polysaccharides from Lachnum sp. alters gut microbiota and reduces inflammation and tumor incidence [549].
Avenanthramide-C, found in oats, is metabolized by gut bacteria into bioactive compounds that show anti-tumor effects [550]. Nutmeg can prevent colon cancer by modulating gut microbiota and inflammation [551]. Eicosapentaenoic acid (EPA) is a type of omega-3 fatty acid that inhibits inflammation and colitis-associated CRC [552]. Investigations on the effects of fructo-oligosaccharides, xylo-oligosaccharides, polydextrose, and resistant dextrin on the gut microbiota of perioperative patients with CRC found an increase in the abundance of Bifidobacterium and Enterococcus and a reduction in Bacteroides [553]. Recent studies have found that berberine, found in Berberis, affects the proliferation, migration, and invasion of CRC cells and induces their apoptosis [554], decreases inflammatory modulators [555], and the expression of NF-κB [556] and increases fecal butyrate, acetate and propionate levels [557]. Ginsenoside compound K, produced from ginseng saponins, suppresses tumor growth and increases the abundance of A. muciniphila in an AOM/DSS-induced colitis-associated CRC Balb/c mouse model [558].

Chemotherapy
Many studies have shown that the gut microbiota influences the activity of various chemotherapeutic drugs and, thus, the efficacy of cancer treatment [559,560]. The gut microbiota modulates the host response by the 'TIMER' mechanistic framework: translocation, immunomodulation, metabolism, enzymatic degradation, and reduced diversity and ecological variation [479]. Gut microbial metabolites can have an enhanced killing effect on CRC [561]. Cyclophosphamide (CTX), a chemotherapeutic drug, can alter the gut microbiota and promote the translocation of Gram-positive bacteria into secondary lymphoid organs, promoting the generation of Th17 cells [560,562]. The mouse model, where Gram-positive bacteria were killed by antibiotics, was resistant to the antitumor effects of CTX [560]. Restoring the microbiota can improve the efficiency of CTX treatment. Studies carried out on mouse models have shown that the anti-tumor effect of CTX is enhanced by bacteria, such as Enterococcus hirae, Lactobacillus johnsonii, and Barnesiella intestinihominis [560,563].
Gut microbiota can influence the anti-tumor activity of oxaliplatin by affecting the production of ROS by immune cells [559]. A recent study explored the effect of gut microbial metabolites on the efficacy of oxaliplatin in combination with fluorouracil and leucovorin [564]. The study found that butyrate stimulated the anti-tumor cytotoxic CD8 + T cell response, and patients that responded to oxaliplatin had higher serum butyrate levels than non-responding patients. Gut microbes, particularly F. nucleatum, promote resistance to chemotherapy by modulating autophagy and inducing selective loss of miR-18a and miR-4802 [565]. A study showed that the effect of chemotherapy could be enhanced by administering irinotecan-loaded dextran nanoparticles that were covalently linked to azide-modified phages against F. nucleatum [566].
Bacterial genotoxins are also being targeted for CRC treatment. As described previously, colibactin produced by enterogenic E. coli plays an important role in CRC progression. The synthesis of colibactin requires a serine enzyme, ClbP [567,568], which can be inhibited by boronic acid, thus suppressing the genotoxicity of colibactin-producing E. coli [293]. The use of boronic acid is effective in preventing cell proliferation in a CRC mouse model. In vitro studies have found that healthy gut microbiota can increase the response to capecitabine or TAS-102 [569]. Gammaproteobacteria were found to metabolize the chemotherapeutic drug gemcitabine in a colon cancer mouse model [570]. Therefore, targeting these bacteria through antibiotics may promote the efficacy of gemcitabine treatment. A recent study found that the antibiotic vancomycin, which targets Gram-positive bacteria, potentiated the radiotherapy-induced antitumor immune response and tumor growth inhibition [571]. A recent study found that the patients that responded to radiochemotherapy showed an enrichment of butyrate-producing bacteria in fecal microbiota and had significantly higher levels of SCFAs, such as acetate, butyrate, and isobutyrate [572].

Immunotherapy
Immunotherapy has been used for the treatment of various cancers. Immunotherapy for CRC was approved by the FDA as a second-line therapy for tumors positive for deficient mismatch repair/microsatellite-high (dMMR/MSI-H) due to increase in overall survival [573]. Immune checkpoint inhibitors (ICIs) are widely used in immunotherapy. ICIs activate T cells to enable them to mount anti-tumor responses [574]. ICIs are usually monoclonal antibodies that prevent programmed cell death protein 1 (PD-1) from interacting with its ligand, PD-L1, or enable cytotoxic lymphocyte-mediated attack on tumor cells by targeting cytotoxic T-lymphocyte antigen 4 (CTLA-4) [575]. Compelling evidence sug-gests that gut microbiota can modulate cancer immunotherapy [512,559,560,576,577], and several studies have found that certain bacteria are positively correlated with immunotherapeutic response, including Bifidobacterium [512], Faecalibacterium [578], and Akkermansia [579] although not necessarily in the CRC context. Tanoue et al. [577] found 11 bacterial strains that could enhance the therapeutic effects of ICI. The oral administration of Bifidobacterium alone can influence the immune response against CRC, by maturing dendritic cell and promoting their function, upregulating cytokine secretion, and activating tumor-specific T cells [577]. The gut microbiota is also associated with ICI-induced colitis [580]. Administration of probiotics that include Bacteriodales and Burkholderiales, as well as fecal microbiota transplantation (FMT), improves ICI-induced colitis [581].

Fecal Microbiota Transplantation
Fecal microbiota transplantation (FMT) is an emerging biotherapeutic procedure that aims to restore normal gut microbial ecology to ameliorate various gastrointestinal disorders, including IBDs [582,583]. FMT involves the transplantation of a microbial population from a donor to a recipient. The prospects of using FMT to enhance the treatment of CRC are largely unexplored, and only a few studies have been carried out in this direction. A study carried out by Rosshart et al. [584] found that fecal microbiota transplantations from wild mice to laboratory mice could provide resistance against DSS/AOM-induced colorectal tumorigenesis. However, the logistics, safety, and potentially limited efficacy of FMT have precluded its wider use. Some patients that received FMT developed adverse effects, such as diarrhea, constipation, and abdominal distension [585]. Moreover, one possible risk with FMT is the transmission of multi-drug-resistant bacteria, potentially leading to life-threatening infections such as Escherichia coli bacteremia [586,587]. Stringent protocols for donor screening can prevent the occurrence of such infections. Another risk associated with FMT is the transmission of microbiome-associated chronic diseases, such as gastrointestinal, cardiometabolic, and autoimmune disorders [588]. In a study, it was found that transplanting human feces from obese individuals to germ-free mice induced obesity [589]. Similar results were found in humans where a woman developed obesity after receiving an FMT from an overweight donor [590]). Gregory et al. [591] reported the transmission of atherosclerosis after FMT.

Conclusions and Future Perspectives
A growing body of evidence suggests that the gut microbiota is strongly associated with the development and progression of CRC. The study of changes in the gut microbiome with CRC progression is critical to obtain a link between the gut microbiota and CRC. The presence of certain bacterial species may serve as a biomarker to assess the risk of developing CRC and the patient's response to chemotherapy and immunotherapy. To determine the likelihood of developing CRC, intestinal microflora profiles may be used in conjunction with other factors, such as age, diet, family history, body mass index, and geographic location. The indiscriminate use of antibiotics should be stopped to prevent any disharmony in the intestinal microecology, which has been linked to IBDs and CRC. Dietary interventions can reshape the gut microbiota and may help in preventing and treating CRC. Gut-microbiota-based diagnostic tests can provide reliable and accurate identification of risk factors for developing CRC. This can also help in developing patient-specific probiotic therapy for CRC treatment.