Role of Bacteria in the Development of Colorectal Cancer

Abstract

Colorectal cancer (CRC) is the second leading cause of cancer-related death in the United States. Once limited to older populations, the incidence of CRC in patients under the age of 50 years is increasing and the etiology for this is uncertain. One hypothesis lies on the impact of the intestinal microbiome. The intestinal microbiome, composed primarily of bacteria but also viruses, fungi, and archaea, has been shown to regulate CRC development and progression both in vitro and in vivo. In this review, the role and intersection of the bacterial microbiome in various stages of clinical CRC development and management are discussed beginning with CRC screening. Various mechanisms whereby the microbiome has been shown to modulate CRC development including the influence of diet on the microbiome, bacterial-induced injury to the colonic epithelium, bacterial-produced toxins, and alteration of normal cancer immunosurveillance by the microbiome are discussed. Finally, the influence of microbiome on the response of CRC to treatment is discussed while highlighting ongoing clinical trials. The complexities of the microbiome and its role in CRC development and progression have become apparent and will require ongoing commitment to translate laboratory findings into meaningful clinical results that will aid more than 150,000 patients that develop CRC every year.

The importance of the microbiome, the collection of microbes (bacteria, viruses, fungi, and archaea) that have a symbiotic, commensal, and pathobiont relationship with its host, in cancer development has been clarified and expounded upon in recent years. Notably, the influence of the microbiome on cancer development, progression, and treatment response has been documented for a variety of cancers. However, the role of microbiome in colorectal cancer (CRC), the second leading cause of cancer-related death in the United States, remains the seminal body of work on this topic. The reason is likely because of the logical reasoning that local microbes could have an impact on CRC and the ease from which samples can be collected to test this hypothesis.

While only Helicobacter pylori (gastric cancer, gastric lymphoma), Human papillomavirus (cervical, oropharyngeal, and anogenital cancers), Hepatitis B and C viruses (hepatocellular cancer), Epstein-Barr virus (nasopharyngeal cancer and Burkitt lymphoma), Human herpesvirus type 8 (Kaposi sarcoma), Schistosoma haematobium (bladder carcinoma), Human T-cell lymphotropic virus (adult T-cell leukemia and lymphoma), Opisthorchis viverrine (cholangiocarcinoma), and Clonorchis sinensis (cholangiocarcinoma) have been recognized by the World Health Organization as directly contributing to cancer development, ¹ microbes have been implicated in the development of CRC through direct cellular injury, production of toxic substances, and alterations of the host immune response as well. Determining what mechanism(s) is the primary driver, as opposed to a single causative microbe, is likely to be more impactful as it is likely that the status of the bacterial community structure will be the instigator of CRC development rather than a single microorganism.

In this review, the preceding topics are explored and presented with a potential clinical focus and applicability in mind. As the majority of studies involve the bacterial microbiome, this will be the focus of discussion, recognizing that the influence of the virome, fungome, and other lesser microorganisms remains relatively unstudied but may yield just as important information as the microbiome. Given that the incidence of CRC has been increasing and that the median age at diagnosis has been decreasing, affecting younger patients, this is an important and germane topic for clinicians to be aware of. ^{2 3}

Microbiome and Colorectal Cancer Screening

Current recommendations for CRC screening take into account risk factors for CRC development based on age, family history, prior polyps, and inflammatory bowel disease (IBD), among others. ³ For low-risk individuals, the use of tumor DNA detection techniques has provided a patient-friendly method of screening and early detection but does rely on the presence of malignancy. However, the false positive rate of these tests is upward of 76%, necessitating a diagnostic colonoscopy. ⁴ Determining associations of microbiome community structure changes or the presence of specific microbes may provide another screening methodology to detect high-risk individuals who have yet to develop CRC. Colorectal carcinoma is accepted to develop in a stepwise fashion beginning with hyperplastic polyps that accumulate genetic aberrations in a progressive fashion to cancer. ⁵ Prior research has demonstrated that as these polyps progress into carcinoma, the microbial community structure changes and provides a potential opportunity to detect by non-invasive means premalignant lesions before the onset of frank carcinoma. ^{6 7} For example, species richness was reduced in stool samples from patients with conventional adenomatous polyps compared with controls without any polyps. However, there was no difference in diversity or overall composition in stool from patients with sessile serrated adenoma or hyperplastic polyps did not differ from control, albeit the authors admitted they had a small sample size. ⁸ Additionally, Rezasoltani and colleagues reported that specific bacterial species ( Fusobacterium nucleatum, Enterococcus faecalis, Streptococcus bovis, Enterotoxigenic Bacteroides fragilis [ETFB], and Porphyromonas spp ) were more prevalent in adenomatous polyp cases compared with sessile serrated polyp, hyperplastic polyp, and normal control cases. ⁹ Fusobacterium spp. has also been shown to be more prevalent in adenomas compared with adjacent normal tissue control. ¹⁰ The theoretical ability to detect cancer at its earliest stages has also been confirmed recently in pancreatic cancer. ¹¹ Several clinical trials seek to exploit these noted changes to identify the process of colorectal carcinogenesis earlier ( Table 1 ). How polyps develop and accumulate their mutational burden is not well-delineated, but these studies provide some initial insight. The microbiota community structure is dictated by many influences, diet being one of them. Dietary differences between individuals and geographical locations may therefore explain differences in CRC incidence through alterations of the microbiome. ¹²

Table 1. Selected trials evaluating the role of the microbiome in colorectal cancer screening.

Study name	Clinical trials id	Study type	Microbiome source
Pilot study of the fecal microbiome in the Shanghai population	NCT01778595	Observational	Feces
Analysis of intestinal microflora combined with DNA methylation in stool to detect colorectal cancer (IMMDC)	NCT04302363	Observational	Feces
Gut microbiota-based tool for the detection of colorectal cancer in positive patients for the fecal occult blood test	NCT04662853	Observational	Feces
Microbiome test for the detection of colorectal polyps and cancer	NCT02141945	Observational	Feces

Dietary Influence on the Microbiome and CRC Development

Much work has focused on the role of diet in CRC development. ^{13 14 15} Dietary intake secondarily impact the intestinal microbiome and likely is an upstream mediator of CRC development, acting through modulation of gut microbiota. Additionally, dietary differences associate with microbiome changes, giving credence to the theory that dietary causation is likely upstream to the actual inciting carcinogenic event of the microbiome. For example, microbes can metabolize dietary sulfur into hydrogen sulfide (H ₂ S), a known carcinogen ¹⁶ and capable of disrupting the protective colonic mucosal layer. ¹⁷ Nguyen and colleagues reported on the effect of a high microbial sulfur diet, that is predicted to enrich for sulfur-metabolizing bacteria, on colonic polyp formation. The high microbial sulfur diet was defined by intake of processed meats, liquor, and low-calorie drinks. They demonstrated that a high sulfur microbial diet was associated with an increased risk to develop tubulovillous/villous and tubular adenomas (age-adjusted odds ratio [OR] of 1.61 [ p  = 0.04] and 1.27 [ p  = 0.05], respectively) before the age of 50. ¹⁸ Additionally, this high sulfur diet was associated with increased risk of conventional adenoma formation in the proximal colon (OR 1.6, p  = 0.009) but not distal colon or rectum. A follow-up study demonstrated that a high microbial sulfur diet resulted in an increased risk of CRC (age-adjusted OR 1.21, p  = 0.008) which was driven by an increase in distal colon and rectal cancers (age-adjusted OR 1.53, p  < 0.0001). Interestingly, Bilophila wadsworthia and Erysipelotrichaceae bacterium 21_3 , both sulfur-metabolizing bacteria and shown to be associated with CRC, were enriched in higher sulfur microbial diets. ^{15 19 20 21}

An inverse relationship has been shown between dietary fiber intake and the incidence of CRC and colon adenoma formation. ^{22 23} A notable increase in the incidence of CRC in patients under the age of 50 is suspected to be partly related to reduced fiber intake. This appears secondary to the effects that fiber has on the gut microbiota. ²⁴ Bacteria ferment dietary fiber into the short-chain fatty acids (SCFA): butyrate, propionate, and acetate. These SCFAs have critical roles in colonic mucosal health, immune function, and serve as a colonocyte energy source. ^{25 26 27} Notably, butyrate has been demonstrated to have potent anti-cancer properties through inhibition of proliferation and induction of apoptosis in vitro. ^{28 29} Similar to butyrate, the SCFAs acetate and propionate have similar, although to a lesser extent, anti-inflammatory properties and the ability to inhibit histone deacetylase which results in inhibition of cancer cell proliferation and apoptosis induction. ³⁰ Interestingly, compared with non-cancer controls, patients with CRC had a decrease in bacteria capable of fermenting fiber into butyrate, notably Clostridium , Roseburia , and Eubacterium species ( p <0.001), and a concomitant decrease in SCFA levels. ³¹ Recently, the butyrate-producing bacteria, Clostridium butyricum , was demonstrated to inhibit colonic tumor formation in a spontaneous model of CRC, the APC ^min/+ mouse model. ³² This appears likely through inhibition of the oncogenic Wnt/β-catenin pathway and alteration of the microbiome to increase the richness of SCFA-producing bacteria. Given the above findings, non-pharmaceutical manipulation of the microbiome through dietary modulation or probiotic/bacterial supplementation is an appealing intervention to reduce CRC incidence that could have broad population health implications. Ongoing clinical trials seek to modify the diet for the sake of microbiome manipulation to reduce CRC risk ( Table 2 ).

Table 2. Selected trials studying the effects of diet on the microbiome and colorectal cancer.

Study name	Clinical trials ID	Study type
Meat-based versus Pesco-vegetarian diet and colorectal cancer (MeaTIc)	NCT03416777	Interventional Randomized
Exploring the effects of a high chlorophyll dietary intervention to reduce colon cancer risk in adults (M3G)	NCT03582306	Interventional Randomized
Diet modulation of bacterial sulfur and bile acid metabolism and colon cancer risk	NCT03550885	Interventional Randomized
Fiber to reduce colon cancer in Alaska native people	NCT03028831	Interventional Randomized

Microbiome-Mediated Injury to the Intestinal Mucosal Barrier

The colonic epithelium and associated mucosal lining are in constant contact with ingested toxins and aim to help establish a symbiont and pathobiont relationship with the host and its microbiota. The colon mucus layer is actually composed of two layers, with the inner layer being devoid of bacteria and in direct contact with the colonic epithelium. ^{33 34} Disruption of this barrier can result in repeated epithelial injury that over time can lead to genetic aberrations that are likely to progress to CRC. The role of the microbiota in this process has gained much attention recently as changes in the microbiome of patients with IBD have been shown to induce inflammation. ³⁵ Interestingly, germ-free mice (mice born and raised without exposure to microbes) do not develop IBD ³⁶ and transplantation of human stool from healthy volunteers into rats with dextran sodium sulfate-induced colitis reduced the inflammatory state of the colon and restored the natural diversity of the intestinal microbiota. ³⁷ Building off the importance of SCFAs, butyrate has been shown to activate the mucin-encoding gene, MUC-2, through regulation of prostaglandin synthesis. This increased mucin production provides a protective barrier to the colonic epithelium and alterations in the intestinal microbiota result in reduced mucosal protection and allowance of bacteria and antigens to cross the epithelial barrier. ³⁸ Dietary emulsifiers, present in processed foods, have been shown to increase the ability of Escherichia coli to translocate across an in vitro model of the membranous barrier of Peyer's patches (known as M cells). ³⁹ Additionally, the dietary emulsifiers, carboxymethylcellulose, and polysorbate-80, induced colitis in mice predisposed to this condition. ⁴⁰ Even in wild-type mice, these emulsifiers were able to increase gut permeability as confirmed with increased serum antibody levels of lipopolysaccharide (LPS) and flagellin, both components of gram negative bacteria. Finally, the bile acid deoxycholic acid (DCA) is increased in patients consuming a high fat diet, a recognized risk factor for CRC development. Not only is DCA capable of disrupting the integrity of the colonic mucosal and accelerating carcinogenesis in the APC ^min/+ mouse model of colon cancer, ⁴¹ but elevated levels of DCA are associated with microbiota changes found in CRC. ⁴² Taken together, the intestinal microbiome interacts directly with the protective mucosal layer of the colonic epithelium. Disruptions to either homeostasis of the microbiome or integrity of the mucosa can lead to increased inflammation and colon cancer formation, demonstrating the fine balance between these host factors.

Interplay of the Microbiome on Immunosurveillance of Colorectal Cancer

An important role of the host immune system is malignant cell surveillance but a hallmark of cancer is the ability of neoplastic cells to avoid immunologic detection and destruction. ⁴³ To balance these antagonistic processes, the intestinal microbiome has been shown to have a multifaceted impact on the immune system. The intestinal wall is rife with immune cells, particularly in Peyer's patches located within the lamina propria. In this location, both innate and adaptive immune cells reside and participate in immunosurveillance by secretion of antimicrobial peptides and pattern recognition receptors (such as Toll-like receptor and Nod-like receptor) that recognize pathogen-associated molecular patterns and damage-associated molecular patterns, such as LPS and flagellin mentioned previously. ^{44 45 46} The response of the local colonic immune system to resident bacteria is dependent on multiple factors that can alter microbiome-specific T-cell responses, including specific bacteria. Yu and colleagues demonstrated that two mouse colonies with distinct microbiome communities developed colon cancer at different rates using the azoxy-methane-dextran sodium sulfate (AOM-DSS) model of colon cancer secondary to alterations in the intratumoral immune environment. ⁴⁷ In these distinct tumors, it was noted the intratumoral T cells in mice with increased tumors had increased exhaustion. Furthermore, these mice had increased CD8 ⁺ IFNγ ⁺ T cells in the lamina propria prior to tumor formation but reduced CD8 ⁺ IFNγ ⁺ T cells in tumors and adjacent tissues compared with mice with fewer tumors. In a hallmark study, Ivanov and colleagues demonstrated that segmented filamentous bacteria (SFB) were able to induce Th17 cells (CD4 ⁺ T-cells characterized by IL-17 and IL-22 production) in the lamina propria of the small intestine. ⁴⁸ Colonization with SFB was associated with increased expression of inflammatory and anti-microbial defensive genes. This study demonstrated the ability of bacteria to regulate mucosal immunity and inflammation. Recently, Helicobacter hepaticus has been recognized as an immunogenic intestinal bacterium capable of mitigating CRC formation in a mouse model. ⁴⁹ Mice colonized with this bacterium had reduced colon cancer formation in the AOM-DSS model of colon cancer. The mechanism for this reduction appears related to the ability of H. hepaticus to induce T follicular helper cell infiltration into colon tumors and expansion of local lymphoid structures adjacent to colon tumors to facilitate clearance. The intestinal microbiome therefore has the capability to alter the immune surveillance of cells in the lamina propria as well as modulate intratumoral immune cell infiltration and activity. Such information will likely prove instrumental in identifying tumors that will be responsive to immunotherapy.

Toxigenic Bacteria in Colorectal Cancer Development

Given the abundance of bacteria in intestinal lumen and their direct contact to the colonic epithelium, the ability of microbes to cause direct injury to epithelium is a natural theoretical extension and one that has been well studied. Three specific bacteria have been identified that have unique mechanisms of direct cellular injury. First, E. coli is prevalent but of low abundance in the gastrointestinal tract. While a variety of strains exist that cause a human disease, E. coli that harbor the polyketide synthase ( pks ) gene island encodes 19 genes responsible for production of colibactin, a potent genotoxin. Colibactin is comprised of dual cyclopropane motifs which induce double-strand DNA breaks and cross links through alkylating adenine residues. ⁵⁰ This induction of DNA damage results in colibactin-mediated mutagenesis. E. coli harboring the pks genotoxin island have accelerated colon carcinogenesis in a mouse model of CRC. ^{51 52} Recently, exposure of colonic organoids with pks ⁺ E. coli has been shown to result in a distinct mutational signature that mirrors the mutational signature from patient CRC specimens. ⁵³ Of note, the adenomatosis polyposis coli ( APC ) gene, the most commonly mutated gene in CRC, harbored the most mutations that matched this signature (>5%).

Second, ETBF has been shown to function to promote colon carcinogenesis through its B. fragilis toxin (BFT). ⁵⁴ While there are both non-toxigenic and toxigenic strains of B. fragilis , the pathogenicity of ETBF is based on its BFT which is a matrix metalloprotease consisting of three isoforms. Notably, isoforms BFT-1 and BFT-2 have been detected in CRC tumor specimens and impact colon carcinogenesis by three distinct mechanisms. ^{55 56} The BFT is also capable to induce reactive oxygen species that subsequently cause direct DNA damage. Next, BFT has been shown to upregulate the transcription factors STAT3 and NF-κB. This results in immunoregulation of anti-tumor function, through IL-17 mediated tumorigenesis, upregulation of TNFα and IL-8, and increased recruitment of protumorigenic myeloid cells that suppress antitumor immunity. Finally, BFT directly activates the known oncogenic pathways of Ras/mTOR, p38, and c-myc resulting in increased intestinal cell proliferation, tumor growth, decreased tumor apoptosis, and increased intestinal cell permeability.

Third, Fusobacterium nucleatum , while not specifically secreting toxic compounds as pks ⁺ E. coli or BFT-producing ETBF, has been associated with a variety of cancers, including CRC. ^{57 58 59} The adhesion molecule, FadA, expressed by F. nucleatum appears to direct the carcinogenic profile of this bacteria. Notably, the FadA adhesion molecule is capable of binding E-cadherin whereby activation of the β-catenin and Wnt pathways occurs, resulting in increased cellular proliferation. ⁶⁰ Additionally, Fap2 is part of a family of secretion systems that gram negative bacteria, such as F. nucleatum , utilize to secrete proteins. There are nine families of secretions systems but F. nucleatum only possesses type V, and not all strains express it. ⁵⁶ Recruitment of F. nucleatum to cancer cells is facilitated by the expression of d-galactose-β(1–3)- N -acetyl-d-galactosamine (Gal-GalNAc ) on cancer cells whereby Fap2 is able to bind to and incite a proinflammatory response. ⁶¹ Additionally, Fap2 is capable of binding to the immune checkpoint inhibitor, TIGIT. Binding to this receptor results in impaired anti-tumor function of immune cells that express TIGIT: CD4, CD8, and natural killer cells. This results in decreased immune-mediated cytotoxicity to CRC and increased immune cell apoptosis. ⁵⁷ Finally, LPS from F. nucleatum and gram negative bacteria in general, can act as a potent virulence factor for carcinogenesis. Utilizing colon cancer cell lines in vitro and a CRC mouse model, LPS interacts with the Toll-like receptor 4 (TLR4) pattern recognition receptor to upregulate microRNA-21, resulting in increased cancer cell and tumor proliferation and growth. ⁶² The LPS-TLR4 interaction also been shown to increase autophagy and chemotherapeutic resistance in a CRC model. ⁶³ The toxigenic properties of F. nucleatum thus appear to bridge the gap of many carcinogenic processes including anti-tumor immunosuppression, activation of oncogenic pathways, autophagy, and cellular recruitment.

Microbiome and Response of Colorectal Cancer to Treatment

Much interest has been generated recently regarding the role of the microbiome in response to treatment of cancers. Hallmark studies demonstrating the role of microbiome in immunotherapy response to CTLA4 and PD-1/PD-L1 inhibition in melanoma and lung cancer support the critical interaction between these systems. ^{64 65 66} From a clinical standpoint, immune checkpoint blockade has limited use in CRC, as microsatellite-stable tumors (the majority of CRC) do not respond to these therapies in contrast to microsatellite-instability high (MSI ^high ) tumors. ⁶⁷ How the microbiome can influence anti-tumor response in all CRC, independent of MSI status, is an area of active interest ( Table 3 ). Recent research has demonstrated that Bifidobacterium pseudolongum , isolated from mouse tumors that responded to immune checkpoint blockade, was able to increase intratumoral IFNγ-expressing CD4 cells in mice with colon cancer tumors. ⁶⁸ In the mice colonized with B. pseudolongum , metabolite analysis revealed an increase in inosine which was also seen in mice colonized with an 11-member consortium of bacteria isolated feces from healthy human donor feces that was able to elicit an increase in IFNγ CD8 lymphocytes. This consortium resulted in improved response to immune checkpoint blockade of MC38 colon cancer xenografts with concomitant increased inosine in the stool of these animals. These studies demonstrate that the microbiome can alter response of colon cancer to immunotherapy and may provide an entry point for future studies. The microbiome has also been shown to impact the response to more traditional chemotherapy used in CRC. ⁶⁹ The chemotherapy irinotecan has been shown to have activity in CRC and a component of the common FOLFIRI chemotherapy regimen. It has been demonstrated that the gut microbiome can convert byproducts of irinotecan metabolism into their original active form. This results in dose-limiting diarrhea that prevents appropriate dosing and timing of therapy for patients with CRC. ^{70 71} In a more direct manner, F. nucleatum has been shown to be involved in resistance to both 5- f luorouracil (5- F U) and ox aliplatin therapy, components of both the FOL F OX and FOL F IRI CRC chemotherapeutic regimens. The mechanism of this resistance is based on activation of autophagy through the Toll-like receptor and MyD88 pathways. ^{56 63} These examples are a growing list of members of the intestinal microbiome that can impact treatment efficacy in CRC. Many other examples exist in non-CRC malignancies which demonstrate the important interaction that the microbiome has with medications, an emerging field of research termed pharmacomicrobiomics. ⁷² This active area of investigation seeks to harness the microbiome for purposes of drug metabolism, efficacy, and side effect mitigation.

Table 3. Selected trials studying the interaction of the microbiome in treatment of colorectal cancer.

Study name	Clinical trials Id	Study type
Microbiotic product to promote microbiome health and improve chemotherapy delivery	NCT05296681	Interventional Randomized
FMT combined with immune checkpoint inhibitor and TKI in the treatment of CRC patients with advanced stage	NCT05279677	Interventional
COLON-MD: Colon cancer longitudinal study (COLON-MD)	NCT04751448	Observational
Gut microbiome in colorectal cancer (GO)	NCT04054908	Observational
Treatment of colorectal liver metastases with immunotherapy and bevacizumab (CLIMB)	NCT03698461	Interventional

Conclusion

The importance of the microbiome–cancer relationship, termed as oncobiome, ⁷³ is no longer an area of debate given the variety of cancers that the microbiome has been shown to play a role in. Progress in a variety of technologies and reductions in their cost, such as sequencing, has facilitated the rapid growth in this arena. Beyond mere associations, the intestinal microbiome is now accepted to have an intimate relationship with CRC development. While a single causative microbe is not likely to be identified that results in CRC development, a better understanding of the intestinal microbiome community structure will yield important details on the pathogenesis of this disease. From a clinical standpoint, it is important to be familiar with the concepts presented in this review as rapid progress is being made to harness and modify the microbiome of each patient for CRC screening, prevention, and treatment. Additionally, clinicians will care for patients who take probiotics but must realize that this unregulated field has not, at present, shown benefit in CRC prevention or treatment. In fact, some probiotics contain the E. coli strain, Nissle 1917 , which harbors a pks island that is responsible for colibactin as described earlier. Current gaps in knowledge regarding the microbiome and the care of CRC patients include a greater insight into bacterial metabolites that modulate CRC development, progression, and treatment response. Additionally, progress in bioreactor technology is needed to recapitulate the complex microbiome communities that potentiate or mitigate CRC. With these bioreactors, a greater understanding of the community interaction will be acquired for patient microbiome manipulation and potential treatment. Finally, success of immunotherapy in other cancers has not necessarily applied to CRC despite the known involvement of the immune system. Further knowledge is needed regarding how the microbiome alters the CRC tumor microenvironment and immune response. Taken together, research into the role of microbiome and CRC development has made enormous strides over the past 20+ years and aims to be one of the first cancers whereby treatment, and cure, is dictated by individual microbiomes.