Targeting Dietary and Microbial Tryptophan-Indole Metabolism as Therapeutic Approaches to Colon Cancer

Over a century ago (1901), English chemist Frederick Gowland Hopkins made a significant contribution to the field of nutrition by discovering the essential amino acid Tryptophan (Trp) from casein, a milk protein. As an essential amino acid, Trp can only be obtained through diet, mainly meat, dairy, and seeds. Trp is required for protein and niacin biosynthesis and is a precursor of serotonin and melatonin. Apart from these fundamental functions, Trp is appreciated for its influence on both host and microbial metabolism via two distinct pathways: the Kynurenine (Kyn) Pathway and Indolic Pathway. These pathways metabolize Trp into metabolic and neuroactive compounds that influence microbial composition and host physiology in an enzyme-dependent manner. Indole has a bicyclic ring formed by benzene and pyrrole groups. This indole structure is part of Trp and makes it unique in its structure and function. Indole can be kept intact or cleaved from the Trp structure, generating bioactive molecules [1]. Specific taxa among the microbiota also take advantage of this unique property of Trp to produce indoles for signaling and defense [2]. Microbial and dietary indoles can also trigger or suppress immune function thereby maintaining a symbiotic relationship with the host [3,4]. However, both Kyn and indolic processes can become dysfunctional in the pathogenesis of colon cancer.

In this review, we begin by describing the different metabolic pathways of Trp and their impact on the host physiology. We then discuss the alterations that occur in these pathways during colon cancer. Next, we introduce the significance of the microbial degradation of Trp in healthy conditions, along with the repercussions of microbial alterations in carcinogenesis. We conclude by highlighting the gaps in knowledge regarding microbial-host Trp metabolism and propose dietary, microbial and drug targets that could have therapeutic applications.

There are four known metabolic pathways that Trp can enter once in the upper gastrointestinal (GI) tract; Kyn pathway, serotonin pathway, indolic pathway (bacterial degradation) and tryptamine pathway [5]. These distinct pathways compete for the available free Trp pool and specifically convert Trp into Kyn, serotonin, indoles, and tryptamine as the end products, respectively [6]. About 90–95% [7,8] of Trp is converted into Kyn and downstream metabolites (Figure 1) via the Kyn pathway. These metabolites include L-kynurine acid, 3-hydroxykynurine, anthralinic acid, quinolinic acid, 3-hydroxyanthralininc acid, picolinic acid, most of which produce NAD+ and ATP in host cells, and play a vital role in inflammation, immune tolerance, and neurotransmission [5,9,10,11].

About 4–6% of unabsorbed Trp then moves along the gastrointestinal tract and is metabolized by the microbiome into indole and indolic compounds including indole-3 pyruvate, indole-3-acetamide, indole-3-acetaldehyde, indole-3-acetic acid, indole-3-lactic acid, and indole-3- propionic acid [8,12,13,14,15,16]. These indolic compounds can bind to pregnane X receptors (PXR) and aryl hydrocarbon receptor (AhR) to promote intestinal homeostasis, enhance barrier function and tight junctions, reduce permeability, regulate intestinal immune tolerance [5,13,17,18]. Additionally, indoles can act as antioxidants and neuroprotective compounds [19,20]. Overall, indole production has an important impact on host health as a result of Trp metabolism by the microbiota [5].

In addition to Kyn pathway and microbial degradation, 1–2% of Trp can be converted into 5-hydroxytryptamine (serotonin) in the enterochromaffin cells of the intestinal mucosa. Serotonin is primarily involved in regulating gut motility, vasodilation, and maintenance of mood [21]. A minor contribution of Trp (about 1%) is towards the production of tryptamine, which can be initiated by both the host and microbiota [22]. Microbial tryptamine, a ligand for serotonin receptors, can also affect gut motility and transit time, given the abundance of such receptors in the GI tract [23]. Contribution of the gut microbiota towards serotonin and tryptamine production is an understudied field of research and will need further investigation in the context of carcinogenesis.

The Kyn pathway (90–95% of ingested Trp) is the major Trp degradation pathway that produces Kyn and other neuroactive metabolites either in the liver or in the extrahepatic tissue. In addition to neuroactive metabolites, the Kyn pathway also regulates systemic Trp levels, availability of Trp for serotonin synthesis, vitamin B3, and hepatic heme synthesis [9]. About 90% of Trp metabolized via the Kyn pathway takes place in the liver where all the enzymes for complete transformation of Trp into NAD+ are present. The hepatic Kyn pathway is mainly induced by signaling from glucocorticoids, as well as estrogen and glucagon [5,24,25]. The remaining 10% of the Trp transformation takes place via the Kyn pathway in the extrahepatic tissues, and is induced by cytokines (Interferon-γ (IFN-γ), Interleukin-1 (IL-1), Interluekin-6 (IL-6)), lipopolysaccharides, prostaglandins and amyloid peptides [7]. However, the metabolites produced in the extrahepatic tissues do not contain all the enzymes for complete conversion of Trp into NAD+. Availability, or lack thereof, of all enzymes to completely transform Trp determines the intermediates that are produced, ultimately affecting the functional outcome [9,26,27,28].

The array of metabolites produced via the Kyn pathway have led many researchers to investigate Trp and the role of these metabolically active metabolites in immune function, behavioral disorders including mood, anxiety and depression, inflammatory conditions, gut homeostasis, inflammatory bowel disease (IBD), neurodegenerative disorders such as Alzheimer’s disease, and cancer. All these disease states appear to have an altered or amplified Kyn pathway activity [1,10,29,30,31,32,33,34]. Under inflammatory conditions, common to most of these disease states, Trp is shunted into the Kyn pathway, especially in the extrahepatic tissue (mostly immune cells), while the hepatic pathway activity is reduced [9,25,35]. This shunting of Trp into Kyn pathway, reduces Trp availability for other pathways especially serotonin and indolic pathways; the two pathways which have shown to be beneficial for host health in the presence of Trp [34,36,37]. Furthermore, reduced Trp and increased Kyn, alters the activation and balance of innate and adaptive immune cells towards a tolerogenic milieu [37]. Therefore, Kyn pathway activity is amplified under inflammatory conditions, in part due to an NAD+ requirement for increased energy demands in the immune cells and to implement immunomodulatory functions [35].

The alterations in Trp metabolism begin early on in colon carcinogenesis and permits immune evasion in tumor microenvironment (Figure 2). Recently, the oncogene c-MYC was shown to contriubte to this immune evasion through alteration of Trp metabolism. Specifically, c-MYC accelerated Trp uptake by upregulating Trp transporters SLC7A5 and SLC1A5 in colon cancer cells and tissues [43]. The study also showed an increase in the cytoplasmic levels of IDO1, arylformamidase (hydrolyzes N-formyl-L-Kyn to L-Kyn), and Kyn, which drive T cell inactivation and increase protein synthesis, in pathogenesis of colorectal cancer (CRC). Other studies have also demonstrated increased levels of Kyn in colon cancer patients when compared to healthy individuals [11]. Interestingly, the levels of enzymes involved in further degradation of Kyn into downstream metabolites were not elevated, suggesting Kyn to be the predominant metabolite elevated in CRC [43]. Our data (unpublished) also indicate an alteration in Trp metabolism in the microbiome of individuals with CRC compared to healthy individuals, which creates additional complexity in the relationship with the host Kyn pathway. Thus, understanding the contribution of the microbial Trp-Kyn pathway to colon carcinogenesis will be important for future drug discovery and treatment.

Excessive Kyn levels in the tumor microenvironment is one of the adaptive mechanism for the tumors to escape immune surveillance and metastasize via T-cell inactivation (Figure 3) [11,64,65]. Kyn drives tumor escape by acting as a ligand and constitutively activating AhR to express genes involved in cell-growth. Collectively, the alterations in Trp transporters and degrading enzymes leads to an increased Kyn pool within the tumor microenvironment. The excessive Kyn levels aid in tumorigenesis in two ways; 1), a portion of the Kyn produced can directly induce T-cell inactivation and apoptosis leading to immune evasion; 2) the remaining Kyn can constitutively activate AhR, transcribing genes for not only tumor escape but also for proliferation and metastasis [26,43,53,66,67]. Thus, the Kyn-AhR signaling pathway is one of the major contributors to colon cancer [33,34,36,38,40,43,57].

Over stimulation of the Kyn pathway can also be induced by stress and inflammation [68]. Recent evidence indicates that pro-inflammatory cytokines, including IFN-γ, can induce IDO1 gene expression due to presence of IFN response element. Increased IDO1 expression, especially in immune cells can enhance the activity of Tregs and suppress effector T-cells leading to immune tolerance [69]. Most individuals with cancer, including hepatocellular carcinoma, colon cancer, kidney cancer lung cancer among other, often exhibit increased Kyn levels and decreased serum Trp levels, or increased kyn/Trp ratio, which in part is explained by excessive inflammatory signaling molecules [11,70,71,72]. Evidence of this increased kyn/Trp ratio was recently shown in a study of individuals with colon cancer. Fecal metabolites from these patients demonstrated both an increased kyn/Trp ration, but also a decreased indole/Trp ratio (Venkateswaran & Conacci-Sorrell, 2020), suggesting loss of either indole producing microbes and/or an increase in microbes producing kynurinene metabolites. Indeed, Pseudomonas aeruginosa produces kynurenic acid and 3-OH-Kyn, which may disrupt the Trp metabolism of the host, and thus the immune response [73]. Additionally, administration with PEGylated kynureninase, a therapeutic enzyme, has shown to inhibit tumor growth by degradation of Kyn into non-toxic metabolites [74]. Therefore, it is becoming widely known, but not completely understood, that alterations in Trp metabolism is one of the key players in overexpression of IDO1 and Kyn. This alteration further leads to constitutive activation of AhR, perpetuating cell-growth. However, what is less well known is how the gut microbiota and their metabolites contribute to IDO1 expression, AhR activation, and immune cell balance. Though recently, the short chain fatty acid butyrate, produced by several commensal bacteria, was shown to decrease expression of IDO1 through reduction of STAT1 and its histone deacetylases inibitory activity, indicating that bacterial metabolites are able to control Trp metabolism [75].

The versatility of AhR binding has increased the research interest in its ability to regulate the immune system, intestinal homeostasis, and cancer progression. Under normal conditions, AhR activation results in transcription of genes, CYP1A1, CYP1B1 and AhRR (Aryl-Hydrocarbon Receptor Repressor), that control AhR activity in negative feedback loop. However, dysregulation of this feedback loop allows uncontrolled AhR activation, which is correlated with inflammatory processes including IBD, multiple sclerosis, cardiovascular conditions, and allergic responses, and carcinogenesis [76]. The multifaceted role of AhR in carcinogenesis is complex and only beginning to be understood. This, in part, is due to the ability of AhR to bind to many different ligand types, resulting in either enhancing or suppressing carcinogenesis [11,77]. Loss of AhR has been shown to enhance colon carcinogenesis, while re-establishing normal AhR activation inhibits the formation of polyps [48,62]. Reduced AhR expression has been seen in individuals with IBD, colitis, Crohn’s disease and CRC [78]. Alterations in Trp metabolism in part explain the reduced AhR availability; a hallmark of inflammatory disease states including CRC. Indeed, Crotti et al. demonstrated that alterations in Trp metabolism occur early in the pathogenesis of colon cancer preceding malignancy [36]. Specifically, it is understood that colon epithelial cells undergo changes in Trp metabolism within the tumor microenvironment as an adaptive mechanism to escape immune surveillance. While under normal physiology the AhR ligands exhibit a protective role by either inducing or by suppressing inflammation, in the context of carcinogenesis, loss of AhR or constitutive activation abrogates this protective function. Therefore, the metabolic end products of the Kyn pathway control the inflammatory process, in part through activation of AhR [36,79,80,81,82,83,84]. However, the mechanism that leads to these alterations in Trp metabolism and loss or dysfunctional AhR activity in colon carcinogenesis is not well understood.

The gut microbiome forms a symbiotic relationship which affects host physiology in several ways. These effects include maintaining gut homeostasis, activating, or suppressing immune function, metabolic and neurological homeostasis. Microbial communities promote host health not only by producing vitamins, degrading bile, and oxalates, but also by synthesizing anti-inflammatory metabolites. These bacterial metabolites can act as neurotransmitters, signaling and secretory molecules, immunoregulators, neurotoxic and antimicrobial molecules directly affecting host physiology. One such metabolite group are indoles, first discovered and isolated by a German scientist Adolf Von Baeyers in 1866 in an indigo reduction process [85]. Indoles are produced through bacterial degradation of dietary Trp by the action of bacterial enzyme tryptophanase (TnaA) or degradation of phenylalanine by phenyllacetate dehydratase (fldAIBC), though other undiscovered pathways may be involved [86,87,88,89]. Over 85 bacterial species express TnaA, with some Peptostreptococcus and Clostridium species containing fldAIBC gene clusters [2,85,88]. Multiple types of indole derivatives can be produced from Trp due to expression of species-specific enzymes. The most prominent bacterial indoles include indole-3-acetamide, indole-3-acetaldehyde, indole-3-pyruvate, indole-3-aldehyde, indole-3-aceticacid, tryptamine, indole-3-propionic acid, and indole acrylic acid. Indole and indole derivatives play an important signaling role between the host and the microbiota and are a critical component of bacterial function. Within the microbial communities in the gut, indole production affects spore formation, plasmid stability, biofilm formation, antibiotic tolerance, cell division, and virulence [13]. Within the host, indoles activate signaling pathways that result in changes in intestinal epithelial barrier function, reduce permeability, promote immune tolerance, evict pathogens, reduce inflammation, and, control mucin production [12,85,90,91,92,93,94,95]. Colonization of germ-free mice for 4 weeks with C. sporogenes or a mutant (fldC) lacking the an enzyme necessary for indole (indole-3-propionic acid (IPA)) production, resulted in serum concentrations of 80 μM in mice colonized with wild-type but undetectable levels in mice colonized with the mutant strain. Further, colonization with the fldC mutant increased gut permeability, and lead to higher levels of circulating neutrophils, T cells (CD4+ and CD8+) and sIgA (lumen) [86]. Together, these results indicate that microbiota are capable of producing biologically relevant levels of indoles, and inducing changes in innate and adaptive immune response that may be important in colon cancer pathogenesis.

Indolic compounds can also act as AhR ligands to promote both pro- and anti-inflammatory effects [17,96]. Indoles can reduce expression of pro-inflammatory signals, IL-8 and NF-κB, and promote expression of anti-inflammatory cytokines including IL-10 [95]. In addition, indolic compounds regulate intestinal homeostasis through induction of IL-22, which improves barrier function, however, in the context of cancer (later stages) IL-22 production can promote tumor progression [97,98]. Microbial indole metabolites are normally a consistent source of host AhR ligands, unlike dietary sources that are transient [99]. The majority of indoles and indole derivatives have demonstrated a protective effect against inflammatory diseases including CRC, with the exception of indoxyl sulfate, a uremic toxin involved in chronic kidney and vascular diseases [100]. Tight epithelial junctions, adequate mucin production and anti-microbial defenses are indispensable for a healthy host physiology; functions all governed via AhR activation by indoles [4,19,90]. Though a more thorough investigation of microbial indoles in early vs. later stages of colon carcinogenesis is warranted given the complex effects on downstream signaling pathways the can both suppress and promote carcinogenesis.

Multiple species of bacteria can produce AhR ligands. Lactobacillus reuteri and acidophilus produce 3IAld, which stimulates innate lymphoid cells to release AMP’s to defend against pathogens [57]. Clostridium Sporogenes can produce IPA, inhibiting the action of pro-inflammatory signaling molecules, NF-κB and TNF-α, and free radicals [94]. Clostridium sporogenes, Peptostreptococcus russellii, Peptostreptococcus anaerobius, and Peptostreptococcus stomatis can produce indoleacrylic acid (IA), shown to attenuate inflammatory response and improve barrier function by mucus production and goblet cell differentiation, potentially via AhR activation [88,101]. Bacteroides ovatus, Bacteroides fragilis, Bifidobacterium pseudolongum, Clostridium bartlettii, Clostridium difficile, and Clostridium lituseburense, among others can produce IA which aids in strengthening intestinal mucosal integrity, possible by activating AhR, and production of IL-22, by mechanisms not well understood [101,102]. Besides indolic ligands, bacteria are also capable of producing non-indole AhR ligands. Pseudomonas aeruginosa and mycobacterium tuberculosis are capable of activating AhR by releasing virulence factors including phenazines and phthiocol resulting in pathogen clearance [99,103]. Therefore, bacterial contribution to the pool of AhR ligands should not be overlooked. The mechanistic explanation to the microbial contribution may be limited, but the functional outcome of this contribution is significant, and is evident in the attention the field has received in the recent years.

As a result of disturbances in host physiology and microbial composition during carcinogenesis, the commensals can take on a pathogenic behavior resulting in an increased inflammation in the host as well as biofilm formation [104,105,106]. About 16–20% of all cancers are the result of pathogenic infections [107]. Patients with Crohn’s disease harbor pathogenic Escherichia coli that contribute to a pro-inflammatory state [108]. This pathogenic behavior can directly or indirectly influence changes in host IDO1 expression and cell proliferation [109]. Increases in Enterococcus faecalis and Escherichia coli enhance the production of intestinal inflammatory signaling molecules, IFN-γ and IL-4, capable of inducing increased expression of IDO1 and altering Trp metabolism [99,110,111]. Another species associated with CRC and IBD, is Fusobacterium nucleatum. F. nucleatum species isolated from patients with IBD are more invasive and more frequently found in adenomas and adenocarcinomas of the colon [112,113]. Further F. nucleatum elicits substantially greater amounts of pro-inflammatory TNF-α gene expression, which can lead to increased IDO1 activation [99,106,114,115]. Both F. Nucleatum and Peptostreptococcus anaerobius can attach to the cancer cells through cell surface attachment proteins, activating the PI3k-Akt pathway leading to cell proliferation [116,117]. Intriugingly, both of the species can also produce high levels of indoles from Trp metabolism. Therefore, the microbiome is a key contributor to inflammation through altering Trp metabolism [11] which is an important contributing step in colon carcinogenesis.

Changes in microbial indole production is also a characteristic of colon cancer. A reduction in indolic pathway activity was observed in the fecal samples of individuals with CRC. The study demonstrated a lower indole/Trp ratio in CRC when compared to healthy individuals, as well as a higher kyn/Trp ratio [109]. This alteration in the indolic pathway can lead to an increased inflammatory response via TNFα, IL-1β, and IL-6 in colon carcinogenesis, subsequently altering AhR activity [109]. AhR activation is reduced in diseases including IBD, liver disease, metabolic syndrome, autoimmune disease, and cancer, suggesting that alteration in Trp metabolism or indole production my contribute to AhR dysfunction [39,54,55,79]. Fecal samples from individuals with metabolic syndrome, obesity, Type-2 diabetes, and chronic intestinal inflammation also have significantly lower levels of microbial Trp metabolites, potentially contributing to reduced AhR activation. Specifically, individuals with obesity have a significantly higher concentration of Kyn and IDO1 in adipose tissue, which is associated with an imbalance in Th17/Treg cells; suggesting a mechanistic link between altered tryptophan metabolism, obesity and CRC pathogenesis [52,118]. Additionally, administration of microbial indolic metabolites, diindolylmethane or I3C (AhR ligands), reduces tumor formation in Apc^Min/+AhR^+/+ and Apc^Min/+AhR^+/- mice indicating that specific microbial indoles are protective against carcinogenesis [119]. Treatment with 6-Formylindolo [3,2-b] carbazole (FICZ), a Trp derived AhR ligand, is also effective at restoring intestinal integrity [52]. Intriguingly, Lactobacillus reuteri is capable of differentiating T cells into Tregs through AhR activation via indolic compounds, implying additional mechanism to reestablish epithelial barrier function [120]. Similarly, restoration of AhR activation, using L. reuteri, reestablished AhR activity in murine model of impaired metabolic syndrome by increasing AhR ligand availability [52]. Collectively, it is implied that a disrupted microbial Trp-indole-AhR pathway significantly contributes to the pathogenesis of CRC. Therefore, identifying the effects of bacterial indoles in the pathogenesis of colon carcinogenesis is important in understanding the impact of the microbiome and potentially developing therapeutics.

Apart from the Kyn and the indolic pathway, 1–2% of Trp can undergo hydroxylation to produce serotonin via the serotonin pathway. As a potent signaling molecule, having 14 classes of receptors, serotonin exhibit diverse effects on the immune function, nervous and endocrine system, psychological processes, blood clotting, metabolic homeostasis, bone metabolism, hematopoiesis, and epigenetic control [121,122]. While serotonin is well recognized as a neurotransmitter of the brain, about 95% of the serotonin is synthesizes and released by the enterochromaffin cells of the intestines, the largest neuroendocrine organ. With majority of serotonin produced in the gut, it plays an important role in maintaining normal gut function, including gut motility, absorption, and vasodilation [121,123]. Therefore, the role of serotonin in a healthy physiology is indispensable.

While serotonin is essential in healthy physiology, studies have demonstrated both protective and detrimental role of serotonin in colon carcinogenesis [124], where a detrimental role is taken over in response to an altered or impaired serotonin activity [125]. Serotonin exhibits both pro- and anti-inflammatory signaling activity in the intestines via activation of 5-HT 7 and 5-HT4 receptors, respectively [121]. Studies have also confirmed the role of serotonin in tumor growth, cell invasion, angiogenesis, and metastasis in CRC [126,127,128]. A β-catenin mutation (CtnnB1) occurring during early phases of stem cell differentiation into enteroendocrine cells, has shown to induce tumors expressing serotonin [127,129]. Activation of serotonin receptors, 5-HTR1B and 5-HTR2B stimulate tumor angiogenesis and cell proliferation [130,131]. Receptor 5-HT1D, by activating Axin1/β-catenin/MMP-7 pathway, also promote tumor cell invasion. The same study demonstrated the inhibition of tumor metastasis by administering 5-HT1D antagonist, GR127935↗, in a mouse model [125]. Early interventions with Mirtazapine, inhibitor of serotonin 2 C receptor (HTR2C), has shown to reduce tumor growth and prolong survival rate in tumor-bearing mice. This reduction in tumor growth was due to activation of immune response and restoration of serotonin levels [132]. The above evidence supports the dual role of serotonin in tumor prevention and proliferation, due in part to altered serotonin pathway, mutations, and inflammation.

The microbiota plays a significant role in regulating serotonin levels, synthesized and released by the enterochromaffin cells of the intestines [121]. Short chain fatty acids produced by the microbiota in response to saccharides in the diet, stimulate the enterochromaffin cells to produce and release serotonin [133]. Spore-forming bacterial have shown to produce metabolites that promote serotonin synthesis, mechanistically unknown. This microbiota-induced serotonin production can affect gut motility and intestinal homeostasis, suggesting a therapeutic intervention [134]. Several microbial species including Corynebacterium spp., Escherichia coli, and Streptococcus spp., can also produce serotonin [134]. Serotonin-like molecules are also secreted by Rhodospirillum rubrum, Bacillus cereus, Enterococcus faecalis, and Staphylococcus aureus [135]. Collectively, the intestinal and microbial serotonin production is involved in several functions including gut motility, platelet function, and immune response under normal physiological conditions [134,136]. However, serotonin uptake and production can become dysregulated in pathogenesis of several diseases including colon cancer [124,134].

Serotonin is essential in protecting against cellular damage and disease development. Serotonin activity protects the intestines from DNA damage [21,127] and induces signaling (HTr4 receptor; effect-gut motility) that aids in reducing the development of colitis and controlling the development of early CRC [137]. Under the conditions of immune homeostasis and a diverse microbiota, serotonin exerts a protective role [133,134,138]. A disruption in the balance of the immune system and the microbial composition allows serotonin to instead promote cell proliferation and angiogenesis [124,127,128]. Specifically decreased serotonin activity is associated with DNA damage, apoptosis, and constricted arterioles in CRC leading to a promotion of carcinogenesis [127,139,140]. A study comparing the metabolomic profile of cancer and normal tissues demonstrated that Trp metabolites; serotonin and 5-Hydroxy-3-indoleacetic acid [5-HIAA]) were only detected in normal tissue and absent in tumor tissue, indicating that the tumor microenvironment was actively metabolizing serotonin [141]. Collectively, the following evidence demonstrates the dual role of serotonin in promoting intestinal homeostasis and accelerating carcinogenesis.

Decarboxylation of Trp leads to production of tryptamine, a monoamine alkaloid with an indole ring in its structure [142]. Tryptamine is mainly found in the central nervous system, but can also be produced by bacterial, fungi, and plants [143]. Under normal physiology, tryptamine acts as a neurotransmitter, vasoconstrictor and vasodilator, anti-oxidant and antibacterial agent [144]. Monoamine oxidase (MAO)-mediated tryptamine metabolism also provides an endogenous source of AhR ligand, thereby inducing CYP1A1 expression [143]. Tryptamine can also induce the enterochromaffin cells to release serotonin, fluctuations of which have shown to play a significant role in pathology of inflammatory disease, a risk factor for colon carcinogenesis [145].

Tryptamine is also produced by the microbiome, which aids in colonic fluid secretion via activation of serotonin receptor HT-4, subsequently affecting colonic transit in a mouse model. Mechanism by which tryptamine increases colonic fluid secretion remains unclear, due in part to the structural similarities of tryptamine to serotonin and ligand-binding ability of tryptamine to serotonin receptors [23]. Given that ~50% of all patients undergoing chemotherapy for colon cancer experience chemotherapy-induce diarrhea, understanding the impact of microbial tryptamine production would be important to abrogate this serious side effect. Specific bacteria have been identified that can perform decarboxylation of Trp into tryptamine, a common practice in plants, but rare among the bacterial species [146]. These bacteria include Xenorhabdus nematophilus and Bacillus atrophaeus. Lactobacillus bulgaricus can also excrete tryptamine [147,148]. Therefore, engineered tryptamine producing bacteria may be a potential therapeutic intervention to increase serotonin levels and AhR ligand availability, but may have negative effects in the context of colon carcinogenesis and treatment.

Both host and bacterial-Trp metabolites play a significant role in colon cancer initiation and development. Increased expression of Kyn and IDO1, and reduced production of indoles have been detected in variety of cancers including CRC [149,150]. Increased activity of IDO1 in conjunction with Trp depletion, leads to cell cycle arrest in activated T lymphocytes. This increased IDO1 activity further leads to apoptotic T-cell death [151,152,153]. Together, evidence indicates that increased IDO1 activity and Trp depletion promotes immunosuppression in the tumor microenvironment, making IDO1 a promising target for therapy in combination with traditional chemotherapeutics [154,155]. Additionally, evidence demonstrates that engineered microbes targeting the IDO1 pathway have therapeutic benefits in animal models of colon cancer [156,157]. Therefore, small molecule compounds with immunoregulatory effects, including both chemical and microbial IDO1 inhibitors, seem hopeful as therapeutics. Yet, it is unknown whether microbial indole production is protective or deleterious in the context of increased IDO1 or AhR expression in colon cancer. We present the following therapeutic modalities to be considered for study as interventions among individuals with CRC and high IDO1 or AhR expression, and discuss the current understanding and opportunities for future research around Trp metabolism and indole production for therapeutic intervention in CRC.

Trp metabolism and indoles, both dietary and bacterial, are key factors in maintaining normal host physiology, including immune homeostasis, gut barrier function and microbial composition. However, under inflammatory conductions such as colon cancer, a series of alterations that affect host and microbial Trp metabolism occur. One key alteration includes overexpression of IDO1 leading to an increase production of Kyn. Kyn can constitutively activate AhR to enhance Treg activity, promoting immune escape. In contrast, Trp metabolites, including indoles, can activate intestinal AhR to suppress colonic stem and progenitor cell growth and reduce tumorigenesis in vivo; potentially ameliorating cancer stem cell proliferation. Furthermore, the changes in the gut microbiome during colon carcinogenesis also result in a paucity of indoles from Trp metabolizing bacteria. Subsequently, the reduced availability of protective indolic metabolites disrupt the symbiosis among microbial communities leading to an enhanced inflammation response. This heightened inflammatory condition further increases IDO1 activity driving a vicious cycle of tumor escape, proliferation, and metastasis. As such, increasing dietary indoles or promoting the growth of Trp metabolizing bacteria (e.g., Lactobacillus) to produce indoles may act to reduce the availability of Trp to cancer cells.

Harnessing the Trp-indole pathways in bacteria and the host hold the potential to attack the tumor cells through several different facets; Trp starvation, IDO1 inhibition, and indolic AhR activation. Further investigation is required to identify specific ligands that control AhR activation and mechanisms that reduce IDO1 expression or activity. Both dietary and microbial indoles hold promise as therapeutic adjuvants to target these pathways to inhibit colon carcinogenesis.